Lipid Biochemistry - The Journal of Biological Chemistry

HISTORICAL PERSPECTIVES

Lipid Biochemistry

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The Journal of Biological Chemistry TABLE OF CONTENTS

2010 HISTORICAL PERSPECTIVES ON LIPID BIOCHEMISTRY

PROLOGUE

H20 The Prostaglandins, Sune Bergstro¨m and Bengt Samuelsson

H1 JBC Historical Perspectives: Lipid Biochemistry. Nicole Kresge,

H23 Biotin-dependent Enzymes: the Work of Feodor Lynen

Robert D. Simoni, and Robert L. Hill

H25 Acetyl-CoA Carboxylase and Other Biotin-dependent Enzymes:

the Work of M. Daniel Lane

CLASSICS H3 The Biosynthetic Pathway for Cholesterol: Konrad Bloch H6 The ATP Requirement for Fatty Acid Oxidation: the Early Work

of Albert L. Lehninger H8 The Kennedy Pathway for Phospholipid Synthesis: the Work of

Eugene Kennedy H11 Fatty Acid Synthesis and Glutamine Synthetase: the Work of

Earl Stadtman H14 A Role for Phosphoinositides in Signaling: the Work of Mabel R.

H28 The Role of the Acyl Carrier Protein in Fatty Acid Synthesis: the

Work of P. Roy Vagelos H30 How Aspirin Interferes with Cyclooxygenase Activity: the Work

of William L. Smith H33 30 Years of Cholesterol Metabolism: the Work of Michael

Brown and Joseph Goldstein H37 Salih Wakil’s Elucidation of the Animal Fatty Acid Synthetase

Complex Architecture H40 N-Myristoyltransferase Substrate Selection and Catalysis: the

Work of Jeffrey I. Gordon

Hokin and Lowell E. Hokin H16 The Selective Placement of Acyl Chains: the Work of William

E. M. Lands H18 Lipid Storage Disorders and the Biosynthesis of Inositol

Phosphatide: the Work of Roscoe Brady

REFLECTIONS H42 Hitler’s Gift and the Era of Biosynthesis. Eugene P. Kennedy H55 The Biotin Connection: Severo Ochoa, Harland Wood, and

Feodor Lynen. M. Daniel Lane

JOURNAL OF BIOLOGICAL CHEMISTRY

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PROLOGUE

This paper is available online at www.jbc.org © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in the U.S.A.

JBC Historical Perspectives: Lipid Biochemistry* Nicole Kresge, Robert D. Simoni, and Robert L. Hill

Lipids are often broadly, and poorly, defined as biomolecules that are insoluble in water but soluble in organic solvents. They are structurally quite diverse, covering pigments, vitamins, fatty acids, cholesterol, phospholipids, sphingolipids, and many others. The Journal of Biological (JBC) Classic articles selected for this collection fall into two general categories: lipid biosynthesis and lipid signaling. Full accounts of the research and attribution can be found in each Classic.

Fatty Acid Synthesis Early work on fatty acid synthesis was done by Horace A. Barker and Earl R. Stadtman, who, in 1949, published a JBC paper examining the synthesis of short chain fatty acids by a species of bacteria called Clostridium kluyveri. The pair took advantage of the newly available 14C isotope and used it to label acetate and demonstrate that fatty acid synthesis is accomplished by the multiple condensation of 2-carbon molecules. Stadtman later developed an in vitro extract to study the enzymology of fatty acid synthesis. The detailed enzymology of fatty acid synthesis came, in part, from the work of P. Roy Vagelos and Salih Wakil. Using Escherichia coli extracts in which all of the enzymes of fatty acid synthesis were soluble, Vagelos was able to define the individual steps in long chain fatty acid synthesis and found that acyl carrier protein (ACP) was the small protein to which growing acyl chains were attached during the elongation cycle. He published his findings in 1965 as a series of JBC papers, two of which are reproduced as JBC Classics. Wakil, who contributed to the studies using the E. coli soluble system, also went on to examine fatty acid synthesis in animals, where the enzyme activities are part of a multifunctional enzyme complex. This work was published as a series of JBC papers in 1983. Adding to the studies done by Vagelos and Wakil, M. Daniel Lane, initially working with Feodor Lynen, described the first step in fatty acid synthesis, which involves the conversion of acetyl-CoA to malonyl-CoA by the enzyme acetyl-CoA carboxylase. He also explained the role of the vitamin biotin in carboxylation reactions. Phospholipid Synthesis One of the pathways for phospholipid synthesis was determined, in part, by Eugene P. Kennedy and his colleagues. However, Kennedy began his research career working not on lipid biosynthesis but on fatty acid oxidation with Albert Lehninger. In the mid-1940s, they published results in the JBC that showed that ATP was required to “activate” fatty acids for degradation. Later, Kennedy turned his attention to synthesis reactions and made the important discovery that CTP is required for phospholipid synthe* To cite articles in this collection, use the citation information that appears in the upper right-hand corner of the first page of the article.

sis. He and his graduate student Samuel Weiss eventually determined that intermediary formation of CDP-choline was occurring in the reaction. Using 14C to label the cytidine coenzymes, the pair proved that CDP-choline and cytidine diphosphate ethanolamine were activated forms of phosphorylcholine and phosphorylethanolamine and were precursors of lecithin and phosphatidylethanolamine. This scheme of phospholipid synthesis became known as the “Kennedy Pathway.” In 1958, William E. M. Lands, who is best known for his discovery of the phospholipid retailoring or “Lands” pathway, published results in the JBC that suggested “the diglyceride unit of the phospholipids is metabolically different in some respect from that of the triglycerides.” This initial finding led Lands to publish a series of papers in the JBC describing the selective placement of acyl chains by phospholipid acyltransferases.

Cholesterol Synthesis and Regulation The synthesis of cholesterol has a long and impressive history in the JBC. Nobel laureate Konrad Bloch and his colleagues made important contributions to the understanding of the synthetic pathway for cholesterol, which he published in the JBC in the mid-1940s. Eventually, through the combined efforts of Bloch, John Cornforth, and George Popja´k, the origin of each of the 27 individual carbon atoms of cholesterol (from the methyl or carboxyl group of acetate) was established. Bloch also aided in the identification of several important landmarks in the series of more than 30 reactions in the biosynthesis of cholesterol, including the intricate and fascinating cyclization of squalene to lanosterol. The regulation of cholesterol synthesis also has a history that is reflected in the JBC. A 1974 paper by Nobel laureates and long time collaborators Michael S. Brown and Joseph L. Goldstein established the role of LDL as a major regulator of cholesterol synthesis. This pioneering work also revealed the existence of a cell surface receptor for LDL, shed light on the process of receptor-mediated endocytosis, and explained the receptor defect in the human genetic disease, familial hypercholesterolemia. Lipid Signaling Phosphoinostides—The role of lipid molecules in signaling has also enjoyed a lot of coverage in the JBC. Lowell and Mable Hokin’s two papers, published in 1953 and 1958, demonstrated that the acetylcholine-stimulated secretion of amylase from pancreas slices caused the incorporation of 32P into phosphoinositides. This so-called “PI Effect” laid the groundwork for thousands of studies on the role of phosphoinositides in signaling. Roscoe O. Brady studied the synthesis of inositol phosphatides, and in 1958, he published a JBC paper showing that an enzyme system catalyzed the incorporation of inositol into inositol phosphatide in the presence of Mg2⫹ and CDP or cytidine JOURNAL OF BIOLOGICAL CHEMISTRY

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PROLOGUE: Lipid Biochemistry 5⬘-phosphate. From these results, Brady proposed a mechanism for the synthesis of inositol phosphatide in which CDP is transphosphorylated to form CDP-D-␣,␤-diglyceride, which then reacts with the hydroxyl group of inositol to form inositol phosphatide. Prostaglandins—The prostaglandins are another class of important lipid signaling molecules. Nobel laureates Sune Bergstro¨m and Bengt Samuelson were pioneers in establishing the biosynthesis and structures of several of these molecules. They published some of these studies in the JBC in the mid-1960s. One of the more fascinating roles that prostaglandins play is in mediating inflammation. In the early 1970s, John Vane showed that aspirin and other anti-inflammatory drugs inhibit prostaglandin synthesis. JBC Associate Editor William L. Smith went on to show that aspirin blocks cyclooxygenase (COX1) by acylating a serine residue.

Lipid Modification of Proteins Many proteins, particularly membrane proteins, are modified by post-translational covalent lipid attachment. Jeffrey

JOURNAL OF BIOLOGICAL CHEMISTRY H2

Gordon, who has two Classic papers, has made significant contributions to our knowledge about protein myristoylation, the post-translational process by which a myristoyl group is covalently attached via an amide bond to the ␣amino group of an N-terminal glycine residue of a nascent polypeptide.

Reflections This collection also contains two JBC Reflections. The first, titled “Hitler’s Gift and the Era of Biosynthesis,” by Eugene P. Kennedy, discusses how the war brought on a flow of students to America, which caused a surge in biochemistry research and biosynthetic studies there. In the second article, M. Daniel Lane talks about his research and the circumstances that brought him into contact with three great biochemists: Severo Ochoa, Harland Wood, and Feodor Lynen. We hope you enjoy this collection that we have assembled for you.

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 280, No. 10, Issue of March 11, p. e7, 2005 Printed in U.S.A.

Classics A PAPER IN A SERIES REPRINTED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005

JBC Centennial 1905–2005 100 Years of Biochemistry and Molecular Biology

The Biosynthetic Pathway for Cholesterol: Konrad Bloch On the Utilization of Acetic Acid for Cholesterol Formation (Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625– 636) The Utilization of Acetic Acid for the Synthesis of Fatty Acids (Rittenberg, D., and Bloch, K. (1945) J. Biol. Chem. 160, 417– 424) The Biological Conversion of Cholesterol to Pregnanediol (Bloch, K. (1945) J. Biol. Chem. 157, 661– 666) Konrad Emil Bloch (1912–2000) was born in Neisse, eastern Germany (now Nysa in Poland). Growing up he was more interested in engineering and natural sciences than chemistry, but an organic chemistry course taught by future Nobel laureate Hans Fischer at the Technische Hochschule in Munich provided a turning point. Bloch said of Fischer’s class, “As he presented it, the subject matter was fascinating, the organization superb, and the delivery monotonous” (1). Despite Fischer’s monotone delivery, Bloch was influenced enough to become a chemistry student in his laboratory.1 In early 1934, Bloch was told by Nazi authorities, in line with new racial laws, that he could no longer study at the Technische Hochschule. Fischer managed to arrange for Bloch to work at the Schweizerisches Ho¨henforschung’s Institut in Davos. There, he studied the lipids of human tubercle bacilli and was able to show that a previous report of the presence of cholesterol in this organism was erroneous. This was Bloch’s first encounter with cholesterol, a subject in which he would eventually play a great role. In 1936, with the help of R. J. Anderson at Yale University, Bloch immigrated to the United States and started working with Hans Clarke at the College of Physicians and Surgeons (P & S), Columbia University. He received his Ph.D. a year and one-half later, after completing a relatively straightforward piece of research on amino acid chemistry, and was then invited by Rudolf Schoenheimer to join his group. Also in Schoenheimer’s laboratory at this time was a scientist named David Rittenberg who had done his graduate work on deuterium with Harold Urey. Shortly after receiving his degree, Rittenberg approached Schoenheimer with the prospect of using deuterium as a biological tracer. This resulted in the publication of several seminal papers authored by Rittenberg and Schoenheimer on the use of deuterium to study metabolism, which was the subject of a previous Journal of Biological Chemistry (JBC) Classic (2). Schoenheimer was eager for Bloch to apply the isotope tracer method to study the biosynthesis of cholesterol and had him start by investigating whether the hydroxyl oxygen in cholesterol came from water or oxygen. Unfortunately, Bloch was unable to solve this first problem because no method existed at that time for the mass spectrometric analysis of stably bound oxygen in complex organic compounds. Eventually, in 1956, Bloch’s student T. T. Tchen would show that molecular oxygen is the source of the hydroxyl oxygen (3). Schoenheimer died in 1941, and his laboratory’s research projects were divided up among its members. Bloch inherited lipids, and Rittenberg acquired protein synthesis. Although he was still working on the cholesterol oxygen problem at that time, Bloch’s attention was quickly diverted with the publication of a paper by R. Sonderhoff and H. Thomas, which reported that “The nonsaponifiable fraction of yeast grown in a medium supplemented with deuterated 1

All biographical information on Konrad Bloch was taken from Refs. 1, 7, 8, and 9.

This paper is available on line at http://www.jbc.org

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Classics

Konrad Bloch. Photo courtesy of the National Library of Medicine.

acetate had a deuterium content so high that a direct conversion of acetic acid to sterols has to be postulated” (4). Schoenheimer and Rittenberg had also done experiments with D2O that indicated that animal cholesterol is synthesized from small molecules (5). Combining their areas of expertise, Rittenberg and Bloch did the next obvious experiment: they fed labeled acetates to rats and mice. As reported in the first JBC Classic reprinted here, they found that a substantial amount of deuterium was incorporated into cholesterol. However, their results did not tell them how many of the 27 sterol carbon atoms were supplied by acetic acid. The definitive answer came 10 years later when Bloch used an acetateless mutant of Neurospora crassa to show that the mutant’s sterol derived all its carbon atoms from exogenous acetate (6). Over time, Rittenberg added 15N, 13C, and 18O to the isotopes he used to study biological processes. This led to a second labeling experiment with Bloch, this time showing that acetic acid is used in the synthesis of fatty acids. This work is described in the second JBC Classic. Rittenberg and Bloch fed sodium acetate labeled with 13C and deuterium to mice and rats and found that the animals’ lipids and cholesterol contained both labeled carbon and hydrogen. From this they concluded that both carbon atoms in acetic acid were used for the synthesis of fatty acids and cholesterol. Eventually Rittenberg became director of the isotope laboratory at P & S in 1941 and remained there until he retired. His research with isotope tracers encompassed a wide variety of subjects, including the study of hippuric acid metabolism, the dynamics of red blood cell survival in patients with blood abnormalities, the development of a method to assay amino acids in protein hydrolysates, and investigations into the synthesis of porphyrin, which will be the subject of a future JBC Classic. Rittenberg’s many contributions to the isotope tracer technique were recognized when he was awarded the Eli Lilly Award in Biological Chemistry from the American Chemical Society and also with his election to the National Academy of Sciences in 1953.2 In addition to using isotopes to study the biosynthesis of cholesterol, Bloch also used the tracers to examine the precursor role of cholesterol in bile acids and steroid hormones. In the final JBC Classic reprinted here, Bloch demonstrates that cholesterol is converted into progesterone. However, Bloch encountered several logistical problems when starting this experiment. First, labeled cholesterol was unavailable commercially so he had to spend much of his time introducing deuterium into cholesterol by platinum-catalyzed exchange in heavy wateracetic acid mixtures. Second, the only practical source for isolating the progesterone metabolite pregnanediol in sufficient quantity was from human pregnancy urine, and his request to the P & S department of obstetrics and gynecology for permission to administer labeled cholesterol to one of its patients was denied. Bloch eventually managed to obtain his pregnanediol due to a “willingness to cooperate at home” (1) and proved that progesterone was indeed synthesized from cholesterol. In 1946 Bloch moved to the Department of Biochemistry at the University of Chicago and then to Harvard University in 1954. He continued to study fatty acids and cholesterol as well as the enzymatic synthesis of the tripeptide glutathione. Eventually, through the combined 2

All biographical information on David Rittenberg was taken from Ref. 10.

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Classics efforts of Bloch, John Cornforth, and George Popja´k, the origin of each of the 27 individual carbon atoms of cholesterol (from the methyl or carboxyl group of acetate) was established. Bloch also aided in the identification of several important landmarks in the series of more than 30 reactions in the biosynthesis of cholesterol, including the cyclization of squalene to lanosterol. His work on fatty acids and cholesterol was eventually rewarded when he shared the 1964 Nobel Prize in Physiology or Medicine with Feodor Lynen “for their discoveries concerning the mechanism and regulation of the cholesterol and fatty acid metabolism.” The elucidation of the pathway from acetic acid to cholesterol was not only a tremendous achievement for biochemistry but also of great importance to medicine. Knowledge of the biosynthetic pathway for cholesterol eventually aided in the discovery of statins, drugs that interfere with cholesterol synthesis, which are now widely used to treat high cholesterol. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. Bloch, K. (1987) Summing up. Annu. Rev. Biochem. 56, 1–19 2. JBC Classics: Schoenheimer, R., and Rittenberg, D. (1935) J. Biol. Chem. 111, 163–168; Rittenberg, D., and Schoenheimer, R. (1937) J. Biol. Chem. 121, 235–253 (http://www.jbc.org/cgi/content/full/277/43/e31) 3. Tchen, T. T., and Bloch, K. (1956) On the mechanism of cyclization of squalene. J. Am. Chem. Soc. 78, 1516 –1517 4. Sonderhoff, R., and Thomas, H. (1937) Ann. Chem. 530, 195–213 5. Rittenberg, D., and Schoenheimer, R. (1937) Deuterium as an indicator in the study of intermediary metabolism. XI. Further studies on the biological uptake of deuterium into organic substances, with special reference to fat and cholesterol formation. J. Biol. Chem. 121, 235–253 6. Ottke, R. C., Tatum, E. L., Zabin, I., and Bloch, K. (1951) Isotopic acetate and isovalerate in the synthesis of ergosterol by Neurospora. J. Biol. Chem. 189 429 – 433 7. Kennedy, E. P. (2001) Hitler’s gift and the era of biosynthesis. J. Biol. Chem. 276, 42619 – 42631 8. Goldfine, H., and Vance, D. E. (2001) Obituary: Konrad E. Bloch (1912–2000) Nature 409, 779 9. Kennedy, E. P. (2003) Biographical memoirs: Konrad Bloch. Proc. Am. Philos. Soc. 147, 65–72 10. Shemin, D., and Bentley, R. (2001) Biographical Memoir of David Rittenberg, Vol. 80, pp. 256 –275, National Academy of Sciences, Washington, D. C.

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Vol. 280, No. 14, Issue of April 8, p. e11, 2005 Printed in U.S.A.



The ATP Requirement for Fatty Acid Oxidation: the Early Work of Albert L. Lehninger The Relationship of the Adenosine Polyphosphates to Fatty Acid Oxidation in Homogenized Liver Preparations (Lehninger, A. L. (1945) J. Biol. Chem. 157, 363–382) Albert Lester Lehninger (1917–1986) was born in Bridgeport, Connecticut. In 1935 he enrolled at Wesleyan University as an English major. Although his interests soon changed to chemistry, Lehninger would later make use of his writing talents to author three classic textbooks: Biochemistry, The Mitochondrion, and Bioenergetics. Inspired by the work of Otto Warburg and Hans Krebs, Lehninger went on to graduate school at the University of Wisconsin and received his Ph.D. in 1942. His graduate research with Edgar J. Witzemann was on the metabolism of acetoacetate and the oxidation of fatty acids by disrupted liver preparations. The Journal of Biological Chemistry (JBC) Classic reprinted here concerns Lehninger’s work on fatty acid oxidation. At the time, much of what was known about glycolysis and the citric acid cycle had been elucidated from minced tissue and tissue extracts. However, similar studies on fatty acid oxidation had been hampered by the fact that ruptured liver cells lost their ability to oxidize fatty acids. Luis F. Leloir and Juan M. Munõz had some success with liver homogenates at low temperatures in the presence of oxygen, inorganic phosphate, fumarate, cytochrome c, adenylic acid, and magnesium ions (1), but these experiments were not always reproducible. When the reaction was carried out successfully, Leloir and Munõz noted that there was a decrease in ATP phosphorus and phosphopyruvic acid phosphate and an increase in inorganic phosphate, indicating the reaction was somehow coupled with phosphorylation. In examining these early experiments, Lehninger realized that high concentrations of ATP or ADP might be required to activate or facilitate oxidation by the liver extracts. He came to this conclusion for a number of reasons including the fact that ATP is rapidly dephosphorylated when cells are disrupted and that the fumarate needed for the reaction might provide a substrate for oxidations capable of phosphorylating adenylic acid to ATP. In the Classic, Lehninger proves his hypothesis by adding ATP to a homogenized liver preparation and demonstrating that it consistently and reproducibly carried out fatty acid oxidation. Subsequent work would show that fatty acids are activated by the formation of a thioester linkage between the carboxyl group of the fatty acid and the sulfhydryl group of coenzyme A. This reaction is driven by ATP. With the start of the war, Lehninger abandoned his fatty acid studies and joined the wartime research effort of the Plasma Protein Fractionation Program led by Edwin Joseph Cohn, who was the author of a previous JBC Classic (2). During this time, Lehninger discovered several papers on oxidative phosphorylation, and from then on the mechanisms of energy capture and transduction in cells became the central focus of his research. In 1945 Lehninger accepted a faculty position at the University of Chicago. During his 6 years in Chicago, Lehninger and two of his students would make two significant discoveries that would contribute greatly to the study of metabolism. First, Lehninger and Eugene P. Kennedy would discover that virtually all of the cell’s oxidative activity occurred in the mitochondria. Second, Lehninger and Morris E. Friedkin would show that electron transport This paper is available on line at http://www.jbc.org

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Classics

Albert L. Lehninger. Photo courtesy of the National Library of Medicine.

from NADH to oxygen is an immediate and direct energy source for oxidative phosphorylation. Lehninger’s work with Kennedy and the latter part of his research career will be the subject of a future JBC Classic.1 Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. Munõz, J. M., and Leloir, L. F. (1943) Fatty acid oxidation by liver enzymes. J. Biol. Chem. 147, 355–362 2. JBC Classics: Cohn, E. J., Hendry, J. L., and Prentiss, A. M. (1925) J. Biol. Chem. 63, 721–766 (http://www.jbc.org/ cgi/content/full/277/30/e19) 3. Lane, M. D., and Talalay, P. (1986) Albert Lester Lehninger 1917–1986. J. Membr. Biol. 91, 194 –197

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All biographical information on Albert Lester Lehninger was taken from Ref. 3.

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Vol. 280, No. 25, Issue of June 24, p. e22, 2005 Printed in U.S.A.



The Kennedy Pathway for Phospholipid Synthesis: the Work of Eugene Kennedy Oxidation of Fatty Acids and Tricarboxylic Acid Cycle Intermediates by Isolated Rat Liver Mitochondria (Kennedy, E. P., and Lehninger, A. L. (1949) J. Biol. Chem. 179, 957–972) The Function of Cytidine Coenzymes in the Biosynthesis of Phospholipides (Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193–214) Eugene Patrick Kennedy was born in Chicago in 1919. He enrolled at De Paul University in 1937 as a chemistry major and then went to the University of Chicago in 1941 for graduate training in organic chemistry. To pay his tuition, Kennedy also got a job in the chemical research department of Armour and Company, one of the large meat packers in Chicago. As part of the war effort, his job at Armour was to assist in the large scale fractionation of bovine blood to obtain pure bovine serum albumin. It was believed that the bovine serum albumin might be useful for treating shock in soldiers on the battlefield. However, by the end of 1942, hope had faded that bovine serum albumin would be an effective treatment, and the Red Cross started to collect blood from volunteers instead. Armour opened a new facility in Fort Worth, Texas for the fractionation of human blood from donors, and Kennedy was sent to Fort Worth to assist in this effort. He remained in Texas until 1945, when the war was clearly nearing its end and large amounts of human plasma proteins had been stockpiled. Returning to the University of Chicago, Kennedy immediately transferred from the Department of Chemistry to the Department of Biochemistry. His experience on the plasma project had led to a new appreciation of biochemistry. When he was ready to begin research for his dissertation, Kennedy approached Albert Lehninger, a young faculty member whose earlier research was the subject of a previous Journal of Biological Chemistry (JBC) Classic (1). At that time, Lehninger was studying oxidative phosphorylation and fatty acid oxidation. Kennedy writes, “With staggering naivete´, I suggested to him that the proper approach would be to purify the various enzymes undoubtedly involved in fatty acid oxidation and crystallize them. He agreed that this would be desirable, but went on to point out rather gently that fatty acid oxidation had not yet been demonstrated in a soluble extract from which individual enzymes might be isolated. To reach that stage, it would first be necessary to discover the nature of the energy-requiring activation or ”sparking“ of fatty acid oxidation and the special dependence of the process on particulate structures” (2). Despite this initial incident, Lehninger agreed to take Kennedy on as a graduate student, and he began to work on the problem of fatty acid oxidation in 1947. Lehninger had observed that both fatty acid oxidation and oxidative phosphorylation were inhibited in a strikingly parallel fashion when particulate enzyme preparations of homogenized rat livers were exposed to hypotonic buffers. The activity could be preserved by adding either salts or iso-osmotic amounts of sucrose to the buffers. Kennedy’s first project in the laboratory was a detailed study of these effects (3). These studies led Lehninger and Kennedy to surmise that fatty acid oxidation, oxidative phosphorylation, and the Krebs cycle must all be taking place in one organelle, bounded by a membrane impermeable to certain solutes. Although their enzyme preparations were quite crude, they were convinced that the organelle was the mitochondrion, even though functionally and morphologically intact mitochondria had not yet been isolated. This paper is available on line at http://www.jbc.org

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Classics Around this time George Palade and his collaborators were developing methods for the separation and identification of organelles. As reported in a previous JBC Classic (4), Palade worked out a method for the isolation of purified mitochondria by differential centrifugation in 0.88 M sucrose. Kennedy immediately tested mitochondria isolated by this method and obtained convincing evidence that oxidative phosphorylation, fatty acid oxidation, and the reactions of the Krebs cycle did indeed occur in the mitochondria. This is the subject of the first JBC Classic reprinted here. After finishing graduate school, Kennedy went to the University of California, Berkeley, to work with Horace A. Barker. Barker and his graduate student Earl Stadtman, both of whom will be featured in future JBC Classics, had just discovered that soluble extracts of Clostridium kluyveri cells could produce short-chain fatty acids from ethyl alcohol. Although the initial discovery had already been made, there was much to be learned about these extracts and Kennedy aided in this effort. In 1950, Kennedy joined Fritz Lipmann, author of a previous JBC Classic (5), at Harvard Medical School.1 He then returned to the University of Chicago in 1951, after being given a joint appointment in the Department of Biochemistry and the newly organized Ben May Laboratory for Cancer Research. In Chicago, Kennedy started to study the origins of the phosphodiester bond of phosphatidylcholine using labeled choline. He found that free choline, but not phosphocholine, was converted to lipid in a reaction dependent on ATP generated by oxidative phosphorylation (6). At the same time, Kornberg and Pricer (7) reported experiments in which phosphocholine was converted to a lipid (later identified as lecithin) in a reaction that required ATP. Determined to understand why he and Kornberg had obtained contradicting results, Kennedy, along with his graduate student Samuel Weiss, undertook a detailed examination of the differences between the two studies. They discovered that they could reproduce Kornberg’s results using commercially available ATP. However, large amounts of ATP were needed, suggesting that an impurity, rather than ATP, might be involved in the reaction. Kennedy and Weiss’ discovery of the cofactor involved in the conversion of phosphocholine to lecithin is the subject of the second JBC Classic reprinted here. After testing several nucleoside triphosphates, they realized that cytidine triphosphate (CTP) was the active cofactor in the phosphocholine reaction. They formulated a number of schemes to account for the involvement of CTP in phospholipid synthesis and eventually decided that intermediary formation of cytidine diphosphate choline (CDP-choline) was occurring in the reaction. Although they had no evidence for its involvement, they synthesized CDP-choline and cytidine diphosphate ethanolamine and tested their abilities to act as cofactors in lipid biosynthesis. Using 14C to label the cytidine coenzymes, Kennedy and Weiss proved that CDP-choline and cytidine diphosphate ethanolamine were activated forms of phosphorylcholine and phosphorylethanolamine and were precursors of lecithin and phosphatidylethanolamine. They also showed that the two cytidine coenzymes were present in high quantities in liver and yeast. In 1959, Kennedy was invited to become a Hamilton Kuhn Professor and head of the Department of Biological Chemistry at the Harvard Medical School. He continued his research on phospholipid biosynthesis and was able to formulate a detailed picture of the pathways of biosynthesis of the principal glycerophosphatides and of triacylglycerol by 1961. Kennedy’s interests also led him to investigate membrane biogenesis and function in bacteria, the translocation of membrane phospholipids, and periplasmic glucans and cell signaling in bacteria. Kennedy is currently at Harvard as the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology, Emeritus.2 Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. JBC Classic: Lehninger, A. L. (1945) J. Biol. Chem. 157, 363–382 (http://www.jbc.org/cgi/content/full/280/14/e11) 2. Kennedy, E. (1992) Sailing to Byzantium. Annu. Rev. Biochem. 61, 1–28 3. Lehninger, A. L, and Kennedy, E. P. (1948) The requirements of the fatty acid oxidase complex of rat liver. J. Biol. Chem. 173, 753–771 1 2

Please see Ref. 8 for Kennedy’s JBC Reflection on Fritz Lipmann, Rudolf Schoenheimer, and Konrad Bloch. All biographical information on Eugene P. Kennedy was taken from Ref. 2.

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Classics 4. JBC Classic: Hogeboom, G. H., Schneider, W. C., and Palade, G. E. (1948) J. Biol. Chem. 172, 619 – 635 (http://www.jbc.org/cgi/content/full/280/22/e19) 5. JBC Classic: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18) 6. Kennedy, E. P. (1953) The synthesis of lecithin in isolated mitochondria. J. Am. Chem. Soc. 75, 249 –250 7. Kornberg, A., and Pricer, W. E. (1952) Fed. Proc. 11, 242 8. Kennedy, E. P. (2001) Hitler’s gift and the era of biosynthesis. J. Biol. Chem. 276, 42619 – 42631

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Vol. 280, No. 26, Issue of July 1, p. e23, 2005 Printed in U.S.A.



Fatty Acid Synthesis and Glutamine Synthetase: the Work of Earl Stadtman Fatty Acid Synthesis by Enzyme Preparations of Clostridium kluyveri. I. Preparation of Cell-free Extracts That Catalyze the Conversion of Ethanol and Acetate to Butyrate and Caproate (Stadtman, E. R., and Barker, H. A. (1949) J. Biol. Chem. 180, 1085–1093) Allosteric Regulation of the State of Adenylylation of Glutamine Synthetase in Permeabilized Cell Preparations of Escherichia coli. Studies of Monocyclic and Bicyclic Interconvertible Enzyme Cascades, in Situ (Mura, U., Chock, P. B., and Stadtman, E. R. (1981) J. Biol. Chem. 256, 13022–13029) Earl Reece Stadtman was born in 1919 in Carrizozo, a small town in New Mexico. When he was 10, his family moved to San Bernardino, California, where he attended high school. After graduating from high school in 1937, Stadtman enrolled in several science courses at San Bernardino Valley College, hoping to eventually set up a soil-testing laboratory. However, he soon realized that he needed a more rigorous education and enrolled at the University of California, Berkeley. He earned a B.S. in soil science in 1942. After spending a year in Alaska, involved in a wartime project of mapping the AlaskanCanadian (Al-Can) Highway, Stadtman returned to Berkeley looking for work. He paid a visit to Horace A. Barker, a Berkeley biochemist for whom he had worked as a laboratory technician (and author of a future Journal of Biological Chemistry (JBC) Classic). At that time, Barker was directing various war efforts in the Department of Food Technology and offered Stadtman a job as principal investigator on a project studying the “Browning of Dried Apricots,” the goal of which was to find a way to slow the deterioration of dried fruits during storage. Around this time, Stadtman also met his future wife, Thressa Campbell, who was working as a laboratory assistant in the food technology department. After the war, Stadtman started graduate studies in the Department of Biochemistry working in Barker’s laboratory. Barker had spent a year as a postdoc in Albert J. Kluyver’s laboratory at the Technical School in Delft in the Netherlands before coming to Berkeley and had isolated a species of bacteria called Clostridium kluyveri (named after Kluyver) from the Delft canal mud. Since then, Barker had been searching for an explanation for the observation that C. kluyveri could produce short-chain fatty acids from ethyl alcohol. He made a breakthrough when he obtained some 14C and used the isotope to label acetate and demonstrate that fatty acid synthesis is accomplished by the multiple condensation of 2-carbon molecules (1). He deduced that ethanol is first oxidized to “active” acetate (a 2-carbon compound), which is condensed with acetate to form a 4-carbon compound that is reduced to form butyrate. Active acetate can then be condensed with butyrate to form caproate. Barker surmised that acetyl phosphate might be the active acetate formed in the above reaction. It was at this point that Stadtman joined Barker’s laboratory and started working on fatty acid synthesis. Initially, like Barker, Stadtman used 14C to trace the metabolic pathways in whole cell preparations of C. kluyveri. However, he abandoned this approach after a visit to Irwin C. Gunsalus’s laboratory at Cornell University. Gunsalus showed Stadtman how to dry bacterial cells and grind the dried preparations to break open cell walls, producing a cell-free extract. Applying this method to C. kluyveri, Stadtman was able to produce extracts that could catalyze all of the reactions involved in the conversion of ethanol and acetate to fatty acids of This paper is available on line at http://www.jbc.org

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Photo courtesy of the Office of NIH History, National Institutes of Health.

4- and 6-carbon atoms. His preparations also catalyzed the aerobic oxidation of ethanol and butyrate. These experiments are reported in the first JBC Classic reprinted here. This discovery was especially significant because up until that time most biochemists believed that the capacity to make fatty acids was a unique property of specialized cellular systems or particulate organelles. In a series of additional papers (2– 6), all published in the JBC, Stadtman and Barker used the enzyme extracts to study the individual reactions involved in fatty acid synthesis and confirmed that ethanol is oxidized to acetyl phosphate, which condenses with acetate and forms butyric acid. They also discovered that C. kluyveri contained an acetyl-transferring enzyme (phosphotransacetylase) and an enzymatic system for using acetyl phosphate to activate other fatty acids. Stadtman later showed that acetyl-CoA was the source of active acetate in the synthesis of butyric acid from acetyl phosphate (7) while working as postdoctoral fellow with Fritz Lipmann (author of a previous JBC Classic (8)). In 1950, Stadtman began to look for an academic position. However, because his wife Thressa also had a Ph.D., they were looking for an institution at which they could both work at the same professional level. Unfortunately, at that time, most universities had antinepotism rules that did not allow more than one family member to work in the same department. Intended to protect universities from charges of favoritism, the rules often had the effect of discriminating against married women. No one seriously challenged the rules until the 1960s, when the American Association of University Women began to protest their unfairness. Fortunately, these polices were not in effect at the National Institutes of Health (NIH), and in September 1950, the Stadtmans moved to Bethesda, Maryland. Both continue to do research at the NIH today. At the NIH, Stadtman continued his research on fatty acid metabolism. In 1952, he successfully carried out the first in vitro net synthesis of acetyl-CoA using only basic materials (acetyl phosphate, CoA, and phosphotransacetylase). Stadtman and his postdoc P. Roy Vagelos also demonstrated that long-chain fatty acid synthesis is catalyzed by an enzyme complex in which malonyl-CoA is the source of active acetate. Another topic of long term research in Stadtman’s laboratory was glutamine synthetase, the enzyme that catalyzes the conversion of glutamate to glutamine. The activity of glutamine synthetase is subject to feedback inhibition by 7 different end products of glutamine metabolism. Stadtman discovered that this end product inhibition was cumulative (the presence of more end products resulted in more inhibition) and that susceptibility to feedback inhibition H12

Classics only occurred when glutamine synthetase was adenylated by adenylyltransferase (ATase). He later found that adenylation was regulated by uridylyltransferase (UTase), which, depending on the cellular concentration of various metabolites, catalyzed the covalent attachment of a uridylyl group to the regulatory protein, PII. The uridylated form of PII stimulates ATase to catalyze glutamine synthetase deadenylation, whereas the unmodified form of PII stimulates ATase catalysis of the adenylation reaction. In view of these results, Stadtman surmised that glutamine synthetase activity was controlled by a cascade system in which two systems of reversible covalent modification were tightly linked. Each system was composed of two reversible reactions, or two interconvertible enzyme cycles, the linkage of which resulted in the formation of a bicyclic cascade system. This cascade system allowed enzyme activity to be shifted gradually in response to metabolite availability. In the late 1970s and early 1980s, Stadtman and P. Boon Chock carried out a theoretical analysis of this bicyclic cascade system to understand its implications in enzyme regulation. However, it was not until 1981 that they were able to study the cascade in vivo. These experiments are discussed in the second JBC Classic reprinted here. Stadtman had discovered that after a freeze-thaw cycle, treatment of Escherichia coli cells with a nonionic detergent rendered them permeable to small metabolites but allowed the cells to retain the protein components of the cascade system. Furthermore, permeabilized cells from cultures containing 10 mM glutamine retained all their cascade enzymes whereas 5 mM glutamine-grown cells had inactivated UTase. Using these cells, they were able to study the effects of different substrates and allosteric effects on the cascade system and to confirm the previous theoretical and in vitro studies. A more complete description of Stadtman’s work on glutamine synthetase can be found in his JBC Reflections (9). Stadtman has received many awards and honors for his numerous research discoveries including the 1979 National Medal of Science, the 1983 ASBC-Merck Award, and the 1991 Robert A. Welch Award in Chemistry. Stadtman was also President of the American Society for Biological Chemists (now American Society for Biochemistry and Molecular Biology) from 1982 to 1983 and has been a member of the National Academy of Sciences since 1969.1 Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. Barker, H. A., Kamen, M. D., and Bornstein, B. T. (1945) The synthesis of butyric and caproic acids from ethanol and acetic acid by Clostridium kluyveri. Proc. Natl. Acad. Sci. U. S. A. 31, 373–381 2. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. II. The aerobic oxidation of ethanol and butyrate with the formation of acetyl phosphate. J. Biol. Chem. 180, 1095–1115 3. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. III. The activation of molecular hydrogen and the conversion of acetyl phosphate and acetate to butyrate. J. Biol. Chem. 180, 1117–1124 4. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. IV. The phosphoroclastic decomposition of acetoacetate to acetyl phosphate and acetate. J. Biol. Chem. 180, 1169 –1186 5. Stadtman, E. R., and Barker, H. A. (1949) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. V. A consideration of postulated 4-carbon intermediates in butyrate synthesis. J. Biol. Chem. 181, 221–235 6. Stadtman, E. R., and Barker, H. A. (1950) Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. VI. Reactions of acyl phosphates. J. Biol. Chem. 184, 769 –794 7. Stadtman, E. R., Novelli, G. D., and Lipmann, F. (1951) Coenzyme A function in and acetyl transfer by the phosphotransacetylase system. J. Biol. Chem. 191, 365–376 8. JBC Classic: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18) 9. Stadtman, E. R. (2001) The story of glutamine synthetase regulation. J. Biol. Chem. 276, 44357– 44364 10. Park, B. S. The Stadtman way: A tale of two biochemists at NIH. http://history.nih.gov/exhibits/stadtman/ index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart, Lung, and Blood Institute)

1

All biographical information on Earl R. Stadtman was taken from Ref. 10.

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Vol. 280, No. 30, Issue of July 29, p. e27, 2005 Printed in U.S.A.



A Role for Phosphoinositides in Signaling: the Work of Mabel R. Hokin and Lowell E. Hokin Enzyme Secretion and the Incorporation of P32 into Phospholipides of Pancreas Slices (Hokin, M. R., and Hokin, L. E. (1953) J. Biol. Chem. 203, 967–977) Phosphoinositides and Protein Secretion in Pancreas Slices (Hokin, L. E., and Hokin, M. R. (1958) J. Biol. Chem. 233, 805– 810) Mabel R. Hokin (born Mabel Neaverson) and Lowell E. Hokin met in Hans Kreb’s department at the University of Sheffield and married soon after. During their time in Sheffield, the Hokins started investigating what they thought was an increase in the incorporation of 32P into RNA caused by the acetylcholine-induced stimulation of pancreatic slices. However, before they could purify the RNA, they moved to McGill University in Montreál, Que´bec, and brought their radiolabeled samples with them. Once established in Montreál, they continued with their experiments but noticed that as they purified the RNA the radioactivity was lost. Investigating this phenomenon further, they found that most of the radioactivity was incorporated into the phospholipid fraction. This was a surprising discovery as up until then phospholipids were regarded as inert structural components of membranes. The Hokins’ studies on 32P uptake into phospholipids during enzyme secretion in pancreas slices are published in the first Journal of Biological Chemistry (JBC) Classic reprinted here. The Hokins incubated pigeon pancreas slices with various compounds along with 32P to see the effects on phosphate incorporation into phospholipids. They found that when enzyme secretion was stimulated by acetylcholine or carbamylcholine, both of which induce amylase secretion, the incorporation of 32P into phospholipids was on average 7.0 times greater than in control tissue. Separating individual phospholipids for analysis was difficult at that time, but fortunately Rex Dawson devised a method that permitted the analysis of diacylglycerophospholipids by deacylation and two-dimensional separation of the water-soluble backbone (1). The Hokins used this method to show that hormone stimulation of pancreatic slices mainly increased the rate of 32P incorporation into phosphoinositide but that phosphatidylcholine, phosphatidylserine, and phosphatidic acid also contained radiolabeled phosphate (2). This was the first demonstration of receptor-stimulated lipid turnover, and it later became known as the “PI effect.” The second JBC Classic reprinted here presents the details of the Hokins’ study of phosphoinositide metabolism in relation to protein secretion in the pancreas. They incubated pigeon pancreas slices with either NaH2P32O4, [2-3H]inositol, or [1-14C]glycerol and extracted the lipids from the tissue and separated them by paper chromatography. They were able to identify seven phospholipids containing 32P as well as two radioactive monophosphoinositides. From these data they concluded, “the present work indicates that phosphoinositides are involved in the secretion of protein from the inside of the pancreatic acinar cell into the lumen . . . It is tempting to think that the active transport out of the cell of many other types of molecules may involve phosphoinositides.” In 1957 the Hokins moved to Madison, Wisconsin, where they both joined the faculty of the University of Wisconsin-Madison Medical School. There they showed that other tissues exhibit similar responses when provoked to secrete. In 1964 the Hokins suggested that phospholipase C-catalyzed phosphatidylinositol hydrolysis might initiate the PI effect. Later it was confirmed that the initiating event was the phospholipase C-catalyzed hydrolysis of phosphatidylinositol This paper is available on line at http://www.jbc.org

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Classics 4,5-bisphosphate and that 3-kinase-catalyzed formation of phosphatidylinositol 3,4,5-triphosphate was a second widespread signaling reaction. The Hokins’ initial work on stimulated phosphoinositide turnover in secretory tissues motivated a large number of other investigators to focus their research on the PI effect and second messengers. Eventually they would discover that the Hokins’ inositol phospholipids play important roles in transmembrane signaling and many other cell regulatory processes.1 Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. Dawson, R. M. C. (1954) The measurement of 32P labeling of individual kephalins and lecithin in a small sample of tissue. Biochim. Biophys. Acta 14, 374 –375 2. Hokin, L. E., and Hokin, M. R. (1955) Effects of acetylcholine on the turnover of phosphoryl units in individual phospholipids of pancreas slices and brain cortex slices. Biochim. Biophys. Acta 18, 102–110 3. Michell, B. (2003) Obituary: Mabel R. Hokin (1924 –2003). The Biochemist. December 4. Irvine, R. F. (2003) 20 years of Ins(1,4,5)P3, and 40 years before. Nat. Rev. Mol. Cell. Biol. 4, 586 –590

1

All biographical information on Mabel R. Hokin and Lowell E. Hokin was taken from Refs. 3 and 4.

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Vol. 284, No. 20, Issue of May 15, p. e3, 2009 Printed in U.S.A.



The Selective Placement of Acyl Chains: the Work of William E. M. Lands Metabolism of Glycerolipids: A Comparison of Lecithin and Triglyceride Synthesis (Lands, W. E. M. (1958) J. Biol. Chem. 231, 883– 888)

William E. M. Lands

William E. M. Lands was born in Chillicothe, Missouri in 1930. He earned his B.S. in chemistry from the University of Michigan in 1951 and his Ph.D. in biological chemistry from the University of Illinois in 1954. After graduating, he spent a year as a postdoctoral fellow at the California Institute of Technology and then joined the faculty of the University of Michigan as an instructor in biological chemistry. Lands spent the next 25 years at Michigan, eventually becoming professor in 1967. In 1980, Lands left Michigan to head the Department of Biological Chemistry at the University of Illinois. He spent 10 years there and then moved to Bethesda, Maryland to become Senior Scientific Advisor to the Director of the National Institute on Alcohol Abuse and Alcoholism (NIH). In 2002, Lands retired from his position at the NIH. Lands spent the majority of his scientific career studying fatty acids and has made many significant contributions to this field. One such contribution is his discovery of the phospholipid retailoring or “Lands” pathway. His initial paper showing the likelihood of acyl chain turnover is reprinted here as a Journal of Biological Chemistry (JBC) Classic. In the paper, Lands incubates various tissues with [14C]acetate and [14C]glycerol and measures the value R, which is the ratio of [14C]acetate to [14C]glycerol in the diglyceride unit of the triglycerides and phospholipids produced by the tissues. Lands reasoned that if diglycThis paper is available on line at http://www.jbc.org

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Classics eride is the sole precursor of phospholipids, these two compounds should have the same R value. Also, because a third fatty acid molecule is added to the diglyceride unit to form triglycerides, they should have a 3/2 R value (see Fig. 1). However, Lands’ results showed that R is 2– 4 times higher in phospholipids than in triglycerides, sugFIGURE 1 gesting to him that “the diglyceride unit of the phospholipids is metabolically different in some respect from that of the triglycerides.” This initial finding led to a series of papers published in the JBC describing the selective placement of acyl chains by phospholipid acyltransferases (1– 4). In addition to the above research which helped to explain the metabolic process that regulates the mixture of acyl chains found in lipids, Lands is also credited with discovering the beneficial effects of balancing excess ␻-6 fatty acids with dietary ␻-3 fatty acids. In recognition of his contributions to science, Lands received the Glycerine Research Award (1969), the Canadian Society of Nutritional Science Lectureship (1991), and the American Oil Chemists’ Society Supelco Lipid Research Award (1997). The University of Michigan’s Department of Biological Chemistry has also endowed a lectureship in honor of Lands. He has also served on the editorial boards of several journals, including those of the Journal of Lipid Research, Biochimica et Biophysica Acta, Lipids, and Prostaglandins. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. Lands, W. E. M. (1960) Metabolism of glycerolipids. II. The enzymatic acylation of lysolecithin. J. Biol. Chem. 235, 2233–2237 2. Lands, W. E. M., and Merkl, I. (1963) Metabolism of glycerolipids. III. Reactivity of various acyl esters of coenzyme A with ␣⬘-acylglycerol phosphorylcholine and positional specificities in lecithin synthesis. J. Biol. Chem. 238, 898 –904 3. Merkl, I., and Lands, W. E. M. (1963) Metabolism of glycerolipids. IV. Synthesis of phosphatidylethanolamine. J. Biol. Chem. 238, 905–906 4. Lands, W. E. M., and Hart, P. (1965) Metabolism of glycerolipids. VI. Specificities of acyl-CoA:phospholipid acyltransferases. J. Biol. Chem. 240, 1905–1911

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Vol. 280, No. 48, Issue of December 2, p. e45, 2005 Printed in U.S.A.



Lipid Storage Disorders and the Biosynthesis of Inositol Phosphatide: the Work of Roscoe Brady The Enzymatic Synthesis of Inositol Phosphatide (Agranoff, B. W., Bradley, R. M., and Brady, R. O. (1958) J. Biol. Chem. 233, 1077–1083) Roscoe Owen Brady was born in 1923 in Philadelphia. He attended Pennsylvania State University from 1941 to 1943 and then received his medical degree from Harvard Medical School in 1947. After interning at the Hospital of the University of Pennsylvania for 1 year, Brady did a postdoctoral fellowship in the Department of Physiological Chemistry at the University of Pennsylvania School of Medicine (1948 to 1950) and then was a fellow in clinical medicine in the Department of Medicine (1950 to 1952). In 1954, following 21⁄2 years on active duty in the U.S. Naval Medical Corps, he joined the National Institutes of Health (NIH) to become section chief of the National Institute of Neurological Diseases and Blindness. He remained in this position until 1967 when he became assistant laboratory chief of neurochemistry at the National Institute of Neurological Diseases and Blindness. Currently, Brady is Chief of the Developmental and Metabolic Neurology Branch of the National Institute of Neurological Disorders and Stroke, a position he has held since 1972. Early in his career at the NIH, Brady started studying lipids, specifically inositol and the synthesis of inositol phosphatide. This is the subject of the Journal of Biological Chemistry (JBC) Classic reprinted here. Although inositol had been isolated more than 100 years before the Classic was published, little was known about its metabolism. In his Classic, Brady uses tritium-labeled inositol and a preparation from guinea pig kidney mitochondria to study inositol metabolism. He found that the enzyme system catalyzed the incorporation of inositol into inositol phosphatide in the presence of Mg2⫹ and cytidine diphosphate-choline (CDP) or cytidine 5⬘-phosphate. From these results, Brady proposed a mechanism for the synthesis of inositol phosphatide in which CDP is transphosphorylated to form CDP-D-␣,␤-diglyceride, which then reacts with the hydroxyl group of inositol to form inositol phosphatide. These early studies stimulated Brady’s interest in lipid storage disorders, in particular, Gaucher disease. In 1967, he showed that people with Gaucher disease had low levels of glucocerebrosidase and thus were unable to break down the lipid glucocerebroside and clear it out of their bodies. He also developed a diagnostic test for Gaucher disease, which worked by measuring glucocerebrosidase activity in white blood cells. A year later, Brady suggested a therapy for Gaucher disease based on replacing the enzyme. Using human placentas, his team isolated a tiny sample of purified glucocerebrosidase and gave it to two patients. The patients’ health improved, and Brady soon developed large scale purification and targeting methods for glucocerebrosidase to use in further clinical trials. Eventually, in 1991, Brady’s macrophagetargeted glucocerebrosidase enzyme replacement therapy was approved as a specific treatment for Gaucher disease by the Food and Drug Administration. In addition to studying Gaucher disease, Brady has discovered the metabolic basis of Niemann-Pick disease, Fabry disease, and the specific biochemical defect in Tay-Sachs disease. He has applied this knowledge to developing diagnostic tests, carrier identification procedures, and the prenatal detection of such conditions. Currently his research is focused on examining enzyme replacement therapy and gene therapy for patients with these other hereditary metabolic disorders. This paper is available on line at http://www.jbc.org

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In recognition of his scientific achievements, Brady received the Gairdner International Award (1973), the Cotzias Award from the American Academy of Neurology (1980), the Passano Foundation Award (1982), the Lasker Foundation Clinical Medical Research Award (1982), and the Kovalenko Medal from the National Academy of Sciences (1991). He is a member of the National Academy of Sciences and a member of the Institute of Medicine of the National Academy of Sciences. He has served on the editorial boards and advisory boards of many journals and organizations. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

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Vol. 281, No. 9, Issue of March 3, p. e9, 2006 Printed in U.S.A.



The Prostaglandins, Sune Bergstro¨m and Bengt Samuelsson Prostaglandins and Related Factors. 15. The Structures of Prostaglandin E1, F1␣, and F1␤ (Bergstro¨m, S., Ryhage, R., Samuelsson, B., and Sjo¨vall, J. (1963) J. Biol. Chem. 238, 3555–3564 On the Mechanism of the Biosynthesis of Prostaglandins E1 and F1␣ (Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336 –5343) Sune Karl Bergstro¨m (1916 –2004) was born in Stockholm, Sweden. Upon completing high school he went to work at the Karolinska Institute as an assistant to Erik Jorpes where he did research on the biochemistry of fats and steroids. Jorpes was sufficiently impressed with Bergstro¨m that in 1938 he sponsored a year-long research fellowship for him at the University of London. Then, in 1940, Bergstro¨m received a Swedish-American Fellowship, which allowed him to study for 2 years at Columbia University and to conduct research at the Squibb Institute for Medical Research in New Jersey. He returned to Sweden in 1942 and received doctorates in medicine and biochemistry from the Karolinska Institute 2 years later. He was then appointed assistant in the biochemistry department of Karolinska’s Medical Nobel Institute. Bergstro¨m’s involvement with prostaglandins started in 1945 at a meeting of the Physiological Society of the Karolinska Institute. There he met Ulf von Euler who had been doing research on prostaglandins. Von Euler asked Bergstro¨m if he might be interested in studying some of his lipid extracts of sheep vesicular glands. Using Lyman Craig’s countercurrent extraction device, which was the subject of a previous Journal of Biological Chemistry (JBC) Classic (1), Bergstro¨m was able to purify the crude extract about 500 times. However, his work was interrupted for a few years when he was appointed chair of physiological chemistry at the University of Lund in 1948. When Bergstro¨m resumed his research on prostaglandins, he was aided by his graduate student Bengt Ingemar Samuelsson. Samuelsson, who was born in Halmstad, Sweden, in 1934, had enrolled at the University of Lund to study medicine when he came under the mentorship of Bergstro¨m. Using countercurrent fractionations and partition chromatography, Bergstro¨m was able to isolate small amounts of prostaglandin E1 and F1␣ by 1957 (2). A year later, Bergstro¨m was appointed professor of chemistry at Karolinska, and he moved his research group with him to Stockholm. Samuelsson received his doctorate in medical science from the Karolinska Institute in 1960 and his medical degree in 1961. At Karolinska, Bergstro¨m started to collaborate with Ragnar Ryhage who had built a combination gas chromatograph and mass spectrometer. Using this instrument, Bergstro¨m, Samuelsson, and Ryhage were able to deduce the structures of prostaglandins E1, F1␣, and F1␤ from mass spectrometric identification of the products formed when the prostaglandins were treated with a weak acid or base. These structure determinations are discussed in the first Classic reprinted here. By 1962, Bergstro¨m and his colleagues had isolated and determined the structures of six different prostaglandins. After completing the structural work on the prostaglandins, Samuelsson spent a year as a postdoctoral fellow with E. J. Corey in the Department of Chemistry at Harvard University, where he was able to study theoretical and synthetic organic chemistry. He returned to the This paper is available on line at http://www.jbc.org

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Sune Karl Bergstro¨m. Photo courtesy of the National Library of Medicine.

Bengt Ingemar Samuelsson. Photo courtesy of the National Library of Medicine.

Karolinska Institute as assistant professor of medical chemistry and resumed work on the prostaglandins. The second JBC Classic deals with some of Samuelsson’s research on the biosynthesis of prostaglandins, an area in which he contributed considerable knowledge. In the paper, Samuelsson follows the conversion of 8,11,14-eicosatrienoic acid to prostaglandin E1 and prostaglandin F1␣, using 3H and 14C labeling, focusing especially on the initial step of the process. In 1967, Samuelsson joined the faculty of the Royal Veterinary College in Stockholm as Professor of Medical Chemistry to explore the veterinary and livestock breeding applications of prostaglandins. However, he returned to the Karolinska Institute in 1972 to become Professor and Chairman of the Department of Physiological Chemistry. He was Dean of the Medical Faculty from 1978 to 1983 after which he was appointed Rector of the Karolinska Institute. Bergstro¨m remained at Karolinska, serving as dean of its medical school from 1963 to 1966 and as Rector of the Institute from 1969 to 1977. He was chairman of the Nobel Foundation’s Board of Directors from 1975 to 1987, and from 1977 to 1982 he served as chairman of the World Health Organization’s Advisory Committee on Medical Research. He retired from teaching in 1981, choosing to devote his full time to research at Karolinska. Independently, both Bergstro¨m and Samuelsson continued to investigate prostaglandins and related compounds throughout their scientific careers. In honor of their contributions to this field, they were awarded the 1982 Nobel Prize in Physiology or Medicine with John R. Vane “for their discoveries concerning prostaglandins and related biologically active substances.” Samuelsson’s research has been recognized by numerous awards and honors in addition to the Nobel Prize. These include the A. Jahres Award in Medicine from Oslo University (1970), the Louisa Gross Horwitz Prize from Columbia University (1975), the Albert Lasker Medical Research Award (1977), the Ciba-Geigy Drew Award for biomedical research (1980), the Gairdner Foundation Award (1981), the Bror Holberg Medal of the Swedish Chemical Society (1982), and the Abraham White Distinguished Scientist Award (1991). Samuelsson was elected to the National Academy of Sciences in 1984.1 Bergstro¨m also received many awards, including the Albert Lasker Award in 1977, Oslo University’s Anders Jahre Prize in Medicine in 1970, and Columbia University’s Louisa Gross Horwitz Prize in 1975. He was a member of the Royal Swedish Academy of Science (and served 1

Biographical information on Bengt Samuelsson was taken from Ref. 3.

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Classics as its president from 1983 to 1985), the American Philosophical Society, the National Academy of Sciences (1973), and the American Academy of Arts and Sciences.2 Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. JBC Classics: Craig, L. C. (1943) J. Biol. Chem. 150, 33– 45; Craig, L. C. (1944) J. Biol. Chem. 155, 519 –534 (http://www.jbc.org/cgi/content/full/280/7/e4) 2. Bergstro¨m, S., and Sjo¨vall, J. (1957) The isolation of prostaglandin. Acta Chem. Scand. 11, 1086 3. Samuelsson, B. I. (1993) Studies of biochemical mechanisms to novel biological mediators: prostaglandin endoperoxides, thromboxanes and leukotrienes. In Nobel Lectures, Physiology or Medicine 1981–1990 (Fra¨ngsmyr, T., ed) World Scientific Publishing Co., Singapore 4. Bergstro¨m, S. K. (1993) The prostaglandins: from the laboratory to the clinic. In Nobel Lectures, Physiology or Medicine 1981–1990 (Fra¨ngsmyr, T., ed) World Scientific Publishing Co., Singapore

2

Biographical information on Sune Bergstro¨m was taken from Ref. 4.

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Vol. 284, No. 23, Issue of June 5, p. e6, 2009 Printed in U.S.A.



Biotin-dependent Enzymes: the Work of Feodor Lynen The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and (ⴙ)-Biotin. I. Purification of the Apoenzyme and Synthetase; Characteristics of the Reaction (Lane, M. D., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239, 2858 –2864) Feodor Felix Konrad Lynen (1911–1979) was born in Munich, Germany. He was undecided about his career during his early education and even considered becoming a ski instructor. Ultimately, he enrolled in the Department of Chemistry at the University of Munich where he studied with Nobel laureate Heinrich Wieland and received his doctorate degree in 1937. Three months later, he married Wieland’s daughter, Eva. After graduating, Lynen remained at Munich University as a postdoctoral fellow. He was appointed lecturer in 1942 and assistant professor in 1947. When World War II broke out, Lynen was exempt from military service because of a knee injury resulting from a ski accident in 1932. However, the war made it difficult to continue to do research in Munich, and Lynen moved his laboratory to the small village of Schondorf on the Ammersee. This was lucky because in 1945 Munich Feodor Lynen University’s Department of Chemistry was destroyed. Lynen continued his work at various laboratory facilities and eventually returned to the rebuilt Department of Chemistry in 1949. During the 1940s, Lynen began studying the biosynthesis of sterols and lipids. He eventually initiated a collaboration with Konrad Bloch, whose cholesterol research was featured in a previous Journal of Biological Chemistry (JBC) Classic (1). Working together, Bloch and Lynen were able to elucidate the steps in the biosynthesis of cholesterol. An especially significant finding made by Lynen was that acetyl coenzyme A (previously discovered by JBC Classic author Fritz Lipmann (2)) was essential for the first step of cholesterol biosynthesis. Lynen later determined the structure of acetyl-CoA. This work on cholesterol resulted in Bloch and Lynen being awarded the 1964 Nobel Prize in Physiology or Medicine. In 1953, Lynen was made full professor at the University of Munich. A year later, he was named director of the newly established Max Planck Institute for Cell Chemistry. He continued to work on fats but also turned his focus to biotin-dependent enzymes. In 1962, he was joined by JBC Classic author M. Daniel Lane (3), who had come to Munich to work with Lynen on a sabbatical leave. Lane was studying the biotin-dependent propionyl-CoA carboxylase and had previously determined that its biotin prosthetic group was linked to the enzyme through an amide linkage to a lysyl ⑀-amino group. Before leaving for Munich, Lane developed an apoenzyme system with which to investigate the mechanism by which biotin became attached to propionyl-CoA carboxylase. This system made use of Propionibacterium shermanii, which expressed huge amounts of methylmalonylThis paper is available on line at http://www.jbc.org

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Classics CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The organism also had an absolute requirement for biotin in its growth medium and produced large amounts of the apotranscarboxylase when grown at very low levels of biotin. As reported in the JBC Classic reprinted here, Lane and Lynen were able to resolve and purify both the apotranscarboxylase and the synthetase that catalyzed biotin loading onto the apoenzyme. Dave Young, a postdoctoral fellow who had recently completed his medical training at Duke University, collaborated with them on these studies. In a second paper reprinted in the Lane Classic (4), Lane and Lynen showed that the synthetase catalyzed a two-step reaction. The first step involved the ATP-dependent formation of biotinyl-5⬘-AMP and pyrophosphate after which the biotinyl group was transferred from the AMP derivative to the appropriate lysyl ⑀-amino group of the apotranscarboxylase. Lane and Lynen also showed that the covalently bound biotinyl prosthetic group, like free biotin, was carboxylated on the 1⬘-N position (5). In 1972, Lynen moved to the recently founded Max Planck Institute for Biochemistry. Between 1974 and 1976, he was acting director of the Institute while continuing to oversee a lab at the University of Munich. He remained at the Institute until his death in 1979. In addition to the Nobel Prize, Lynen received many honors and awards. These include the Neuberg Medal of the American Society of European Chemists and Pharmacists (1954), the Liebig Commemorative Medal of the Gesellschaft Deutscher Chemiker (1955), the Carus Medal of the Deutsche Akademie der Naturforscher Leopoldina (1961), and the Otto Warburg Medal of the Gesellschaft fu¨r Physiologische Chemie (1963). Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. JBC Classics: Bloch, K., and Rittenberg, D. (1942) J. Biol. Chem. 145, 625– 636; Rittenberg, D., and Bloch, K. (1945) J. Biol. Chem. 160, 417– 424; Bloch, K. (1945) J. Biol. Chem. 157, 661– 666 (http://www.jbc.org/cgi/content/full/280/10/e7) 2. JBC Classics: Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 (http://www.jbc.org/cgi/content/full/280/21/e18) 3. JBC Classics: Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239, 2865–2871; Gregolin, C., Ryder, E., Warner, R. C., Kleinschimdt, A. K., Chang, H.-C., and Lane, M. D. (1968) J. Biol. Chem. 243, 4236 – 4245; Guchhait, R. B., Polakis, S. E., Hollis, D., Fenselau, C., and Lane, M. D. (1974) J. Biol. Chem. 249, 6646 – 6656; Polakis, S. E., Guchhait, R. B., Zwergel, E. E., Lane, M. D., and Cooper, T. G. (1974) J. Biol. Chem. 249, 6657– 6667 (http://www.jbc.org/cgi/content/full/281/49/e40) 4. Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) The enzymatic synthesis of holotranscarboxylase from apotranscarboxylase and (⫹)-biotin. II. Investigation of the reaction mechanism. J. Biol. Chem. 239, 2865–2871 5. Lane, M. D., and Lynen, F. (1963) The biochemical function of biotin. VI. Chemical structure of the carboxylated active site of propionyl carboxylase. Proc. Natl. Acad. Sci. U. S. A. 49, 379 –385

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Vol. 281, No. 49, Issue of December 8, p. e40, 2006 Printed in U.S.A.



Acetyl-CoA Carboxylase and Other Biotin-dependent Enzymes: the Work of M. Daniel Lane The Enzymatic Synthesis of Holotranscarboxylase from Apotranscarboxylase and (ⴙ)-Biotin. II. Investigation of the Reaction Mechanism (Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964) J. Biol. Chem. 239, 2865–2871) Liver Acetyl-CoA Carboxylase. II. Further Molecular Characterization (Gregolin, C., Ryder, E., Warner, R. C., Kleinschimdt, A. K., Chang, H.-C., and Lane, M. D. (1968) J. Biol. Chem. 243, 4236 – 4245) Acetyl Coenzyme A Carboxylase System of Escherichia coli. Site of Carboxylation of Biotin and Enzymatic Reactivity of 1ⴕ-N-(Ureido)-Carboxybiotin Derivatives (Guchhait, R. B., Polakis, S. E., Hollis, D., Fenselau, C., and Lane, M. D. (1974) J. Biol. Chem. 249, 6646 – 6656) Acetyl Coenzyme A Carboxylase System of Escherichia coli. Studies on the Mechanisms of the Biotin Carboxylase- and Carboxyltransferase-catalyzed Reactions (Polakis, S. E., Guchhait, R. B., Zwergel, E. E., Lane, M. D., and Cooper, T. G. (1974) J. Biol. Chem. 249, 6657– 6667) Malcolm Daniel Lane was born in Chicago in 1930. He received both his B.S. and M.S. from Iowa State University in 1951 and 1953, respectively. Lane then went to the University of Illinois for graduate school and was awarded his Ph.D. in 1956. He joined the faculty of the Virginia Polytechnic Institute and State University in Blacksburg, Virginia in 1956 as Associate Professor and was promoted to Professor of Biochemistry in 1963. Upon joining the faculty at Virginia Polytechnic Institute, Lane decided to try to determine how propionate was metabolized in the bovine liver. About this time, Journal of Biological Chemistry (JBC) Classics author Severo Ochoa (1, 2) reported that propionyl-CoA was carboxylated to form methylmalonyl-CoA, which was then was converted to succinyl-CoA. Lane was able to purify propionyl-CoA carboxylase from bovine liver mitochondria. Then in 1959 a paper by Lynen and Knappe appeared in Angewandte Chemie (3) indicating that ␤-methylcrotonyl-CoA carboxylase, a biotin-dependent carboxylase, catalyzed the ATPdependent carboxylation of “free” biotin in the absence of its acyl-CoA substrate. Lynen proposed that the free biotin had accessed the active site of the carboxylase and by mimicking the biotinyl prosthetic group it had been carboxylated. Lane determined that propionyl-CoA carboxylase was also a biotin-dependent enzyme and determined that the biotin prosthetic group was linked to propionyl-CoA carboxylase through an amide linkage to a lysyl ⑀-amino group. In 1962 Lane decided to take a sabbatical leave in Munich with Feodor Lynen at the Max-Planck Institu¨t Fu¨r Zellchemie where he continued to work on the enzymatic mechanism by which biotin became attached to propionyl-CoA carboxylase. Before leaving for Munich, Lane developed an apoenzyme system with which to investigate the “biotin loading” reaction. This system made use of Propionibacterium shermanii, which expressed huge amounts of methylmalonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme. The organism also had an absolute requirement for biotin in its growth medium and produced large amounts of the apotranscarboxylase when grown at very low levels of biotin. This paper is available on line at http://www.jbc.org

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M. Daniel Lane

In Munich, Lane was able to resolve and purify both the apotranscarboxylase and the synthetase that catalyzed biotin loading onto the apoenzyme (4). Dave Young, a postdoctoral fellow who had recently completed his medical training at Duke University, and Karl Rominger, a Ph.D. candidate under Lynen’s direction, collaborated with him on these studies. In a second paper, which is the first JBC Classic reprinted here, Lane and his colleagues showed that the synthetase catalyzed a two-step reaction. The first step involved the ATPdependent formation of biotinyl-5⬘-AMP and pyrophosphate after which the biotinyl group was transferred from the AMP derivative to the appropriate lysyl ⑀-amino group of the apotranscarboxylase. Lane and Lynen also showed that the covalently bound biotinyl prosthetic group, like free biotin, was carboxylated on the 1⬘-N position (5). Shortly after he returned from Munich, Lane left Virginia Polytechnic Institute to become Associate Professor of Biochemistry at the New York University School of Medicine. He was later promoted to Professor of Biochemistry in 1969. In New York, Lane and his colleagues isolated acetyl coenzyme A carboxylase from chicken liver (6). The biotin-containing enzyme catalyzes the carboxylation of acetyl-CoA to malonyl-CoA in a 2-step process involving a carboxybiotin intermediate. In an accompanying paper, Lane described the molecular characteristics of the enzyme, including its reversible inter-conversion between protomeric and polymeric forms. The paper is reprinted here as the second JBC Classic by Lane. He determined that the carboxylase has a binding site for citrate and another for acetyl-CoA and that citrate binding might be involved in regulating the enzyme. Lane left New York in 1970 to become Professor of Biological Chemistry at the Johns Hopkins University School of Medicine. Right around the time Lane took up his new post at Johns Hopkins, Thomas C. Bruice and A. F. Hegarty published a paper (7) that called into question Lane’s conclusion that biotin was carboxylated on the 1⬘-N position. They pointed out that carboxylation could occur at the ureido-O and result in the same derivative. In the third JBC Classic, Lane uses the acetyl coenzyme A carboxylase system from Escherichia coli to provide definitive evidence that the ureido-N of biotin is the site of carboxylation. In the final JBC Classic reprinted here, Lane presents a thorough analysis of the acetyl coenzyme A carboxylase system from Escherichia coli. He defines the requirements and properties of isotopic exchange and stoichiometric reactions representative of the two halfreactions in acetyl-CoA carboxylation and also describes studies using prosthetic group and intermediate model derivatives as substrates to elucidate the mechanisms of the partial reactions. Lane was eventually promoted to Director and DeLamar Professor in 1978. He is currently Distinguished Service Professor in the Department of Biological Chemistry at Johns Hopkins. More information about Lane’s early work on biotin can be found in his JBC Reflections (8). Lane’s honors and awards include the American Institute of Nutrition’s Mead-Johnson award in 1966, the American Society of Biological Chemists’ William C. Rose award in 1981, and the Johns Hopkins University School of Medicine Professor’s Award for Distinction in H26

Classics Teaching in 1986. He was elected to the American Academy of Arts and Sciences in 1982, the American Society for Nutritional Sciences in 1996, and the National Academy of Sciences in 1987. In addition to serving as president of the American Society for Biochemistry and Molecular Biology in 1990, Lane served on the Society’s Program Committee, Membership Committee, and Public Affairs Committee. He has served on the Editorial Boards of several journals, including those of the Journal of Biological Chemistry, Biochemistry et Biophysica Acta, the Archives Biochemistry and Biophysics, and Annual Reviews of Biochemistry. He also served on the editorial board and was Executive Editor of Biochemical and Biophysical Research Communications in 1986. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. JBC Classics: Stern, J. R., and Ochoa, S. (1951) J. Biol. Chem. 191, 161–172; Korkes, S., del Campillo, A., Gunsalus, I. C., and Ochoa, S. (1951) J. Biol. Chem. 193, 721–735 (http://www.jbc.org/cgi/content/full/280/11/e8) 2. JBC Classics: Salas, M., Smith, M. A., Stanley, W. M., Jr., Wahba, A. J., and Ochoa, S. (1965) J. Biol. Chem. 240, 3988 –3995 (http://www.jbc.org/cgi/content/full/281/21/e16) 3. Lynen, F., Knappe, J., Lorch, E., Jutting, G., and Ringelmann, E. (1959) Die biochemische Funktion des Biotins. Angew. Chem. 71, 481– 486 4. Lane, M. D., Young, D. L., and Lynen, F. (1964) The enzymatic synthesis of holotranscarboxylase from apotranscarboxylase and (⫹)-biotin. I. Purification of the apoenzyme and synthetase; characteristics of the reaction. J. Biol. Chem. 239, 2858 –2864 5. Lane, M. D., and Lynen, F. (1963) The biochemical function of biotin. VI. Chemical structure of the carboxylated active site of propionyl carboxylase. Proc. Natl. Acad. Sci. U. S. A. 49, 379 –385 6. Gregolin, C., Ryder, E., and Lane, M. D. (1968) Liver acetyl coenzyme A carboxylase. I. Isolation and catalytic properties. J. Biol. Chem. 243, 4227– 4235 7. Bruice, T. C., and Hegarty, A. F. (1970) Biotin-bound CO2 and the mechanism of enzymatic carboxylation reactions. Proc. Natl. Acad. Sci. U. S. A. 65, 805– 809 8. Lane, M. D. (2004) The biotin connection: Severo Ochoa, Harland Wood, and Feodor Lynen. J. Biol. Chem. 279, 39187–39194

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Vol. 280, No. 35, Issue of September 2, p. e32, 2005 Printed in U.S.A.



The Role of the Acyl Carrier Protein in Fatty Acid Synthesis: the Work of P. Roy Vagelos Acyl Carrier Protein. III. An Enoyl Hydrase Specific for Acyl Carrier Protein Thioesters (Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 618 – 621) Acyl Carrier Protein. VII. The Primary Structure of the Substrate-binding Site (Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 4723– 4726) P. Roy Vagelos was born in Westfield, NJ in 1929. He received an A.B. degree from the University of Pennsylvania in 1950 and an M.D. from Columbia University’s College of Physicians and Surgeons in 1954. Following an internship and residency at the Massachusetts General Hospital in Boston, he joined the National Institutes of Health. There he launched a career as a research scientist under the guidance of Earl Stadtman, who authored a previous Journal of Biological Chemistry (JBC) Classic (1). With Stadtman, Vagelos demonstrated that long-chain fatty acid synthesis is catalyzed by an enzyme complex in which malonyl-CoA is the source of active acetate. From 1956 to 1966, Vagelos served as Senior Surgeon and then Section Head of Comparative Biochemistry in the National Heart Institute’s Laboratory of Biochemistry. During this time, he continued to study fatty acid synthesis, focusing on the role of acyl carrier protein (ACP). He discovered that the intermediates in fatty acid synthesis in Escherichia coli are linked to an acyl carrier protein via a thioester linkage. Vagelos published a series of papers on acyl carrier protein in the JBC, two of which are reprinted here as Classics. During fatty acid synthesis, the acyl groups of acetyl-CoA and malonyl-CoA are initially transferred by acetyl and malonyl transacylases to the sulfhydryl group of ACP. Acetyl-ACP and malonyl-ACP are then condensed to form acetoacetyl-ACP, which is reduced to D(⫺)-␤hydroxybutyryl-ACP. The transacylases, condensing enzyme (acyl-malonyl-ACP condensing enzyme), and reductase (␤-ketoacyl-ACP reductase) were characterized by Vagelos. This first Classic focuses on the purification and properties of the enol hydrase (3-hydroxyacyl-ACP dehydratase) that catalyzes the dehydration of D(⫺)-␤-hydroxybutyryl-ACP to crotonyl-S-ACP. The second Classic deals with how substrates are linked to ACP. Vagelos had previously reported that, similar to CoA, substrates are bound to ACP via the sulfhydryl group of 4⬘-phosphopantetheine. However, he noticed that despite this similarity between the two carriers, thioesters of CoA could not substitute effectively for ACP in fatty acid synthesis. Upon further study of the structure of ACP, as reported in the second Classic, Vagelos discovered that 4⬘-phosphopantetheine is bound to ACP through a phosphodiester linkage to the hydroxyl group of a serine residue. In 1966, Vagelos assumed the chairmanship of the Department of Biological Chemistry at Washington University’s School of Medicine in St Louis, MO. He continued to work on fatty acid biosynthesis and metabolism and expanded his research to the synthesis of complex lipids and the role of cholesterol in the biochemistry of the cell. In 1973 he became Director of the University’s Division of Biology and Biomedical Sciences, which he founded. This Division eventually became a model for other universities. It included both the undergraduate Department of Biology and the Medical School in one umbrella unit, which was unheard of at the time. This paper is available on line at http://www.jbc.org

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Vagelos left academia in 1975 to join Merck Sharp & Dohme Research Laboratories as Senior Vice President for Research. In 1984 he was named an Executive Vice President of Merck and was elected to its Board of Directors, and in 1984 he became Merck’s Chief Executive Officer. He served as CEO and Chairman of the Board until 1994. Under his direction, the company expanded its philanthropic efforts as well as its pharmaceutical research. He is perhaps best known for his decision to make Merck’s Invermectin (Mectizan) available free to millions of people in Africa and Central America for the treatment of river blindness, a disease spread by black flies that causes chronic rashes, itching, weight loss, and blindness. In recognition of his contributions to science, Vagelos received the American Chemical Society’s Enzyme Chemistry Award in 1967. He was elected to both the National Academy of Sciences and the American Academy of Arts and Sciences in 1972 and to the American Philosophical Society in 1993. In 1989 he received the Thomas Alva Edison Award from then New Jersey Governor Thomas Kean. He is currently Chairman of the Board of Regeneron Pharmaceuticals, Inc. as well as a member of the Board of Directors of the Prudential Insurance Company.1 Vagelos’ coauthors on several of the JBC acyl carrier protein papers, including the two reprinted here, are Philip W. Majerus and Alfred W. Alberts. Majerus went on to become a Professor at Washington University School of Medicine and has been a leader in phosphoinositide metabolism and signaling, platelet physiology, and blood coagulation. He is a member of the National Academy of Sciences and has won numerous awards for his research, including the 1998 Bristol-Myers Squibb Award for Distinguished Achievement in Cardiovascular/ Metabolic Research. Alberts moved from Washington University to Merck with Vagelos and was the lead scientist in Merck’s development of the statin drugs Lovastatin and Zocor. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. JBC Classics: Stadtman, E. R., and Barker, H. A. (1949) J. Biol. Chem. 180, 1085–1093; Mura, U., Chock, P. B., and Stadtman, E. R. (1981) J. Biol. Chem. 256, 13022–13029 (http://www.jbc.org/cgi/content/full/280/26/e23) 2. Hawthorne, F. (2003) The Merck Druggernaut, John Wiley & Sons, Inc., Hoboken, NJ 3. Park, B. S. The Stadtman Way: a Tale of Two Biochemists at NIH. http://history.nih.gov/exhibits/stadtman/ index.htm (An online exhibit produced by the Office of NIH History in collaboration with the National Heart, Lung, and Blood Institute) 1

All biographical information on P. Roy Vagelos was taken from Refs. 2 and 3.

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Vol. 283, No. 20, Issue of May 16, p. e12, 2008 Printed in U.S.A.



How Aspirin Interferes with Cyclooxygenase Activity: the Work of William L. Smith Stimulation and Blockade of Prostaglandin Biosynthesis (Smith, W. L., and Lands, W. E. M. (1971) J. Biol. Chem. 246, 6700 – 6702) The Aspirin and Heme-binding Sites of Ovine and Murine Prostaglandin Endoperoxide Synthases (DeWitt, D. L., el-Harith, E. A., Kraemer, S. A., Andrews, M. J., Yao, E. F., Armstrong, R. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 5192–5198) William L. Smith, Jr. was born in Tulsa, Oklahoma in 1945. He and his family moved to the Chicago area when he was an infant and then to Fort Collins, Colorado when he was in high school. After graduating from high school, Smith enrolled in a premed program at the University of Colorado in the fall of 1963, even though he had no idea what sort of career he wanted to pursue. However, this changed during the first couple years of college when he took several chemistry courses that were taught by excellent teachers. His admiration for these professors led to his decision to attend graduate school. Originally, he planned to go into physical organic chemistry but chose biological chemistry when he was told that physical organic chemistry was already a highly populated field. “Little did I know that the same thing would happen in biochemistry by the time I was searching for a position,” recalls Smith. After graduating with a B.A. in 1967, Smith decided to attend the University of Michigan and work with William E. M. Lands. This decision William L. Smith was influenced by three papers by Mats Hamberg and Bengt Samuelsson (1–3) published in the Journal of Biological Chemistry (JBC) that Lands gave him to read.“These papers dealt with the mechanism of oxygenation of dihomo-␥-linolenic acid and other fatty acids by soybean lipoxygenase and sheep seminal vesicle cyclooxygenase (i.e. prostaglandin H2 synthase 1 or PGHS-1),” recalls Smith. “In the course of these studies they labeled the ␻8 carbon (C-13) of the substrate with tritium in the This paper is available on line at http://www.jbc.org

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Classics proS and proR orientations and found a distinct kinetic isotope effect in the removal of the proS hydrogen. This indicated that the rate determining step was abstraction of this hydrogen from the fatty acid. To this day, I find this to be a brilliant experiment and an exciting outcome. Almost 25 years later we provided evidence that Tyr-385 of PGHSs abstracted this hydrogen atom from the fatty acid (4).” More information on Samuelsson’s work on the prostaglandins can be found in his JBC Classic (5). Just as Smith was finishing up his thesis work with Lands, John Vane reported that acetylsalicylic acid (aspirin) and another well known nonsteroidal anti-inflammatory drug called indomethacin blocked the biosynthesis of prostaglandins from arachidonic acid (6, 7). Smith had just set up an O2 electrode assay to measure prostaglandin production in acetone powder preparations of sheep seminal vesicle microsomes, and he and Lands immediately tested aspirin and indomethacin in this assay. As reported in the first JBC Classic reprinted here, they found that aspirin and indomethacin blocked arachidonic acid-induced O2 uptake and concluded that these drugs were blocking oxygenase activity. They also observed that the two drugs acted in a time-dependent manner, suggesting that they were causing a chemical modification of their target. Subsequently, it was shown that the acetyl group of aspirin was incorporated into a protein (8) that Smith later purified (9) in his first JBC paper as an independent scientist. The protein is now known as prostaglandin endoperoxide H synthase-1 (PGHS-1) or cyclooxygenase-1 (COX-1). Smith completed his Ph.D. work in under 4 years, and in 1971 went to the University of California at Berkeley to work with Clinton E. Ballou. There he changed the focus of his research and worked on polysaccharide structures. Although he enjoyed this work, he decided he was more interested in solving problems that he felt were more biomedically relevant. He also realized that he still had an intense interest in prostaglandins. In 1974, Smith took a job as a senior scientist at Mead Johnson in Evansville, Indiana where he spent his time studying platelet aggregation, prostaglandin formation, and arachidonic acid mobilization. A year later, he accepted a position in the Department of Biochemistry at Michigan State University where he remained for 28 years and served as chair for the last 8 years of his time there. At Michigan State, Smith continued to work on prostaglandins, focusing, among other things, on the acetylation of PGHS-1. Work in other laboratories suggested that aspirin was acetylating a serine residue on the enzyme (10, 11). In 1988 Smith and his long time colleague David DeWitt were able to clone and sequence PGHS-1 cDNA derived from seminal vesicles (12). They observed that the acetylated serine corresponded to Ser-530. Two years later, as reported in the second JBC Classic reprinted here, Smith and his colleagues showed that substitution of Ser-530 with alanine rendered the protein refractory toward aspirin but had relatively little effect on the kinetic properties of the cyclooxygenase. They concluded that the Ser-O-acetyl protrudes into the cyclooxygenase active site thereby interfering with arachidonic acid binding. This is the most definitive biochemical work on how aspirin works at the molecular level to interfere with cyclooxygenase activity. Smith later showed that substitutions of Ser-530 with bulkier residues such as threonine and asparagine gradually reduced cyclooxygenase activity (13). In 2002 Smith decided to step down as Chair of Biochemistry at Michigan State, saying “My opinion is that administrators should serve no longer than American presidents.” Shortly thereafter he was given the opportunity to become Chair of the Biological Chemistry Department at the University of Michigan. Still at the University of Michigan today, he continues to work on prostaglandins. In recognition of his many contributions to science, Smith has received several awards and honors. These include the 1991 Treadwell Award from George Washington University, the 1992 Distinguished Faculty Award from Michigan State University, the 1996 Abraham White Distinguished Scientific Achievement Award from George Washington University, the 1997 Senior Aspirin Award from Bayer Corporation, the 1999 Michigan Universities Association of Governing Boards Award, the 2004 State of Michigan Scientist of the Year Award, the 2004 Berzelius Lectureship from the Karolinska Institute, the 2004 Avanti Award from the American Society for Biochemistry and Molecular Biology, the 2006 William C. Rose Award from the American Society for Biochemistry and Molecular Biology, and the 2006 Hayaishi Lectureship from Hamamatsu University.1 1

We thank William L. Smith for providing background information for this introduction.

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Classics Smith’s co-author on the first Classic, William E. M. Lands, was a young faculty member at the University of Michigan when Smith joined his laboratory. Lands subsequently left Michigan in 1980 to become Chair of Biochemistry at the University of Illinois, Chicago, and later moved to the National Institutes of Health in 1990, where he served as the Senior Scientific Advisor to the Director of the National Institute on Alcohol Abuse and Alcoholism. Lands is the discoverer of the “retailoring” pathway for membrane phospholipid synthesis. He is an authority on essential fatty acids and is credited with recognizing the beneficial effects of balancing excess ␻-6 fatty acids with dietary ␻-3 fatty acids. In recognition of his work, the University of Michigan’s Department of Biological Chemistry endowed a lectureship in nutritional biochemistry in honor of Lands. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. Hamberg, M., and Samuelsson, B. (1967) On the specificity of the oxygenation of unsaturated fatty acids catalyzed by soybean lipoxidase. J. Biol. Chem. 242, 5329 –5335 2. Hamberg, M., and Samuelsson, B. (1967) On the mechanism of the biosynthesis of prostaglandins E1 and F1␣. J. Biol. Chem. 242, 5336 –5343 3. Hamberg, M., and Samuelsson, B. (1967) Oxygenation of unsaturated fatty acids by the vesicular gland of sheep. J. Biol. Chem. 242, 5344 –5354 4. Shimokawa, T., Kulmacz, R. J., DeWitt, D. L., and Smith, W. L. (1990) Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis. J. Biol. Chem. 265, 20073–20076 5. JBC Classics: Bergstro¨m, S., Ryhage, R., Samuelsson, B., and Sjo¨vall, J. (1963) J. Biol. Chem. 238, 3555–3564; Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336 –5343 (http://www.jbc.org/cgi/content/full/ 281/9/e9) 6. Vane, J. R. (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat. New Biol. 231, 232–235 7. Ferreira, S. H., Moncada, S., and Vane, J. R. (1971) Indomethacin and aspirin abolish prostaglandin release from the spleen. Nat. New Biol. 231, 237–239 8. Roth, G. J., Stanford, N., and Majerus, P. W. (1975) Acetylation of prostaglandin synthase by aspirin. Proc. Natl. Acad. Sci. U. S. A. 72, 3073–3076 9. Hemler, M., Lands, W. E., and Smith, W. L. (1976) Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme. J. Biol. Chem. 251, 5575–5579 10. Van Der Ouderaa, F. J., Buytenhek, M., Nugteren, D. H., and Van Dorp, D. A. (1980) Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid. Eur. J. Biochem. 109, 1– 8 11. Roth, G. J., Machuga, E. T., and Ozols, J. (1983) Isolation and covalent structure of the aspirin-modified, active-site region of prostaglandin synthetase. Biochemistry 22, 4672– 4675 12. DeWitt, D. L., and Smith, W. L. (1988) Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc. Natl. Acad. Sci. U. S. A. 85, 1412–1416 13. Shimokawa, T., and Smith, W. L. (1992) Prostaglandin endoperoxide synthase. The aspirin acetylation region. J. Biol. Chem. 267, 12387–12392

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Vol. 281, No. 31, Issue of August 4, p. e25, 2006 Printed in U.S.A.



30 Years of Cholesterol Metabolism: the Work of Michael Brown and Joseph Goldstein Binding and Degradation of Low Density Lipoproteins by Cultured Human Fibroblasts. Comparison of Cells from a Normal Subject and from a Patient with Homozygous Familial Hypercholesterolemia (Goldstein, J. L., and Brown, M. S. (1974) J. Biol. Chem. 249, 5153–5162) Joseph Leonard Goldstein was born in 1940 in Sumter, South Carolina. He attended Washington and Lee University in Lexington, Virginia and received a B.S. degree in chemistry in 1962. Goldstein then went to the Southwestern Medical School at the University of Texas Health Science Center in Dallas where he was inspired to pursue a career in academic medicine by the Chairman of the Department of Internal Medicine, Donald W. Seldin. During Goldstein’s last year in medical school, Seldin offered him a future faculty appointment if he agreed to specialize in genetics and return to Dallas to establish a division of medical genetics in the Department of Internal Medicine. Goldstein initially declined, and after receiving his M.D. in 1966, he moved to Boston where he was an intern and resident at the Massachusetts General Hospital. It was there that he first met and developed a friendship with Michael Stuart Brown, his long term scientific collaborator. Brown, who was born in 1941 in Brooklyn, New York, graduated in 1962 from the University of Pennsylvania, with a major in chemistry. He received his M.D. degree from the University of Pennsylvania School of Medicine in 1966, after which he became an intern and resident at the Massachusetts General Hospital. After they completed their training in 1968, both Brown and Goldstein obtained positions at the National Institutes of Health in Bethesda, Maryland. Brown joined the Laboratory of Biochemistry at the National Heart, Lung, and Blood Institute (NHLBI). Goldstein worked with Nobel laureate Marshall W. Nirenberg and also worked as a clinical associate at NHLBI, serving as physician to the patients of Donald S. Fredrickson, who was an expert on disorders of lipid metabolism. Several of these patients had homozygous familial hypercholesterolemia, a condition that causes severe elevations in cholesterol levels. Goldstein discussed these patients extensively with Brown and, in view of their common interest in metabolic disease, convinced Brown to join him as a faculty member at the University of Texas Health Science Center at Dallas, where they would work collaboratively on the genetic regulation of cholesterol metabolism. In 1971 Brown joined the division of Gastroenterology in the Department of Internal Medicine at the University of Texas Southwestern Medical School. Before going back to Dallas, Goldstein spent 2 years with Arno G. Motulsky at the University of Washington in Seattle, studying human genetics. He returned to the University of Texas Health Science Center in 1972 and was appointed Assistant Professor in the Department of Internal Medicine and head of the medical school’s first Division of Medical Genetics. Together, Brown and Goldstein began to address the task of identifying the genetic defect in familial hypercholesterolemia. They started by observing tissue cultures of fibroblasts harvested from healthy individuals and individuals with familial hypercholesterolemia. They set up a microassay to measure the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-determining enzyme of cholesterol biosynthesis, in the fibroblasts. Soon it became clear that the cholesterol transport protein, low density lipoprotein (LDL), suppressed the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Because high density lipoproThis paper is available on line at http://www.jbc.org

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Fig. 1

tein (HDL) was unable to do this, Brown and Goldstein suspected that a receptor might be involved in the control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The existence of an LDL receptor was confirmed when Brown and Goldstein radiolabeled LDL with 125I and incubated it with normal and familial hypercholesterolemial fibroblasts. As reported in the JBC Classic reprinted here, their studies showed that normal cells had high affinity binding sites for 125I-LDL whereas familial hypercholesterolemial cells did not. Binding of LDL to the high affinity membrane receptor sites suppressed the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase and also facilitated the degradation of LDL when it was present at low concentrations. Cells from subjects with familial hypercholesterolemia not only lacked the binding sites but were also resistant to suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by LDL and were deficient in high affinity degradation of LDL. However, the question of how LDL generated the signal that suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase still remained. The answer to this question came from studies of surface-bound 125I-LDL. Brown and Goldstein found that the receptor-bound LDL remained on the cell’s surface for less than 10 min. Within this time most of the surface-bound LDL particles entered the cell. Within another 60 min the protein component of 125I-LDL was digested completely. The only cellular organelle that could have degraded LDL so completely and rapidly was the lysosome. This was eventually confirmed, and Brown and Goldstein also showed that the cholesterol that was generated from lysosomal degradation of LDL acted as the second messenger responsible for suppressing 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. As is often the case with truly novel, groundbreaking discoveries, the work presented in this JBC Classic was not met with great enthusiasm by journal reviewers. The initial review of this paper by the JBC is presented in Fig. 1, and the decision letter from Associate Editor Eugene Kennedy is shown in Fig. 2. This paper, the basis of the Nobel Prizes awarded to Brown and Goldstein, was eventually accepted. By 1974 Brown and Goldstein had merged their laboratories. They continued their work on the LDL receptor and eventually purified and sequenced it. In recognition of their work, they were awarded the 1985 Nobel Prize in Physiology or Medicine “for their discoveries concerning the regulation of cholesterol metabolism.” Goldstein eventually became Associate Professor of Internal Medicine at the University of Texas Southwestern Medical School (1974) and then Professor (1976). In 1977, he was made Chairman of the Department of Molecular Genetics at the University of Texas Health Science Center and Paul J. Thomas Professor of Medicine and Genetics, a position that he still holds today. In 1985, he was named Regental Professor of the University of Texas, and in 1983 he became a Non-resident Fellow of The Salk Institute for Biological Sciences. H34

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Fig. 2

In 1974, Brown was promoted to the rank of Associate Professor of Internal Medicine at the University of Texas Southwestern Medical School. He became a Professor in 1976. In 1977 he was appointed Paul J. Thomas Professor of Medicine and Genetics and Director of the Center for Genetic Disease at the same institution. In 1985, Brown was named Regental Professor of the University of Texas. In addition to the Nobel Prize, Brown and Goldstein’s work has been recognized by their receipt of numerous awards, including the Heinrich Wieland Prize for Research in Lipid Metabolism (1974), the American Chemical Society’s Pfizer Award for Enzyme Chemistry (1976), the Passano Award (1978), the National Academy of Sciences’ Lounsbery Award (1979), the Gairdner Foundation International Award (1981), the Lita Annenberg Hazen Award (1982), the Association of American Medical Colleges’ Distinguished Research Award (1984), the American Heart Association’s Research Achievement Award (1984), the FASEB 3M Life Sciences Award (1985), the Albert D. Lasker Award in Basic Medical Research (1985), the U. S. National Medal of Science (1988), the Albany Medical Prize in Biomedical Sciences (2003), and the Herbert Tabor/Journal of Biological Chemistry Lectureship (2005). Both Brown and Goldstein were elected to the National Academy of Sciences and the American Academy of Arts and Sciences. Goldstein is, or has been, a member of the editorial board of several journals including the Annual Review of Genetics, Arteriosclerosis, Cell, the Journal of Biological Chemistry, the Journal of Clinical Investigation, and Science. Brown has served on the editorial boards of the Journal of Lipid Research, the Journal of Cell Biology, and Arteriosclerosis and Science.1,2 Nicole Kresge, Robert D. Simoni, and Robert L. Hill 1 2

All biographical information on Joseph L. Goldstein was taken from Refs. 1 and 2. All biographical information on Michael S. Brown was taken from Refs. 2 and 3.

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Joseph L. Goldstein

Michael S. Brown

REFERENCES 1. Goldstein, J. L., and Brown, M. S. (1993) A receptor-mediated pathway for cholesterol homeostasis. From Nobel Lectures, Physiology or Medicine 1981–1990 (Fra¨ngsmyr, T., ed) World Scientific Publishing Co., Singapore 2. Goldstein, J. L. (1986) Joseph L. Goldstein—Biography. In The Nobel Prizes 1985 (Odelberg, W., ed) Nobel Foundation, Stockholm 3. Brown, M. S. (1986) Michael S. Brown—Biography. In The Nobel Prizes 1985 (Odelberg, W., ed) Nobel Foundation, Stockholm

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Vol. 281, No. 5, Issue of February 3, p. e5, 2006 Printed in U.S.A.



Salih Wakil’s Elucidation of the Animal Fatty Acid Synthetase Complex Architecture The Architecture of the Animal Fatty Acid Synthetase. I. Proteolytic Dissection and Peptide Mapping (Mattick, J. S., Tsukamoto, Y., Nickless, J., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15291–15299) The Architecture of the Animal Fatty Acid Synthetase. II. Separation of the Core and Thioesterase Functions and Determination of the N-C Orientation of the Subunit (Mattick, J. S., Nickless, J., Mizugaki, M., Yang, C. Y., Uchiyama, S., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15300 –15304) The Architecture of the Animal Fatty Acid Synthetase. III. Isolation and Characterization of Beta-Ketoacyl Reductase (Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15305–15311) The Architecture of the Animal Fatty Acid Synthetase Complex. IV. Mapping of Active Centers and Model for the Mechanism of Action (Tsukamoto, Y., Wong, H., Mattick, J. S., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15312–15322) Salih Jawad Wakil was born in 1927 in Kerballa, Iraq. Because he placed third in the nation on the baccalaureate examination out of high school, he received a scholarship to the American University in Beirut. While at the American University he met Stanley Kerr, who introduced him to biochemistry and gave him the opportunity to work in his laboratory. After graduating in 1948, Wakil was accepted at the University of Washington, which he assumed was located in the U. S. capital. However, he arrived in the United States only to learn that he would have to take a 3-day train journey from New York to his university in Washington State. In Seattle, Wakil worked with Donald Hanahan and finished his graduate studies in biochemistry in 31⁄2 years. Next, he decided to do postdoctoral training at the Enzyme Institute of the University of Wisconsin, where he began to work on fatty acid oxidation. It was there that he helped to elucidate the steps by which fatty acids are oxidized and showed that fatty acids are synthesized and oxidized by different pathways. Wakil was named assistant professor in 1956, but joined the Department of Biochemistry at the Duke University School of Medicine in 1959 and rose to the rank of professor there (1965). At Duke, Wakil investigated fatty acid synthesis in Escherichia coli. He and Roy Vagelos, who was featured in a previous Journal of Biological Chemistry (JBC) Classic (1), independently studied the role of acyl carrier protein as well as several of the individual reactions of fatty acid elongation. Wakil left Duke in 1971 to become professor and chairman of the Verna and Marrs McLean Department of Biochemistry and Molecular Biology at Baylor College of Medicine in Houston,Texas. At Baylor, Wakil studied the multifunctional enzyme, fatty acid synthetase. The characterization of this enzyme complex is the subject of the four JBC Classics reprinted here. In vertebrates, the fatty acid synthetase complex exists as a dimer of what Wakil believed were identical subunits derived from a single large mRNA. The complex contains the seven enzymatic activities needed for the assembly of fatty acids: (i) acetyl transacylase, (ii) malonyl transacylase, (iii) ␤-ketoacyl synthetase, (iv) ␤-ketoacyl reductase, (v) ␤-hydroxyacyl dehyThis paper is available on line at http://www.jbc.org

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Salih J. Wakil

dratase, (vi) enoyl reductase, and (vii) palmitoyl thioesterase, as well as an acyl carrier peptide to which the nascent chain is attached. These Classics, which were printed as a back-to-back series in one issue of the JBC, present Wakil’s comprehensive proteolytic analysis of chicken fatty acid synthetase in which he assigned relative locations for the enzymatic activities in the complex. In the first Classic, Wakil and his colleagues used seven different proteases to digest the synthetase. They found that the sum of the molecular weights of each set of fragments generated by the proteases corresponded to the size of the synthetase subunit rather than the native dimer, indicating that the synthetase was indeed a homodimer. The researchers also reported that the subunit is arranged into three major domains of Mr ⫽ 127,000, 107,000, and 33,000. Wakil describes the cleavage of chicken fatty acid synthetase by ␣-chymotrypsin in the second Classic. The complex was cleaved into two fragments. The larger 230-kDa fragment contained all the core activities involved in the assembly of the fatty acyl chain whereas the smaller 33-kDa fragment retained the thioesterase activity which releases the complete product. Using amino acid sequence analysis, Wakil showed that the thioesterase domain is located at the carboxyl terminus of the synthetase monomer. In the third Classic, Wakil used trypsin and subtilisin to cleave fatty acid synthetase and isolated a polypeptide containing only ␤-ketoacyl reductase activity. Using a kallikrein/subtilisin double digestion, Wakil and his colleagues also isolated another fragment containing ␤-ketoacyl reductase activity as well as the phosphopantetheine prosthetic group. From this, Wakil concluded that the acyl carrier protein moiety is located in the 15-kDa segment that separates the ␤-ketoacyl reductase from the thioesterase domain. In the fourth and final Classic, Wakil presents an architectural model for the synthetase based on his results from the previous three papers. In Wakil’s model, domain I functions as a site for acetyl and malonyl substrate entry and acts as the site of carbon-carbon condensation. Thus, this domain contains the amino terminus of the polypeptide and the ␤-ketoacyl synthetase and acetyl and malonyl transacylases. Domain II, the reductive domain, contains the ␤-ketoacyl and enoyl reductases, probably the dehydratase, and the 4⬘-phosphopantetheine prosthetic group of the acyl carrier protein. Finally, domain III contains the thioesterase activity. Based on his observations, Wakil concluded that even though each subunit contains all the activities needed for fatty acyl synthesis, the actual synthesizing unit consists of one-half of one subunit interacting with the complementary half of the other subunit. This is shown in the model in Fig. 2. Wakil, along with Bornali Chakravarty, Ziwei Gu, Subrahmanyam S. Chirala, and Florante A. Quiocho, subsequently solved the crystal structure of the H38

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Fig. 2

thioesterase domain of human fatty acid synthetase (2). Recent crystallographic analysis of both the animal and fungal fatty acid synthases demonstrates that the structure is of a head-to-head coiled dimer (3, 4). Today, Wakil remains at Baylor where he is Distinguished Service Professor and Bolin Professor in the Department of Biochemistry and Molecular Biology. Most recently, his focus has been on acetyl-CoA carboxylase (ACC), which exists in two forms, ACC1 and ACC2. He has developed a transgenic mouse, which does not produce ACC2, and as a result can eat 20 –30% more food and weighs 10% less than mice that produce the enzyme. In honor of Wakil’s contributions to the field of fatty acid metabolism, he has received many awards and honors. These include the Paul Lewis Award from the American Chemical Society (1967), the Chilton Award of the University of Texas Southwestern Medical Center (1985), the Kuwait Prize of the Kuwait Foundation for the Advancement of Sciences (1988), the Yamanouchi USA Foundation Award (2001), and the Bristol-Myers Squibb Freedom to Discover Award (2005). In 1990, Wakil was the first Baylor College of Medicine faculty member to be elected to the National Academy of Sciences. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. JBC Classics: Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 618 – 621; Majerus, P. W., Alberts, A. W., and Vagelos, P. R. (1965) J. Biol. Chem. 240, 4723– 4726 (http://www.jbc.org/cgi/ content/full/280/35/e32) 2. Chakravarty, B., Gu, Z., Chirala, S. S., Wakil, S. J., and Quiocho, F. A. (2004) Human fatty acid synthase: structure and substrate selectivity of the thioesterase domain. Proc. Natl. Acad. Sci. U. S. A. 101, 15567–15572 3. Smith, S. (2006) Architectural options for a fatty acid synthase. Science 311, 1251–1252 ˚ resolution. Science 4. Maier, T., Jenni, S., and Ban, N. (2006) Architecture of mammalian fatty acid synthase at 4.5 A 311, 1258 –1262

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Vol. 283, No. 2, Issue of January 11, p. e2, 2008 Printed in U.S.A.



N-Myristoyltransferase Substrate Selection and Catalysis: the Work of Jeffrey I. Gordon Myristoyl CoA:Protein N-Myristoyltransferase Activities from Rat Liver and Yeast Possess Overlapping Yet Distinct Peptide Substrate Specificities (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon, J. I. (1988) J. Biol. Chem. 263, 1784 –1790) Isothermal Titration Calorimetric Studies of Saccharomyces cerevisiae MyristoylCoA:Protein N-Myristoyltransferase. Determinants of Binding Energy and Catalytic Discrimination among Acyl-CoA and Peptide Ligands (Bhatnagar, R. S., Jackson-Machelski, E., McWherter, C. A., and Gordon, J. I. (1994) J. Biol. Chem. 269, 11045–11053) Jeffrey I. Gordon obtained his A.B. from Oberlin College in 1969 and his M.D. from the University of Chicago in 1973. He then became an intern and junior assistant resident at Barnes Hospital in St. Louis before spending 3 years as a research associate in the Laboratory of Biochemistry at the National Cancer Institute (National Institutes of Health). Gordon returned to Barnes Hospital as a Senior Assistant Resident in 1978 and was concurrently a Chief Medical Resident at John Cochran VA Hospital in St. Louis. He became an assistant professor at Washington University in St. Louis in 1981 and has remained there ever since. He is currently Director of the Center for Genome Sciences as well as the Dr. Robert J. Glaser Distinguished University Professor. Gordon is probably best known for his research on gastrointestinal development and how gut bacteria affect normal intestinal function and predisposition to health and to certain diseases. However, he has also made significant contributions to our knowledge about myristoylation, the posttranslational process by which a myristoyl group is covalently attached via an amide bond to the ␣-amino group of an N-terminal Jeffrey I. Gordon glycine residue of a nascent polypeptide. Some of Gordon’s work on N-myristoyltransferase, the enzyme responsible for myristoylation, is the subject of the two Journal of Biological Chemistry (JBC) Classics reprinted here. Gordon started doing research on N-myristoyltransferase when Luis Glaser, a fellow scientist at Washington University, accepted a position at the University of Miami and transferred This paper is available on line at http://www.jbc.org

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Classics the project, along with his student Dwight Towler, to Gordon’s laboratory. Glaser and Towler had already partially purified N-myristoyltransferase and were using peptides with altered sequences to test as enzyme substrates in an in vitro assay system (1, 2). From their studies they had determined that residues occupying positions 1, 2, and 5 in peptide substrates (where the myristoylated glycine is residue 1) have important effects on protein-ligand interactions. In Gordon’s laboratory, Towler worked out a purification scheme for N-myristoyltransferase from yeast (3) and continued to investigate the primary structural characteristics of the enzyme’s peptide substrates. As reported in the first JBC Classic, Towler and Gordon screened over 80 synthetic peptides and discovered that a substrate hexapeptide contains much of the information necessary for recognition by N-myristoyltransferase. They also identified a number of potential N-myristoyl proteins from searches of available protein data bases. In the second JBC Classic, Gordon and his colleagues report on the kinetics of the reaction in which N-myristoyltransferase transfers myristate from myristoyl-CoA to the amino-terminal glycine nitrogen of its substrate. Using isothermal titration calorimetry they quantified the effects of varying acyl chain length and removing the 3⬘-phosphate group of CoA on the energetics of interaction between N-myristoyltransferase and acyl-CoA ligands. From these studies, they were able to gain insights into how the enzyme selects its substrates and about its catalytic mechanism. In recognition of his contributions to science, Gordon has received many awards and honors. These include the 1990 American Federation for Clinical Research Young Investigator Award, the 1990 National Institute of Diabetes and Digestive and Kidney Diseases Young Scientist Award, the 1992 American Gastroenterological Association (AGA) Distinguished Achievement Award, the 1994 Marion Merrell Dow Distinguished Prize in Gastrointestinal Physiology, the 2003 Janssen/AGA Award for Sustained Achievement in Digestive Sciences, and the 2003 Horace W. Davenport Distinguished Lectureship from the American Physiological Association. Gordon is also a member of the National Academy of Sciences and the American Academy of Arts and Sciences. Luis Glaser, who was responsible for initiating Gordon’s investigations of N-myristoyltransferase, has also led a fruitful career in science. Glaser, who grew up in Mexico, attended the University of Toronto for his undergraduate education and earned his Ph.D. from Washington University. After graduating, he joined the faculty of Washington University where he remained for the next 30 years, spending the last 10 years as Chairman of the Department of Biology and Chemistry and Director of the Division of Biology and Biomedical Science. In 1986, Glaser accepted an offer to become Executive Vice President and Provost at the University of Miami. He retired from that position in July of 2005 and is currently a Professor of Biology and Special Assistant to the President. Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES 1. Towler, D., and Glaser, L. (1986) Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide. Proc. Natl. Acad. Sci. U. S. A. 83, 2812–2816 2. Towler, D. A., Eubanks, S. R., Towery, D. S., Adams, S. P., and Glaser, L. (1987) Amino-terminal processing of proteins by N-myristoylation. Substrate specificity of N-myristoyltransferase. J. Biol. Chem. 262, 1030 –1036 3. Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon, J. I. (1987) Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase. Proc. Natl. Acad. Sci. U. S. A. 84, 2708 –2712

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Vol. 276, No. 46, Issue of November 16, pp. 42619 –42631, 2001 Printed in U.S.A.

Reflections A PAPER IN A SERIES COMMISSIONED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005


Hitler’s Gift and the Era of Biosynthesis Published, JBC Papers in Press, September 14, 2001, DOI 10.1074/jbc.R100051200

Eugene P. Kennedy‡ From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Before the Second World War biochemistry in the United States had a strong flavor of clinical chemistry. It was much occupied with problems of analysis of blood and tissues and the determination of the structures of body constituents. This was important and indeed essential work, but American students had to go abroad to Germany or to England for training in what came to be called dynamic aspects of biochemistry. After the war, the flow of students was largely reversed. This transformation was in considerable part the result of new insights and new approaches brought to America by immigrant scientists. It is a remarkable fact that as late as 1945 when I began graduate studies in biochemistry at the University of Chicago almost nothing was known about the linked reactions leading to the biosynthesis of any of the major types of cell constituents, carbohydrates, lipids, proteins, or nucleic acids. However, this picture was about to change with dramatic rapidity. The latter half of the 20th century became the era of biosynthesis. Now, in 2001, we know in great detail the patterns of reactions leading to the formation of each of these classes of cellular materials, although to be sure much remains to be learned about the regulation and integration of biosynthetic processes in living organisms. The achievements of three biochemists, Fritz Lipmann, Rudolf Schoenheimer, and Konrad Bloch, greatly stimulated this flowering of biosynthetic studies in the United States at the mid-20th century. Each had been driven out of Germany by the brutal anti-Semitism of the Nazi regime. Each was an important part of what has been called Hitler’s gift (1) to American and British science. In helping to bring about the transition to the era of biosynthesis, Fritz Lipmann made clear the crucial role of “energy-rich” phosphates in driving biosynthetic reactions and showed how this principle operated in the formation of the much sought and highly elusive “active acetate” involved in so many pathways. Rudolf Schoenheimer helped put into the hands of biochemists their most subtle and versatile approach, that of the isotope tracer technique, and with its aid revealed the dynamic state of body constituents. Konrad Bloch’s work on the formation of cholesterol illustrated how the insights of Lipmann and Schoenheimer could be combined in a masterpiece of biochemistry to solve a problem of great medical as well as biological significance. Fritz Lipmann: The Energetics of Biosynthesis Fritz Lipmann (Fig. 1), who helped to shape the development of modern biochemistry, was born in Koenigsberg, East Prussia in 1899 into a Jewish family of the professional class (2). In 1917, he began the study of medicine. In 1918, while still a medical student, he was drafted into the German army and spent the rest of the war in the medical corps in France. Released from the army, Lipmann resumed his medical studies and received the M.D. degree in 1921. He soon abandoned plans for the practice of medicine in favor of biochemical research, but he always valued the broad view of biology his medical education had given him, concluding: “The ‡ To whom correspondence may be addressed. E-mail: [email protected]. This paper is available on line at http://www.jbc.org

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Reflections: Hitler’s Gift and the Era of Biosynthesis

FIG. 1. Fritz Lipmann. Photo by John Brook, made available through the courtesy of Ms. Freda Hall Lipmann.

biological education to which the observant student is exposed in medicine is a superior preparation for any career.” Indeed, the study of medicine offered the most comprehensive view of biology then available. Many of the greatest figures in biochemistry early in the 20th century, including Warburg, Meyerhof, and Krebs, were trained as physicians. The breadth of his background helped give Lipmann the confidence that nothing in biology was beyond his range. Again and again, he proved ready to tackle new problems, no matter how far removed from previous work in his laboratory. Turning to a career in research rather than the practice of medicine, Lipmann realized that the most fruitful approach to biological problems was through chemistry. He began a program leading to a Ph.D. in chemistry. His work for the dissertation, begun in 1927, was carried out in the laboratory of Otto Meyerhof. Meyerhof, whose work on glycolysis in muscle earned him a Nobel Prize, had a laboratory on the first floor of the Kaiser-Wilhelm Institute for Biology in Berlin, a city that was then the leading center of science in the world. Lipmann felt that his experience in Meyerhof’s laboratory was in many ways the origin of all his later work. His most intense admiration, however, was reserved for Warburg. As Lipmann later recalled (3): “At the top of everything, on the uppermost floor, was Otto Warburg. Warburg already had a mystery about him. We admired him boundlessly but saw little of him . . . ” In Meyerhof’s laboratory, Lipmann worked on the role of creatine phosphate in muscle contraction. It was of course known that muscle contraction, with its attendant production of lactic acid, is intimately linked to glycolysis. The energetics of this linkage, however, remained obscure. Lipmann (3) commented on “ . . . the vagueness of the understanding, then prevalent, of both the intermediary path of glycolysis and the mechanism of action of energy-rich phosphate.” This work did much to turn Lipmann’s thinking to the role of phosphorylated intermediates in energy transduction. In 1930, Lipmann was already aware that a career for a Jewish scientist in Germany was fraught with difficulty and peril. There began a period of Wanderjahre before he finally found a position that offered both independence and scope. However, he wished to remain in Berlin at least for a time to be near his fiancee Freda Hall (3). He became an assistant to Albert Fischer, working on problems of tissue culture. In 1931 during a hiatus in the work of Fischer’s laboratory caused by its move from Berlin to Copenhagen, Lipmann, newly married to Freda Hall, traveled to the United States to work at the Rockefeller Institute in New York in the laboratory of Phoebus A. Levene on the biochemistry of phosphoproteins. Here he succeeded in isolating phosphoserine from partial acid hydrolysates of egg phosphoprotein. H43

Reflections: Hitler’s Gift and the Era of Biosynthesis In 1932, Lipmann rejoined Fischer’s group in its new quarters in the Carlsberg Laboratories in Copenhagen, where he was to remain until 1939. He was free to work independently in pursuit of his own ideas. At about this time, Otto Warburg was making his great discoveries elucidating the central mechanisms of glycolysis. Why the splitting of glucose involved phosphorylated intermediates had long been a great puzzle, which Warburg now solved. In 1905 Arthur Harden, working in London, had discovered that glycolysis requires a heat-stable, organic cofactor, which he termed “cozymase.” This cofactor proved to be remarkably elusive. In 1929, more than two decades later, Hans von Euler received a Nobel Prize for his work on its isolation and characterization, but it is clear from his Nobel lecture that he had at that time no real idea of its true structure and function. It was Warburg and his collaborators (4) who isolated “cozymase” and showed that it contains a pyridine ring that undergoes alternate reduction and re-oxidation. It is of course the famous coenzyme NAD now known to function in many hundreds of enzyme-catalyzed redox reactions. Warburg also discovered that the oxidation of 3-phosphoglyceraldehyde by NAD is linked to the uptake of orthophosphate and the formation of 1,3-diphosphoglyceric acid. This acyl phosphate may then react with ADP to form ATP. For the first time, the bioenergetic function of glycolysis became clear. A portion of the free energy released during the breakdown of glucose is made available to the cell as ATP. Lipmann followed these developments closely and they deeply influenced his thinking. In 1939, he turned to an investigation of the role of phosphate in the oxidation of pyruvate in extracts of the organism then called Bacterium acidificans longissimum (Delbrueckii). He discovered (5) that the oxidation of pyruvate was coupled to the uptake of orthophosphate and the phosphorylation of AMP (presumably with the formation of ATP). By analogy with the role of 1,3-diphosphoglyceric acid in glycolysis, he formulated the following reactions. Pyruvate ⫹ phosphate ¡ acetyl phosphate ⫹ 2关H] Acetyl phosphate ⫹ AMP ¡ “adenosine polyphosphate” REACTIONS 1 AND 2

The isolation from these enzyme preparations of highly labile acetyl phosphate present only in very small amounts was really not feasible with the methods then available. Lipmann neatly got around this difficulty by synthesizing acetyl phosphate from acetyl chloride and trisilver phosphate. He then showed that this synthetic compound, like the presumed intermediate, was effectively utilized for the formation of “adenosine polyphosphate” in these extracts. (Much later, when I worked in Lipmann’s laboratory and read his early papers, I was greatly taken by this strategy. I learned from it that it is sometimes easier to synthesize a suspected intermediate in an enzyme system than to isolate it, a lesson that led me to synthesize CDP-choline first and then demonstrate its role as coenzyme.) The work on acetyl phosphate marked the beginning of Lipmann’s long and productive engagement with both the role of phosphate esters in energy transduction and the problem of “active acetate.” In July of 1939, with the Nazi menace ever more threatening, Fritz and Freda Lipmann left Copenhagen for the United States. There followed a difficult period in which he sought without success a position that would offer him security and scope commensurate with his talents. In 1940, he was invited to present a talk in a symposium at the University of Wisconsin in Madison attended by many of the leading figures in American biochemistry. Lipmann, never a facile or polished speaker, vastly underestimated the time needed for the material he wished to present and finally, midway through his discourse, had to be interrupted by the chairman of the session (3). Later he felt that this painful episode was one of the factors that made it difficult for him to secure a suitable position. In 1940, Lipmann was invited by F. F. Nord to contribute a chapter to the first volume of the series, Advances in Enzymology. As Lipmann later (3) wrote: “ . . . I was happy when he accepted my suggestion that I write about the role of phosphate bonds as carriers in energy transformations and in biosynthesis. This had begun to impress me as an extension of my experience with acetyl phosphate. Some of the propositions made in that article must have been more novel than I realized.” Now, in 2001, it is very difficult to realize the impact of this article (6), particularly on American biochemists who had not closely followed the work in European laboratories. H44

Reflections: Hitler’s Gift and the Era of Biosynthesis Lipmann clearly distinguished between two classes of phosphate compounds in living cells. The first class, phosphate esters of alcohols such as glycerophosphate with a free energy of hydrolysis of 2– 4 kcal, was termed by Lipmann as “energy-poor” phosphates, designated in the shorthand which he introduced as (⫺ph). These were to be sharply distinguished from another class comprising pyrophosphates, acyl phosphates, enol phosphates, and nitrogen-linked phosphates such as phosphocreatine. The free energy of hydrolysis of phosphates of this class is of the order of 8 –12 kcal. In Lipmann’s terminology these are energy-rich phosphate bonds, designated with a symbol that was to become famous as the “wiggle bond” (⬃ph). A great generalization was stressed in his essay. Photosynthesis and the breakdown of organic foodstuffs provide energy to living cells, some part of which is captured in useful form as “energy-rich” phosphates, leading to the formation of ATP. Lipmann pointed out: “Indications are found that the phosphate current can be utilized to carry out mechanical work . . . (and) to synthesize protoplasmic material as lecithin, nucleic acid, and so forth.” Lipmann made it clear that the energy needed to drive biosynthetic processes must come from ATP either directly or, as was soon to be found, indirectly. Before this time biosynthetic processes could be studied only in intact animals or in preparations such as tissue slices (developed by Warburg) in which cellular structure remained intact. The principal conceptual barrier to the study of cell-free systems was now removed. Biochemists began to add ATP (of varying degrees of purity!) to their enzyme systems when searching for biosynthetic reactions. The 1941 essay is revealing in many ways of Lipmann’s style, which had a personal flavor even when dealing with chemical thermodynamics. These were problems about which he had thought deeply, and he conveyed his ideas in striking and forceful metaphors. Thus he spoke of a “phosphate potential” in analogy to an electrical potential and a “phosphate current” that conveys energy as “energy-rich” phosphates are hydrolyzed. Critics pointed out that his use of the term “bond energy” to denote the energy released in breaking a bond was the opposite of the conventional use to denote the energy of bond formation, but Lipmann (3) tended to wave aside such criticism. “The physical chemist remains aloof. He may be forced to accept the usage, but he usually refrains from referring to the dilettante who originated it.” Lipmann’s search for a suitable position now found a happy outcome in a rather unusual way. In 1941 Dr. Oliver Cope offered him an appointment in the Department of Surgery at the Massachusetts General Hospital. Although the space made available was at first quite limited, he was given complete freedom to follow his own ideas. Lipmann’s years at the Massachusetts General Hospital were highly productive and led him to a Nobel Prize in 1953. In 1941 the identity of “active acetate,” also described as the “two-carbon unit,” was one of the most pressing problems in intermediary metabolism. A growing body of evidence suggested that “active acetate” was the fundamental building block for the synthesis of sterols and fatty acids. Derived from the oxidation of pyruvate or of fatty acids, it could also react with oxalacetate to form citrate and thus enter the Krebs cycle for the final common pathway of oxidative metabolism. Strongly encouraged by his success in identifying acetyl phosphate as an intermediate in the bacterial oxidation of pyruvate, Lipmann set out to examine its possible role as the elusive “active acetate” in animal tissues. He chose to study the acetylation of sulfanilamide, known to occur in liver, because of the ease with which this aromatic amine could be diazotized and coupled with a chromogen to form an intensely colored dye. The conversion of sulfanilamide to the unreactive N-acetyl derivative could thus be easily measured. He succeeded in obtaining preparations from pigeon liver that actively acetylated sulfanilamide but to his considerable disappointment found that acetyl phosphate did not stimulate acetylation but instead was rapidly hydrolyzed (7). Significantly, however, he found that ATP as well as acetate was required for acetylation and further reported that enzyme preparations, inactivated by storage overnight at 7 °C, could be restored to activity by the addition of boiled liver extract. Nachmansohn and Machado had previously described a cofactor needed for the acetylation of choline. With the arrival of Kaplan in his laboratory, Lipmann’s cofactor was purified about 100-fold and shown to be active also in the acetylation of choline (8). It appeared to be a general coenzyme for acetylation and hence the designation coenzyme A or CoA. The next step was the discovery (9) in 1947 that CoA, by then purified about 700-fold, contains the vitamin pantothenic acid. This was a very great advance. A little later, in 1950, recommended by H. A. Barker, I entered Lipmann’s laboratory as a postdoctoral fellow following the footsteps of Earl Stadtman, who had just departed to take up H45

Reflections: Hitler’s Gift and the Era of Biosynthesis a position at the National Institutes of Health. Stadtman had also come to Lipmann from Barker’s laboratory. Lipmann’s group at this time included David Novelli, John Gregory, Morris Soodak, Harold Klein, Charles Du Toit, and Lipmann’s research assistant, Ruth Flynn. We were crammed into a single, tiny laboratory in the Massachusetts General Hospital next to the famous Ether Dome, scene of the first (or so it was claimed) use of diethyl ether as an anesthetic. In the course of the year we were to move into spacious, even rather elegant, quarters in a newly constructed research building. With abundant hair just turning gray and usually wearing a soft bow tie and a dark blue shirt, Lipmann presented a figure closer to that expected of an artist rather than a scientist. He spoke softly, and his sentences often trailed off into the distance. Lipmann’s manner toward those who worked in his laboratory was rather formal. He was friendly but a little aloof. He inspired nevertheless not only loyalty and admiration but also lasting affection in those who worked under his direction. At this time, Lipmann’s chief goal was the final purification of coenzyme A, which was proving very difficult, and the determination of the structure of “active acetate,” the intermediate with so many crucial roles in metabolism. Because acetyl phosphate, shown to be an activated form of acetate in bacteria, was so labile, we surmised that acetyl-CoA, whatever its structure might be, would be even more labile, and this supposed lability was assumed to explain the failure of our efforts to isolate it. One day in 1951, I came upon an article in Angewandte Chemie (10) from the laboratory of Feodor Lynen. He and his student Ernestine Reichert reported evidence for an essential sulfhydryl residue in CoA. They had isolated acetyl-CoA and proved it to be a thioester! I brought the article at once to Lipmann who had not learned previously of this development. He was generous in praise of the work although Lynen had stolen some of his thunder. He was particularly impressed by the fact that in isolating acetyl-CoA from yeast, they had begun by boiling the yeast. We should have realized, Lipmann pointed out, that an intermediate that plays such varied roles is unlikely to be so extremely labile as we had feared. Lipmann also noted that thioesters must be added to the list of biologically active “energy-rich” compounds. “Yes,” he mused in a discussion at this time, “there is a world of sulfur, like the world of phosphorus, only smaller!” In 1953 Lipmann shared the Nobel Prize with H. A. Krebs. Although the citation for the prize emphasized his work on CoA, Lipmann placed greater stress on his contributions to bioenergetics. “In my own judgment,” he wrote (3), “there was greater scope in the recognition that ⬃P, as I had dubbed it, was acting as a biological energy quantum, carrying energy packages to metabolic function and biosynthesis.” In 1957, he moved to the Rockefeller Institute. He continued to be remarkably productive in a wide variety of biosynthetic problems, further developing his grand themes of group activation and the energetics of biosynthesis until his death in 1986 at the age of eighty-seven. Rudolf Schoenheimer and the Dynamic State of Body Constituents The single most important technical advance that transformed biochemistry in the 20th century was the isotope tracer technique. Without it, the rapid growth of our knowledge of biosynthesis would be simply inconceivable. Georg Hevesy was the first to explore the biological usefulness of radioactive tracers in studies of the uptake of radiolead and its movement into tissues of plants (11). It is to Rudolf Schoenheimer (Fig. 2), however, that we owe the brilliant exploitation of the concept of isotopic tagging, that is the introduction of isotopes into specific positions of organic molecules, whose metabolic transformations could then be traced. Valuable accounts of Schoenheimer’s career have been published by Kohler (12) and by Young and Ajami (13). He was born in Berlin in 1898 (12). Like Lipmann, he studied medicine and received the M.D. degree from the University of Berlin in 1922. Again like Lipmann, he recognized the need for deeper knowledge of chemistry and spent 3 years in the laboratory of Karl Thomas in Leipzig, working largely on problems such as the chemical synthesis of peptides. In 1926, Schoenheimer went to the Institute of Pathological Anatomy in Freiburg as assistant to Ludwig Aschoff, a leading expert on atherosclerosis (12). Schoenheimer began an investigation on the deposition of cholesterol into the arteries of rabbits fed a high level of cholesterol in the diet. He was to pursue his interests in cholesterol metabolism for the rest of his life. H46


FIG. 2. Rudolf Schoenheimer. From Ref. 13 with permission. Photo made available through the courtesy of Mrs. Peter Klein.

It was here in Freiburg in 1930 that Schoenheimer encountered Hevesy, who wished to study the partition of labeled lead between normal and tumor tissue (12). Realizing his inadequate background in biology, Hevesy asked Aschoff to suggest a collaborator for this work. Aschoff suggested Schoenheimer. Later, Hevesy (14) wrote: “It was in the course of these investigations that Schoenheimer became familiar with the method of isotopic indicators, which he applied several years later with such great success . . . Never were more beautiful investigations carried out with isotopic indicators than those of the late Professor Schoenheimer . . . ” Although the collaboration with Hevesy was undoubtedly significant for Schoenheimer’s thinking, his development of the use of isotopes was to go far beyond the scope of Hevesy’s approach. In 1933 Schoenheimer, like so many others, was forced to leave Germany. The Josiah Macy Foundation in the United States had begun in 1931 to support Schoenheimer’s research, and the director of the foundation, Ludwig Kast, now arranged an appointment for Schoenheimer in the Department of Biological Chemistry at Columbia, with salary and research funds supplied by the Foundation (12). Hans T. Clarke, an organic chemist by training, had assumed the direction of the Department of Biological Chemistry in 1928, and he proceeded to make it the finest department in the United States. In an account of his career (15), Clarke stated: “Among the many benefits which accrued to Columbia University from the racial policy adopted by the Germans under the Third Reich was the arrival in our laboratory of various European-trained biochemists, notably Erwin Chargaff, Zacharias Dische, Karl Meyer, Rudolf Schoenheimer and Heinrich Waelsch. Erwin Brand, who joined our group during the same period, reached this country somewhat earlier. The scientific achievements subsequently made by these men are so well known that their enumeration is unnecessary.” Clarke modestly omitted to mention that his own vision and humane instincts in welcoming these gifted refugees were by no means to be found in every American academic institution. H47

Reflections: Hitler’s Gift and the Era of Biosynthesis In 1932, also at Columbia University in the Department of Chemistry, Harold Urey discovered deuterium, the heavy isotope of hydrogen, by demonstrating the presence of new bands in the positions predicted for a form of hydrogen of mass 2, in the spectrum of a sample of hydrogen enriched in the heavier isotope by fractional distillation of liquid hydrogen. In 1934, Urey received a Nobel Prize for this work. Because separation of the isotopes of an element is a function of the ratio of their masses, isotopes of the heavier elements are very difficult to separate. Deuterium, however, has twice the mass of ordinary hydrogen, and its preparation in pure form or as D2O (immediately dubbed “heavy water”) is comparatively straightforward and was very soon undertaken in the laboratories of Urey and G. N. Lewis among many others. The discovery of a completely new form of a substance of such universal importance as water immediately attracted great public interest all over the world. When Urey received his Nobel Prize in 1934, Palmer, in his laudatory introduction of Urey, mentioned that large amounts of heavy water were already being produced by an electrolytic process at the Norsk Hydro Concern in Norway at the rate of about a half-liter per day (16). In 1940 after a more sinister use of heavy water as the moderator for atomic piles had emerged, this Norwegian heavy water production facility was taken over by the German army of occupation. It then became the target for heroic and tragic efforts of Norwegian patriot saboteurs and the allied air forces to destroy it. The Germans finally dismantled it in 1945. The first biological experiments with D2O were relatively crude. For example, Lewis (17) reported that tobacco seeds suspended in pure D2O failed to germinate, and flatworms died when placed in water containing more than 90% D2O. In these and other early experiments, the emphasis was on replacement of H2O as a medium for growth by D2O and not on the specific replacement of hydrogen by deuterium in molecules of biological importance. Urey, a physical chemist, stated that he was a biologist at heart. Indeed, at a later stage of his career at the University of Chicago he turned to fundamental biological research. With his gifted collaborator Stanley Miller, he designed experiments that demonstrated the ready synthesis (under conditions that simulated the atmosphere of the early earth) of molecules that might plausibly be considered to be building blocks for the formation of cell substances. These studies greatly influenced many later investigations of the origin of life. To promote the applications of the deuterium isotope to biological research, Urey persuaded Warren Weaver, head of the Rockefeller Foundation, to provide funds to permit David Rittenberg, a recent Ph.D. in physical chemistry in Urey’s department, to come to the Department of Biological Chemistry (12). As Hans Clarke commented (15): “In 1934, Schoenheimer made a new contact which proved to exert a fundamental influence on the nature of his work . . . David Rittenberg came from Urey’s group to the laboratory in which Schoenheimer had been working for a year. From their association there developed the idea of employing a stable isotope as a label in organic compounds, destined for experiments in intermediary metabolism, which should be biochemically indistinguishable from their natural analogs . . . ” This new conception of Schoenheimer and his collaborators was a far cry from the simple measurement of the movement of a radioactive ion from one part of a plant or animal to another, as had been done by Hevesy. In the new approach, the fate of the molecule into which the isotope had been incorporated was studied, not simply the isotope itself. Perhaps the nearest intellectual predecessor of this idea was the approach of Knoop, who in 1904 “labeled” fatty acids by the attachment of a phenyl residue to the ␻-carbon atom. Knoop found that if the fatty acid had an even number of carbon atoms, phenylacetic acid (linked to glycine in a so-called detoxification reaction) was recovered from the urine of dogs to which it had been fed. If on the other hand, the fatty acid had an odd number of carbon atoms, benzoic acid was similarly recovered. Knoop concluded that the phenyl residue could not be cleaved from the ␻ carbon to which it was linked and more significantly correctly concluded that fatty acid oxidation in animal tissues must involve oxidation at the ␤-position. This result strongly influenced later studies of fatty acid oxidation, but the work was subject to the objections that phenyl-substituted fatty acids are very different from natural fatty acids, and a more serious limitation was that this type of labeling was not generally suitable for substances other than fatty acids. Schoenheimer was well aware of Knoop’s work. In a brief review in 1935 (18), Schoenheimer and Rittenberg pointed out: “Many attempts have been made to label physiological substances by the introduction of easily detectable groups such as halogens and benzene nuclei. However, the physical and chemical properties of the resulting compounds differ so markedly from those H48

Reflections: Hitler’s Gift and the Era of Biosynthesis of their natural analogs that they are treated differently by the organism. The interpretation of metabolic experiments involving such substances is therefore strictly limited. We have found the hydrogen isotope deuterium to be a valuable indicator for this purpose . . . We have prepared several physiological compounds (fatty acids and sterol derivatives) containing one or more deuterium atoms linked to carbon, as in methyl or methylene groups . . . The number of possible applications of this method appear to be almost unlimited.” At this period, mass spectrometers were still rare and finicky instruments. It was an advantage of these early experiments that the content of deuterium in organic compounds could be determined comparatively simply by combustion of the compound and very precise measurement of the density of the water so produced. In 1935, it was a widely held doctrine that the bodily constituents of an adult animal were quite stable, while foodstuffs in the diet were immediately metabolized to provide energy and the end products excreted. In their earliest experiments, Schoenheimer and Rittenberg found evidence to overturn this doctrine. When fatty acids labeled with deuterium were fed to mice, most of the deuterated fat was first deposited in the fat depots. The fat burned in the body was not taken directly from the diet but from adipose tissue. Schoenheimer (19) concluded: “These first experiments with isotopes showed that the fats of the depots are not inert storage materials but are constantly involved in metabolic reactions.” To study the synthesis of fatty acids, Bernhard and Schoenheimer (20) administered D2O to mice and later measured the isotope content of their fatty acids. The saturated fatty acids were found to contain relatively high levels of deuterium, but the polyunsaturated linoleic and linolenic acids, known to be essential components of the diet, contained only traces. They concluded that the mice carried out a very active de novo synthesis of saturated but not of essential fatty acids. Because the total fat content of the mice did not change, the results indicated a rapid breakdown of body fats, equal to the rate of synthesis. As might be expected, an important objective of Schoenheimer’s new program was an investigation of the metabolism of cholesterol. When cholesterol was isolated from mice given D2O, Rittenberg and Schoenheimer (21) found from the rate of incorporation of deuterium into it that cholesterol must be continually renewed with a half-time of the order of 3 weeks. To account for the extensive incorporation of stably bound deuterium into the cholesterol molecule, it was concluded that its synthesis, like that of fatty acids, must involve the condensation of many small molecules. A major extension of the range Schoenheimer’s investigations came with the concentration of the isotope 15N by Urey and his collaborators in 1937. It was immediately applied to studies of the metabolism of amino acids and proteins. In 1938, Schoenheimer et al. (22) reported the first experiments in which an amino acid in the diet, tyrosine, was labeled with 15N. “The original aim of this exploratory experiment was merely to find out whether in nitrogen equilibrium, the nitrogen in the urine is derived from the food proteins directly, or whether dietary nitrogen is deposited, with liberation of an equivalent amount of tissue nitrogen for excretion . . . The results indicate that in our rat the nitrogen of at least one amino acid, tyrosine, was only partly excreted in the urine, while almost half of it was retained in the body proteins.” Here was another blow at the doctrine that ingested foods were immediately metabolized and the products promptly excreted. Schoenheimer now found this view very naı¨ve. If one puts a penny into a gumball machine, he asked, and a gumball comes out, does the machine turn copper into gum? Schoenheimer had now become the central figure in Clarke’s Department of Biological Chemistry. New and larger laboratory facilities were made available for him. His enthusiasm and vision attracted collaborators and students. As Kohler (12) has pointed out, he had become the leader of perhaps the first multidisciplinary biochemical laboratory. A physicist was needed for the preparation and measurement of isotopes. An organic chemist was employed for the synthesis of isotopically labeled compounds, because of course none were available commercially. Biochemists were required for the separation and analysis of cell constituents. Technicians for animal care were also needed. Schoenheimer’s background in chemistry as well as in biology and medicine made him especially effective in the leadership of this disparate group. Schoenheimer’s investigations of protein metabolism, carried out with amino acids containing 15N in the amino group and deuterium on the carbon chains provided results that had the H49

Reflections: Hitler’s Gift and the Era of Biosynthesis greatest impact on biochemical thought. Briefly summarized (19), body proteins were found to be in a state of continuous turnover. “The peptide bonds have to be considered as essential parts of the proteins and one may conclude that they are rapidly and continually opened and closed in the proteins of normal animals. The experiments give no direct indication as to whether the rupture is complete or partial.” The work thus raised questions that were to challenge the next generation of biochemists. Together with the earlier work on fat metabolism, a new and remarkable picture of the overall metabolism of animals emerged. Schoenheimer summarized his conclusions (19): “The large and complex molecules and their component units, fatty acids, amino acids, and nucleic acids, are constantly involved in rapid chemical reactions. Ester, peptide, and other linkages open; the fragments thereby liberated merge with those derived from other large molecules and with those absorbed from the intestinal tract to form a metabolic pool of components indistinguishable as to origin . . . This idea can scarcely be reconciled with the classical comparison of a living being to a combustion engine nor with the theory of independent exogenous and endogenous types of metabolism . . . The classical picture must thus be replaced by one which takes account of the dynamic state of body structure.” In 1941, Schoenheimer was invited to give the prestigious Dunham Lectures at the Harvard Medical School. The materials and notes that he prepared for the lectures, from which some of the quotations above are taken, were later published (19) under the title “The Dynamic State of Body Constituents.” This lucid summary of his innovative work made a deep impression on the biochemists of the generation to follow. Schoenheimer had apparently been subject to attacks of depression and was undergoing a period of considerable personal stress when tragically in September of 1941 he ended his own life (12). Forty-three years of age at the time of his death, he was at the height of his powers. Fortunately many of the projects that he had begun were carried forward by very able collaborators, one whom took up the cholesterol problem. Konrad Bloch and the Biosynthesis of Cholesterol Konrad Bloch (Fig. 3) was born in 1902 in Neisse, a town in the eastern German province of Silesia, the second child of a prosperous Jewish family (23). In his boyhood, Bloch evinced little interest in science other than nature studies, but his attendance in a course of organic chemistry at the Munich Technische Hochschule taught by Hans Fischer marked a turning point for him. Fischer, later to receive a Nobel Prize, was one of the remarkable group of gifted German chemists who then dominated the study of natural products. Although Fischer’s lectures were delivered in a monotone, Bloch found the material fascinating and he realized that he had found his field (23). In 1934, the brutal Nazification of Germany prevented Bloch from continuing his studies there. Hans Fischer came to his rescue by recommending his appointment at the Schweizerisches Hoehensforschungs Institut in Davos, Switzerland, the scene where Thomas Mann placed the tuberculosis sanitarium in his novel The Magic Mountain. In Davos, Bloch worked for a time on the lipids of the tubercle bacillus. In 1936, however, he was refused permission to continue to reside in Switzerland. Desperate, he applied to R. J. Anderson at Yale, with whom he had some correspondence concerning his research. He promptly received two letters, the first from the Dean of the Medical School of Yale University informing him that he had been appointed assistant in Biological Chemistry and the second from Anderson informing him that there was no salary attached to this position. He showed the first letter, but not the second, to the United States consul in Frankfurt and received a life-saving visa to immigrate to the United States. Upon arrival in New York, Bloch applied to Hans Clarke’s department for admission as a graduate student. The sole formality in those happy days was an interview with Clarke himself. The most important question, Bloch later jested, was: “Do you play a musical instrument?” Fortunately, Bloch could say that he played the cello, an answer agreeable to Clarke, who loved chamber music. Shortly after completion of his work for the Ph.D. degree under Clarke’s supervision, Bloch joined Schoenheimer’s group. In 1940, Schoenheimer suggested that he investigate the origin of the hydroxyl oxygen in cholesterol. Was it water or O2? The thought that it might be molecular oxygen showed the remarkable prescience of Schoenheimer because direct oxygenation was without precedent at that time. Unfortunately, Bloch found the technical problems H50


FIG. 3. Konrad Bloch. Photo made available through the courtesy of Mrs. Konrad Bloch.

of the mass spectrometry of oxygen compounds intractable in the state of technology of 1940 and was forced to give up the project. In 1956, however, he returned to the problem and with his student Tchen (24) showed that molecular oxygen is indeed the source of the hydroxyl oxygen. As Bloch (23) recalled: “Schoenheimer’s untimely death in 1941 left his associates without the leader and the inspired leadership they so admired. We feared that we might have to look for jobs elsewhere, but Hans Clarke encouraged us to continue as heirs to the wealth of projects Schoenheimer had begun and developed . . . How the division of ‘spoils’ came about I do not recall—it may have been by drawing lots. At any rate, David Shemin ’drew’ amino acid metabolism, which led to his classic work on heme biosynthesis. David Rittenberg was to continue his interest in protein synthesis and turnover, and lipids were to be my territory.” Bloch now began his independent studies of the biosynthesis of cholesterol. It was a formidable enterprise. In the era before NMR, infrared, and mass spectroscopy, the determination even of the chemical structure of cholesterol, with its 27 carbon atoms arranged in four rings and with a branched hydrocarbon side chain, had been a challenge to the world’s greatest chemists of natural products. The Nobel Prizes in chemistry for 1927 and 1928 had been awarded to Heinrich Wieland and Adolf Windaus, respectively, for their work on the structure of cholesterol and the closely related bile acids, but it was not until 1932 that the fully correct structure was established. In his 1928 Nobel lecture (25), Windaus stated: “This formula [of cholesterol] is very complicated and has no similarity to the formulae of sugars, fatty acids, or the amino acids which occur in protein. The synthesis of such a substance appears to the chemist particularly difficult, and up to now I have not dared to attempt it, as success is extremely improbable. Furthermore, the majority of physiologists have not been inclined to believe the animal organism capable of such a synthesis, for it is known that other seemingly simpler syntheses— e.g. that of tyrosine and tryptophane— have not succeeded in the animal organism.” H51

Reflections: Hitler’s Gift and the Era of Biosynthesis Bloch of course knew that Windaus’ pessimistic view of the capabilities of the animal organism was unfounded. Schoenheimer and Rittenberg had demonstrated the extensive incorporation of deuterium from D2O into cholesterol in the mouse and concluded that cholesterol must be synthesized by the joining of a number of small molecules. The pathway for the biosynthesis of cholesterol from acetate, involving more than 30 separate enzyme-catalyzed reactions, can now be found in every textbook of biochemistry. A detailed review is beyond the scope of this essay. Here we will consider only the principal landmarks in its three major stages: 1) acetate to “activated isoprene”; 2) “activated isoprene” to squalene; and 3) squalene to cholesterol. Bloch’s studies began with investigations of the overall process of formation of cholesterol in the intact organism. Stimulated by a report from the German workers Sonderhoff and Thomas (26), indicating that acetate is efficiently converted into the sterols of yeast, Bloch began a series of studies demonstrating the incorporation of specifically labeled acetate into cholesterol in the intact animal. These studies were continued and expanded after his move in 1946 to the Department of Biochemistry at the University of Chicago, where his good friend Earl Evans, also a product of Hans Clarke’s department, had become chairman. I was a graduate student in the Department at this time, and so I came to know Konrad Bloch, first as a teacher and later as a colleague and friend. He was a man of personal qualities commensurate with his great abilities. His manner with students was friendly and easy. He was painstakingly generous in acknowledging the research contributions of his colleagues and of other laboratories. He was widely cultured, devoted to music, literature, and art. In the mid-1940s, Bloch (23) was completely convinced of the truth of Lipmann’s dictum that energy-requiring biosynthetic reactions are driven by ATP, directly or indirectly. Before this period the synthesis of peptide bonds had been observed only by reversal of the reactions catalyzed by proteases. In a project quite unrelated to the cholesterol problem, he and his students began to investigate the synthesis of the tripeptide glutathione as a possible model of protein synthesis. They were indeed able to show that the assembly of glutathione requires the successive activation of glutamate and glutamylcysteine by ATP, but unfortunately the mechanism proved to shed little light on the ribosomal synthesis of proteins. Bloch was also very much aware of the potential power of microbial genetics for the analysis of metabolic pathways, and he enrolled as a student in the famous course in microbiology taught by C. B. Van Niel at the Hopkins Marine Station in Pacific Grove, CA. When a mutant of the mold Neurospora crassa was isolated in Tatum’s laboratory that grew only when acetate was added to the medium, Bloch was eager to follow this lead. He and his collaborators found that isotopically labeled acetate was converted to ergosterol in this mutant essentially without dilution of the isotope. Clearly the sterol could be built up entirely from acetate. In the conversion of acetate to cholesterol, which of the carbon atoms of cholesterol were derived from the carboxyl group and which from the methyl group? Studies carried out over a number of years in the laboratories of Cornforth and of Popjak, as well as of Bloch, achieved the ambitious goal of defining the origin of each of the 27 carbon atoms of cholesterol as either the methyl or the carboxyl carbon of acetate. This work placed important constraints on possible structures of intermediates in the scheme. It had been known for some time that squalene (a branched, acyclic hydrocarbon found in abundance in the livers of sharks) when fed to animals increases the levels of cholesterol in their tissues. To test the idea that squalene might be a precursor of cholesterol, Bloch went to the Biological Research Station in Bermuda to attempt the preparation of isotopically labeled squalene in shark liver, but the shark proved to be an intractable subject for study (23). “All I was able to learn was that sharks of manageable length are very difficult to catch and their oily livers impossible to slice.” Back at the University of Chicago, however, his student Robert Langdon was able to prepare labeled squalene by feeding rats labeled acetate along with unlabeled squalene as an isotopic trap. Labeled squalene so obtained was then fed to rats and found to be converted to cholesterol (27). This was an important result. In the dissection of every biosynthetic pathway, it is particularly helpful to identify an intermediate in the middle of the chain of reactions; the researcher can then trace the pathway both backwards and forwards. At this stage in his work, in 1954 Bloch moved to the Department of Chemistry at Harvard, where he was to remain for the rest of his career. Squalene, containing 30 carbon atoms, could plausibly be considered to be built up from 6 units of isoprene, a branched, unsaturated compound containing five carbon atoms. Isoprene H52

Reflections: Hitler’s Gift and the Era of Biosynthesis was already known to be a building block of other naturally occurring hydrocarbons such as rubber, although the nature of the biologically active “isoprene donor” remained unknown. Robinson (28) had suggested that squalene might be folded to form the basic structure of cholesterol directly. Bloch, however, after illuminating discussions with his Harvard colleague Robert Woodward considered that lanosterol, with a structure closely similar to cholesterol but with three “extra” methyl groups, was likely to be an intermediate in this transformation. Up to this point, Bloch’s experimental approach to the cholesterol problem had been largely confined to isotopic tracer studies with intact animals or with tissue slices in which cellular structure was preserved intact, but now he turned increasingly to the study of cell-free enzyme systems. Rat liver homogenates, prepared by the methods developed by Nancy Bucher, were found to catalyze the transformation of labeled squalene to lanosterol and of lanosterol to cholesterol. Although much work remained to be done, Bloch had established the landmarks for the final stages of the biosynthesis of cholesterol (29). The focus now was turned to the first stages of the pathway, the conversion of acetate to the “active isoprene donor.” A mutant strain of Lactobacillus acidophilus had been found to grow only when acetate was added to the medium. A substance that very efficiently replaced the acetate requirement was identified by workers at Merck, Sharpe and Dohme (30) as mevalonic acid (isolated as the lactone). Mevalonic acid was then shown to be a very efficient precursor of squalene and of cholesterol in homogenates of liver (31). These findings opened the way for the elucidation of the reactions leading to the formation of the “active isoprene unit” of which mevalonate was clearly the precursor. Progress in this area now became fast and furious with important contributions from the laboratories of Rudney, Lynen, Cornforth, and Popjak among others. Bloch and his collaborators showed that the overall conversion of labeled mevalonic acid to squalene in extracts of bakers’ yeast required ATP as well as reduced pyridine nucleotide and manganese ions. His colleague Chen then discovered the phosphorylation of mevalonate to a monophosphate. The further conversion of this monophosphate to the important intermediates isopentenylpyrophosphate and dimethylallylpyrophosphate was elucidated largely by work in Lynen’s laboratory. The synthesis of squalene via geranyl pyrophosphate and farnesyl pyrophosphate was next documented. As shown by the early studies of Bloch, squalene is converted in a series of steps to lanosterol, which after several further transformations gives rise to cholesterol. It is impossible, of course, in this highly condensed account to do justice to the vast amount of work, still ongoing in laboratories over the world, that has led to our present knowledge of the biosynthesis of cholesterol. It was Bloch, however, who was a prime mover in all three phases of the problem. For this work he was awarded a Nobel Prize, with Feodor Lynen, in 1964. Working out the pathway for the assembly of the complex structure of cholesterol was an exemplary achievement of the era of biosynthesis, important not only because of the intrinsic interest of its enzymology but also because of its significance for medicine. High levels of blood cholesterol, characteristic of populations in developed countries, strongly increase the danger of heart disease and stroke. An understanding of the detailed route of biosynthesis made it possible to determine that the synthesis of mevalonate from HMG-CoA is a rate-making step in the production of cholesterol. This advance made possible the development of drugs, the family of statins, that reduce levels of blood cholesterol with a minimum of toxic side effects. These drugs are among the most useful in modern medicine. Konrad Bloch made outstanding contributions to fields other than the biosynthesis of cholesterol, including the enzymic synthesis of fatty acids and the mechanism of enzyme action (23). He died on October 15, 2000 at the age of eighty-eight. The development of any field of science is inevitably the work of many hands. Obviously, Lipmann, Schoenheimer, and Bloch cannot be regarded as single handedly transforming American biochemistry. Their work was nonetheless a great gift to their adopted country and a shining manifestation of the international character of science. REFERENCES 1. 2. 3. 4.

Medawar, J., and Pyke, D. (2001) Hitler’s Gift, Arcade Publishing, New York Lipmann, F. (1953) Annu. Rev. Biochem. 54, 1–32 Lipmann, F. (1971) Wanderings of a Biochemist, Wiley-Interscience, New York Warburg, O. (1949) Wasserstoffuebertragende Fermente, Editio Cantor, Freiburg, Germany

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Reflections: Hitler’s Gift and the Era of Biosynthesis 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Lipmann, F. (1939) Nature 144, 33–34 Lipmann, F. (1941) Adv. Enzymol. 1, 99 –162 Lipmann, F. (1945) J. Biol. Chem. 160, 173–190 Lipmann, F., and Kaplan, N. O. (1946) J. Biol. Chem. 162, 743–744 Lipmann, F., Kaplan, N. O., Novelli, G., Tuttle, L. G., and Guirard, B. M. (1947) J. Biol. Chem. 167, 869 – 870 Lynen, F., and Reichert, E. (1951) Angew. Chem. 63, 47– 48 Hevesy, G. (1923) Biochem. J. 17, 439 – 445 Kohler, R. E., Jr. (1977) Hist. Studies Phys. Sci. 8, 257–298 Young, V. R., and Ajami, A. (1999) Proc. Nutr. Soc. 58, 15–32 Hevesy, G. (1948) Cold Spring Harbor Symp. Quant. Biol. 13, 129 –150 Clarke, H. T. (1958) Annu. Rev. Biochem. 27, 1–14 Palmer, W. (1966) in Nobel Lectures Chemistry 1922–1941, pp.333–338, Elsevier Science Publishing Co., Inc., New York Lewis, G. N. (1934) Science 79, 151–153 Schoenheimer, R., and Rittenberg, D. (1935) Science 82, 156 –157 Schoenheimer, R. (1949) The Dynamic State of Body Constituents, Harvard University Press, Cambridge, MA Bernhard, K., and Schoenheimer, R. (1940) J. Biol. Chem. 133, 707–712 Rittenberg, D., and Schoenheimer, R. (1937) J. Biol. Chem. 121, 235–253 Schoenheimer, R., Ratner, S., and Rittenberg, D. (1939) J. Biol. Chem. 127, 333–344 Bloch, K. (1987) Annu. Rev. Biochem. 56, 1–19 Tchen, T. T., and Bloch, K. (1956) J. Am. Chem. Soc. 78, 1516 –1517 Windaus, H. O. (1996) in Nobel Lectures Chemistry 1922–1941, pp. 105–121, Elsevier Science Publishing Co., Inc., New York Sonderhoff, R., and Thomas, H. (1937) Ann. Chem. 530, 195–213 Langdon, R. G., and Bloch, K. (1952) J. Biol. Chem. 200, 129 –144 Robinson, R. J. (1934) J. Chem. Soc. Ind. 53, 1062–1063 Bloch, K. (1965) Science 150, 19 –28 Wolf, D. E., Hoffman, C. H., Aldrich, P. E., Skeggs, H. R., Wright, L. D., and Folkers, K. (1956) J. Am. Chem. Soc. 78, 4499 Tavormina, P. A., Gibbs, M. H., and Huff, J. W. (1956) J. Am. Chem. Soc. 78, 4498 – 4499

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Vol. 279, No. 38, Issue of September 17, pp. 39187–39194, 2004 Printed in U.S.A.

Reflections A PAPER IN A SERIES COMMISSIONED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005


The Biotin Connection: Severo Ochoa, Harland Wood, and Feodor Lynen Published, JBC Papers in Press, May 27, 2004, DOI 10.1074/jbc.X400005200

M. Daniel Lane From the Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Unique circumstances sometimes bring us into contact with individuals who will profoundly influence us, particularly in our formative years. In this article I would like to reflect on the circumstances that brought me into contact with three great biochemists, Severo Ochoa (1), Harland Wood (2), and Feodor Lynen (3). Each entered the field by a different route: Ochoa as a physician with an interest in physiology, Wood as a bacteriologist trained at Iowa State University, and Lynen as an organic chemist trained in the German tradition with Nobel Prize winner, Heinrich Wieland. They entered the field of biochemistry in the late 1930s when the race was on to discover new enzymes, cofactors, and metabolic cycles. Hans Krebs had formulated the tricarboxylic acid cycle in 1937 and ornithine cycle (now known as the urea cycle) in 1932, some B vitamins had been found to function as cofactors or prosthetic groups of enzymes, and Rudolf Schoenheimer (Columbia University College of Physicians and Surgeons) had demonstrated the dynamic state of tissue proteins using heavy isotopes of hydrogen and carbon (mid-1930s). This was where the action was and it attracted many of the brightest young minds into the field. This was the arena in which Ochoa, Wood, and Lynen were early participants. Excited by discovery, they transmitted this excitement to their younger colleagues. I was fortunate to have scientific associations and enduring friendships with each of them. My connection developed through the B vitamin, biotin, and its role in the reactions catalyzed by a family of biotin-dependent enzymes, notably carboxylases. The B vitamin, biotin, has an interesting history not familiar to most scientists who now make use of it. Today, this vitamin is widely used along with avidin (or its cousin, strepavidin), the specific biotin-binding protein from egg white, to probe biochemical phenomena. Biotinylation of proteins and nucleotides and the use of avidin to “fish out” or detect these molecules from/in complex mixtures has found great utility. It is a curiosity that nature has brought together within the hen’s egg the richest source of biotin in the yolk and in the white, a “toxic” factor, avidin, which when fed to animals causes biotin deficiency. In 1936, Ko¨gl and To¨nnis isolated 1.1 mg of biotin from more than 500 pounds of egg yolk. Paul Gyo¨rgy recognized that the distribution, fractionation behavior, and chemical properties of Ko¨gl’s yeast growth factor and the anti-egg white injury factor in egg yolk (then called vitamin H) were similar. When Ko¨gl’s pure biotin methyl ester became available it was found to be extremely potent in protecting rats against “egg white (i.e. avidin) injury.” Within a few years Vincent Du Vigneaud and colleagues determined the structure of biotin, which cleared the way for an attack on the role of biotin at the molecular level. By 1950 biotin had been implicated in a number of seemingly unrelated enzymatic processes including the decarboxylation of oxaloacetate and succinate; the “Wood-Werkman reaction” (discovered by Harland Wood (2)), i.e. the carboxylation of pyruvate; the biosynthesis of aspartate; and the biosynthesis of unsaturated fatty acids. Of course, we now know that biotin This paper is available on line at http://www.jbc.org

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Reflections: The Biotin Connection functions in each of these processes as a mobile “CO2 carrier” bound covalently to a carboxylase. The long sought after link between biotin and enzymatic function was provided by Henry Lardy at the University of Wisconsin. Lardy showed that liver mitochondrial extracts catalyzed the ATP- and divalent cation-dependent carboxylation of propionate (subsequently shown to be propionyl-CoA) to form succinate (4). Later work in the laboratory of Severo Ochoa found that the initial carboxylation product was methylmalonyl-CoA, an intermediate en route to succinyl-CoA. The connection to biotin was made by Lardy with the finding that the propionate-carboxylating activity was lacking in liver mitochondria from rats made biotindeficient by being fed egg white, which of course contained avidin (KD(biotin) ⬃10⫺15). Moreover, the failure of mitochondrial extract to catalyze the carboxylation of propionate was quickly cured by injecting the rats with biotin. Upon joining the faculty at Virginia Polytechnic Institute in Blacksburg, Virginia in 1956, I decided to try to determine how propionate is metabolized in the liver. Because of its unique features, I settled on bovine liver as the tissue source of the enzyme system to address this question, propionate being a major hepatic carbon source in ruminants. Unlike carbohydrate digestion by monogastric animals, ruminants digest carbohydrates in the rumen, the large anaerobic fore compartment of their multi-compartmented “stomach.” Virtually all carbohydrate is fermented in the rumen to short chain fatty acids, primarily acetate and propionate. Thus glucose, the major digestion product of carbohydrates in monogastric animals, is not available for absorption in ruminants. Propionate, produced in abundance by fermentation in the rumen, is absorbed directly into the portal system and transported to the liver where it is the major carbon source for gluconeogenesis, the pathway leading to glucose production. My entry into this area coincided with Lardy’s report that propionate was somehow carboxylated to form succinate. I recall writing to Henry Lardy, and he referred me to Severo Ochoa at New York University School of Medicine. He knew that Ochoa was working on propionate metabolism and had found that propionyl-CoA first became carboxylated to form methylmalonyl-CoA and then was converted to succinyl-CoA. With some trepidation about competing with the Ochoa laboratory, I decided to forge ahead and purify propionyl-CoA carboxylase from bovine liver mitochondria. For the reasons mentioned above bovine liver turned out to be an excellent source of the enzyme. At that point I wrote to Severo Ochoa, and he generously gave me a status report on their progress and put me in contact with the people in his laboratory (Alisa Tietz, Martin Flavin, and later, Yoshito Kaziro) who were working on the enzyme. This initiated what was to be a long relationship with Severo Ochoa and also his colleague, Yoshito Kaziro (now in Tokyo). About that time I applied to the National Science Foundation for a research grant to support my work on propionate metabolism. The grant proposal was rejected because the reviewers felt that I was really “in over my head” competing with the Ochoa laboratory and also because it had been rumored that his laboratory had already crystallized the enzyme from muscle. I knew that this was not true because in my correspondence with Ochoa he had indicated that the crystals turned out to be pyruvate kinase, not propionyl-CoA carboxylase. After much anguish I wrote to the Head of the National Science Foundation Review Committee, Louis Levin, indicating that the Committee was mistaken: “the carboxylase had not been crystallized” and that I thought it was inappropriate for the National Science Foundation to take a position on a grant application based on the size of the laboratory, rather than the merit of the proposal. A few weeks later I received a letter from Lou Levin indicating that the Study Section had reversed its decision and that the grant would be funded. I doubt seriously if that could happen today. Thus began my independent career in research and a developing relationship with Severo Ochoa. In 1959, a paper by Lynen and Knappe appeared in Angewandte Chemie (5) (later published in full in Biochemische Zeitschrift (6)) that created tremendous excitement in my laboratory. The paper described the rather remarkable finding that ␤-methylcrotonyl-CoA carboxylase, a biotin-dependent carboxylase (involved in leucine catabolism in certain bacteria), catalyzed the ATP-dependent carboxylation of “free” biotin in the absence of its acyl-CoA substrate. The product was shown to be a labile carboxylated biotin derivative, later identified as 1⬘-Ncarboxybiotin. Because biotin was believed to be a prosthetic group covalently bound to the enzyme and because free biotin exhibited an extremely high Km, Lynen proposed that the free biotin had accessed the active site of the carboxylase and by mimicking the biotinyl prosthetic group had gotten carboxylated. H56

Reflections: The Biotin Connection

FIG. 1. Harland Wood, circa 1991. (Reprinted with permission of the Cleveland Plain Dealer newspaper.)

Shortly thereafter Don Halenz and I succeeded in purifying a related enzyme, propionyl-CoA carboxylase, from bovine liver mitochondria. After convincing ourselves that it too was a biotin-dependent enzyme, we turned our attention to how the biotinyl group was attached to the carboxylase and what enzymatic reactions were involved in its becoming attached to the carboxylase. Dave Kosow, also in my laboratory at Virginia Tech, had just found that extracts of liver from biotin-deficient rats contained catalytically inactive propionyl-CoA apocarboxylase. Moreover, he demonstrated that a soluble ATP-dependent enzyme system in these extracts from the livers of the biotin-deficient animals catalyzed the covalent attachment of [14C]biotin to the apoenzyme, thereby restoring its ability to carboxylate propionyl-CoA (7). Moreover, Dave Kosow showed (7) that upon treating the 14C-biotinylated carboxylase with Streptomyces griseus protease, biocytin (i.e. ⑀-N-biotinyl-L-lysine) was released. This meant, of course, that the biotin prosthetic group had been linked to propionyl-CoA carboxylase through an amide linkage to a lysyl ⑀-amino group. A few years later it became evident that this long (⬃14 Å) side arm facilitates oscillation of the 1⬘-N-carboxybiotinyl prosthetic group between catalytic centers on the enzyme (7). After completing those experiments I invited Severo Ochoa to visit Virginia Polytechnic Institute and to present two lectures, which he graciously agreed to do. One of these talks dealt with propionyl-CoA carboxylase and the other with the genetic code, the two major projects under way in the laboratory of Ochoa at the time. While he was in Blacksburg Dave and I showed him our results on the site of attachment of biotin to the enzyme. We gave him some of the protease and within a month of his return to New York City he confirmed our findings with the heart propionyl-CoA carboxylase. It was at this point in 1962 that I decided to take a sabbatical leave in Munich with Feodor Lynen (known to his colleagues as “Fitzi”) at the Max-Planck Institu¨t Fu¨r Zellchemie where I could continue the work on the enzymatic mechanism by which biotin became attached to propionyl-CoA carboxylase. Before leaving for Munich Dave Kosow and I developed another more potent apoenzyme system with which to investigate the “biotin loading” reaction. This H57

Reflections: The Biotin Connection system made use of Propionibacterium shermanii that expressed huge amounts of methylmalonyl-CoA:pyruvate transcarboxylase, another biotin-dependent enzyme studied extensively by Harland Wood. Moreover, this organism had an absolute requirement for biotin in the growth medium, which when grown at very low levels of biotin produced large amounts of the apotranscarboxylase. The choice of the P. shermanii system turned out to be a good one. It so happened that my stay in Munich coincided with Harland Wood’s sabbatical leave in Lynen’s Institute. This was a two-fold bonus for me, first because Harland was the world’s expert on this enzyme and second because it began a lasting personal relationship with him. He has been a role model for me ever since that period in Munich. Harland (1907–1991) grew up on a farm near Mankato, Minnesota. He entered Macalester College in Minnesota where he majored in chemistry and worked his way through college. While a student at Macalester, he met Milly Davis and in their third year of college they married (in 1929, the year of the stock market crash and beginning of the great depression of the 1930s). In those days this required a meeting (for approval I presume) with the President of the college, who needn’t have been concerned as she was at his side for the next 62 years. They were an amazing couple, a cooperative inseparable team. My wife and I shared their friendship for more than 30 years. In 1931, Harland became a graduate student in bacteriology in the laboratory of C. H. Werkman at Iowa State University in Ames, Iowa, where he made a discovery that was so controversial, although correct, that it was questioned by his thesis adviser Werkman as well as by leaders in the field of microbial metabolism including C. B. van Niel. Harland had discovered (2) that heterotrophic organisms, such as the Propionibacteria, were able to fix CO2. Prior to this it was believed that only auxotrophs, i.e. chemosynthetic or photosynthetic auxotrophs, could carry out the net synthesis of organic compounds from CO2. His discovery truly opened the area of enzymatic carboxylation in higher organisms. After completing his Ph.D. degree Harland (Fig. 1) did postdoctoral work at the University of Wisconsin with W. H. Peterson and then returned to Iowa State as a faculty member. Harland was an innovator and an improviser. While at Iowa State he decided to conduct CO2 fixation experiments using 13CO2, but because of World War II restrictions he could not gain access to a mass spectrometer nor could he obtain “heavy” 13CO2. In true Woodsian style, he built his own mass spectrometer and constructed a thermal diffusion column in the Science building at Iowa State College (2). In 1946, Harland became Professor and Director of the Biochemistry Department at Western Reserve (now Case-Western Reserve) University. He ran the most democratic department on record in which faculty salaries were determined by the faculty at a meeting where members voted on one another’s salary for the upcoming year! Upon arriving in Munich in August of 1962, I indicated to Lynen that I would like to investigate the P. shermanii “biotin loading” enzyme system, and he agreed with my proposal. Because Harland Wood was already at the Institute, I got his advice on growth conditions and for large scale preparations of the transcarboxylase (actually, the apotranscarboxylase). Both Lynen and Wood were quite enthusiastic about the project. It turned out that by growing P. shermanii in biotin-deficient medium the bacteria produced as much of the apotranscarboxylase as the holotranscarboxylase when the organism was grown on normal/biotin-containing medium. Within a short time I was able to resolve and purify both the apotranscarboxylase and the synthetase that catalyzed loading biotin onto the apoenzyme (7). Dave Young, a postdoctoral fellow who had recently completed his medical training at Duke University, and Karl Rominger, a Ph.D. candidate under Lynen’s direction, collaborated with me on these studies. Finally, we proved that the synthetase catalyzed a two-step reaction in which the first step involved the ATP-dependent formation of biotinyl-5⬘-AMP and pyrophosphate after which the biotinyl group was transferred from the AMP derivative to the appropriate lysyl ⑀-amino group of the apotranscarboxylase. While in the midst of these studies, a controversy developed regarding the site at which biotin became carboxylated during catalysis. It was suggested that HCO3⫺ became incorporated into the 2⬘-position of the ureido ring of the covalently bound biotinyl prosthetic group of biotin-dependent enzymes and that the 2⬘-carbon was then transferred to the acceptor substrate. It was suggested that Lynen’s experiments (referred to above) had been done with free biotin and not the biotinyl prosthetic group covalently linked to the carboxylase. Such a mechanism would have necessitated opening and then closing the ureido ring of biotin during the course of the reaction, which to a chemist like Lynen didn’t make chemical sense. Moreover, this proposal was inconsistent with the known lability of free 1-N-[14C]carboxyH58


FIG. 2. Feodor (“Fitzi”) Lynen, circa 1980. (Reprinted with permission of the Max-Planck Gesellschaft.)

biotin. We knew from my earlier studies that enzyme-14CO2⫺, presumably enzyme-biotin14 CO2⫺ (prepared by incubating propionyl-CoA carboxylase with H14CO3⫺ and ATP-Mg2⫹), was even less stable than free 1-N-carboxy-[14CO2⫺]biotin. so we set out to address the issue head on using propionyl-CoA carboxylase as the source of enzyme-biotin-14CO2⫺. The previous spring before going to Munich, I had found that enzyme-14CO2⫺ (derived from propionyl-CoA carboxylase) could be stabilized by methylation with diazomethane, i.e. enzyme-14CO2⫺ was labile to acid before but was stable after methylation. Moreover, digestion of methylated enzyme-14CO2⫺ (enzyme-14CO2-CH3) with S. griseus protease produced a single radioactive derivative, presumably methoxy-[14C]carbonyl-⑀-N-biotinyl lysine. This product had chromatographic properties similar, but not identical, to ⑀-N-biotinyl lysine. Because I did not have the authentic compound for comparison, these experiments could not be completed at the time. Fortunately, Joachim Knappe, a former member of Lynen’s research group now at the University of Heidelberg, had synthesized the derivative and provided Lynen with a sample. Thus, we were able to verify the presumptive identification. This proved that the covalently bound biotinyl prosthetic group, like free biotin, was carboxylated at the 1⬘-N position (8). Shortly thereafter, Knappe in Heidelberg and Harland Wood on sabbatical in Lynen’s laboratory in Munich showed using a similar approach that the carboxybiotin prosthetic groups of ␤-methylcrotonylCoA and transcarboxylase, respectively, had identical structures (9). Taken together these studies proved unequivocally that the site of carboxylation of biotin was on the 1⬘-N of the biotinyl prosthetic group. By this point in my sabbatical in Lynen’s Institute, I began to recognize certain habits of “the Chief.” For example, he had the habit of working in his office until late in the afternoon. Then, around dusk, i.e. 6:00 – 6:30 p.m., he would emerge to make “rounds” in the Institute, moving from one bench to the next to survey the day’s progress or lack of it. Of course not one of the ⬃30 investigators would consider leaving until after he had passed through. He ran a “tight H59


FIG. 3. Severo Ochoa with colleagues viewing an enlargement of an electron micrograph of acetyl-CoA carboxylase (1966). From right to left: Albrecht Kleinschmidt, Erwin Stoll, Severo Ochoa, and Dan Lane.

ship”! Fitzi had an uncanny memory and could recall details of experiments done weeks earlier. Lynen (1911–1974) (3) (Fig. 2) was born and spent his entire life in Munich and environs. He received his doctoral training in organic chemistry at the University of Munich with Heinrich Wieland (Nobel Prize in Chemistry in 1927), graduating in 1937. He then married Wieland’s daughter, Eva. He was spared the ravages of World War II because of a serious skiing accident, which left him with a persistent limp. Perhaps Lynen’s most important contribution was the discovery of acetyl-CoA, the elusive molecule “active acetate,” sought after by many investigators including Fritz Lipmann, David Nachmansohn, and Severo Ochoa. Ochoa had discovered “condensing enzyme,” now known as citrate synthase, which catalyzed the formation of citrate from “active acetate” and oxaloacetate. These discoveries led to an important collaboration between Lynen and Ochoa in which they proved that citrate synthase used acetyl-CoA, along with oxaloacetate, to form citrate. These findings finally answered the question of how “active acetate” entered the citric acid cycle. In 1964 Lynen received the Nobel Prize (with Konrad Bloch) in Physiology or Medicine for his work on “the mechanism and regulation of cholesterol and fatty acid metabolism.” Lynen had strong connections to the United States. Many Americans came to his Institute to do postdoctoral work or sabbaticals. During the period that Harland Wood and I spent in Munich, the other Americans in the group included Esmond Snell, on sabbatical leave from Berkeley, David Young, Walter Bortz, Dick Himes, Paul Kindel, Martin Stiles, and Ed Wawskiewicz. Although Fitzi Lynen was a hard driving biochemist, he did like to socialize over a beer or a martini. On Friday afternoons Harland would often bring a half-gallon bottle of Gilbey’s gin to the Institute and prepare martinis in the second floor laboratory. Shortly after returning from Munich in the Summer of 1963, I received a phone call from Severo Ochoa, who asked if I might be interested in joining the faculty of his department at New York University School of Medicine in New York City. My wife, Pat, and I had some concern about moving from the bucolic setting of Blacksburg, Virginia (where we could see 20 miles from our living room window) to the big city. Nevertheless, we relished the new challenges ahead and were ready for a change in lifestyle. We loved New York City and never regretted having made the decision. Severo Ochoa helped make it worthwhile. Severo Ochoa (1905–1993) (1, 10) (Fig. 3) was born in Luarca, Spain, the youngest of seven children. His father was a lawyer and businessman. He completed his M.D. degree (with honors) at the University of Madrid. Though never having studied with him, he was inspired by Ramoń y Cajal, the Spanish neuroanatomist and Nobel Prize winner (1906). Following H60

Reflections: The Biotin Connection medical school (1929 –1931) Ochoa joined Otto Meyerhof’s laboratory in Heidelberg where he worked on muscle glycolysis. His early days in science were marked by the upheavals in Europe leading up to World War II. At the time of the Spanish civil war in 1936, he left Spain for Heidelberg for the second and final time. Then in 1938, because of the turmoil in Germany, he moved to Oxford University in England to work in Professor Rudolph Peter’s unit. In 1941 he came to the United States where he joined Carl and Gerty Cori at Washington University in St. Louis. In his comments at the Nobel Prize banquet in 1964, Ochoa spoke of those who had influenced him most. I was deeply influenced by my great predecessor Santiago Ramoń y Cajal. I entered Medical School too late to receive his teachings directly but, through his writings and his example he did much to arouse my enthusiasm for biology and crystallize my vocation. Among the great names that adorn the roll of Nobel prize-winners in Medicine is that of Otto Meyerhof, my admired teacher and friend, to whose inspiration, guidance and encouragement I owe so very much. I was very fortunate to have worked also under the guidance of other great scientists and I wish to acknowledge my indebtedness to Sir Rudolph Peters and to Nobel prize winners Carl and Gerty Cori who did so much to add new dimensions to my scientific outlook and enlarge my intellectual experience.

The seven years (1964 –1970) I spent in Ochoa’s department were among the most exciting of my scientific career. It was a small department with only a handful of faculty, which at that time included Charles Weissman, Bob Warner, Bob Chambers, Albrecht Kleinschmidt, and Severo. Upon arriving at New York University Medical School in August of 1962, Severo asked me to give 15 lectures in the first year medical student biochemistry course the next month. This course was Ochoa’s pride and joy and he and the faculty attended every lecture. (In retrospect, I feel that this is an excellent way to ensure quality control in teaching.) At the time, however, I hadn’t relished the idea of having a Nobel prize winner (1964, with Arthur Kornberg, Ochoa’s first postdoctoral fellow) in the audience for the first 15 lectures in my new scientific home. Despite knowing that my first few lectures at New York University were not particularly good, after the lecture Severo put his hand on my shoulder and said, “That was an excellent lecture, Dan.” I knew that it hadn’t been, but I did appreciate the encouragement. This was typical of Severo’s behavior toward young scientists in whom he had confidence. I suspect that his response reflected the encouragement he had received from his mentors during his development. Every afternoon at 3:00 p.m. we took a break for coffee in the department library where we discussed the latest results of our experiments or a hot new paper. Because the faculty was small, these were informal gatherings, which created a sense of camaraderie. Severo never failed to show up for these sessions. We could always count on Charles Weissman for a good, often slightly “off color” joke. “Have you heard the one about the ——?” Because of his innate ability at story telling, Charles was a favorite lecturer of the medical students. His timing was impeccable. Severo had a princely presence in part because of his carriage, tall stature, and silver hair. At national/international meetings, when he walked into a room he attracted hushed attention. Despite this, he had a warm personality and showed genuine concern for his colleagues, associates, and students. It is natural that we feel a closeness to those to whom we are related through research interests. In Hans Krebs book, Reminiscences and Reflections (11), he illustrates the scientific genealogy leading to Ochoa. We talk rather loosely these days about “impact factor” (and citation index) in evaluating the worth of one’s publications, but it is the excitement and joy of doing science, rather than the recognition itself, that motivates us. Research today moves at great speed. Communication is rapid, publication is rapid, and one is left with the impression that everything of importance was done in the past 10 years. However, science is built stepwise on the shoulders of those who came before us. Little is taught today as to how each of our particular areas of the biological sciences developed. For many students the “important stuff” now goes back into the past for only 7– 8 years. Most online scientific journals go back only 7– 8 years. Fortunately, the Journal of Biological Chemistry is the exception and is to be commended, because it is online all the way back to the point of its origin in 1905. These Reflections may be a sign of recognition that the history of discovery still has importance. H61

Reflections: The Biotin Connection Acknowledgment—I thank my wife, Pat Lane, who assisted with this article and shared these friendships and experiences with me.

Address correspondence to: [email protected].

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Ochoa, S. (1980) Annu. Rev. Biochem. 49, 1–30 Wood, H. G. (1985) Annu. Rev. Biochem. 54, 1– 41 Lynen, F. B. (1964) Information available on the Nobel Museum Web Site: www.nobel.se/ Lardy, H. A., and Adler, J. (1956) J. Biol. Chem. 219, 933–942 Lynen, F., Knappe, J., Lorch, E., Jutting, G., and Ringelmann, E. (1959) Angew. Chem. 71, 481– 486 Knappe, J., Ringelmann, E., and Lynen, F. (1961) Biochem. Z. 335, 168 –176 Moss, J., and Lane, M. D. (1971) Adv. Enzymol. Relat. Areas Mol. Biol. 35, 321– 442 (a review article) Lane, M. D., and Lynen, F. (1963) Proc. Natl. Acad. Sci. U. S. A. 49, 379 –385 Lynen, F. (1967) Biochem. J. 102, 381– 400 Kornberg, A., Horecker, B. L., Cornudella, L., and Oro´, J. (eds) (1975) Reflections on Biochemistry, pp. 1–14, Pergamon Press, New York 11. Krebs, H. A. (1981) Reminiscences and Reflections, Oxford University Press, Oxford, UK

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