Cocatalytic zinc motifs in enzyme catalysis

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2715-2718, April 1993 Biochemistry

Cocatalytic zinc motifs in enzyme catalysis (zinc metalloenzymes/crystaflography/amino acid sequence/metal coordination)

BERT L. VALLEE AND DAVID S. AULD Center for Biochemical and Biophysical Sciences and Medicine and Department of Pathology, Harvard Medical School and Brigham & Women's Hospital, Boston, MA 02115

Contributed by Bert L. Vallee, December 29, 1992

ABSTRACT Cocatalytic zinc binding sites are characteristic of enzyme molecules which contain two or more zinc and/or other metal atoms. In each site an aspartate, glutamate, or histidine residue simultaneously binds to two zinc atoms or a zinc and a different metal atom. In the resultant amino acid bridge, two of the cocatalytic metal atoms bind to the same amino acid. Consequently the participating metal atoms are in close proximity and function as a catalytic unit, typical of this motif. In these functional units aspartate seems to be preferred over glutamate. Serine, threonine, tryptophan, and lysine residues are encountered as zinc ligands, although they have not so far been identified as ligands in monozinc enzymes or DNA-binding zinc proteins. The resultant coordination spheres and their mechanistic implications raise interesting questions for further study.

istics of the cocatalytic motifs. The salient features of catalytic and structural sites will again be summarized briefly for comparison.

MATERIALS AND METHODS Computer and literature searches have ascertained sequences, zinc content, and the functional characteristics of the catalytic, cocatalytic, and structural zinc sites for families ofzinc enzymes corresponding to known structural standards of reference. A family is here defined as a group of proteins related by common ancestry as revealed by their homology and with identical or very similar functions. The National Biomedical Research Foundation Protein Identification Resource, the Swiss-Prot data base, and translations of coding regions of DNA sequences from the GenBank/EMBL data base available at the Molecular Biology Computer Research Resource at Harvard Medical School were used to search for homologous family members. Protein sequences were aligned and families were formed by the use of a patterninduced multialignment algorithm for amino acid sequences

We have detailed the chemical properties of zinc as they pertain to the roles of this metal in catalytic and structural sites of enzymes and in zinc fingers, twists, and clusters of transcription factors, glucocorticoid receptors, and other gene products which are essential in the transmission of genetic information and the metabolic regulation by hormones (1-5). We now direct attention to the cocatalytic (coactive) motif in multizinc enzymes (1) whose characteristics were deferred in our earlier communications in anticipation of sufficient structural data to verify preliminary

(6). RESULTS AND DISCUSSION Catalytic Zinc Sites. The x-ray structure analyses of 11 enzymes containing a single catalytic zinc atom identify their ligands. This metal forms complexes with any three nitrogen and oxygen ligands of histidine and glutamate residues with a binding frequency of His >> Glu (4).* When the catalytic zinc sites include those of the Znlt cocatalytic sites and alcohol dehydrogenase (see below), then the ligands encompass aspartate and cysteine residues, and the overall binding frequency becomes His >> Glu > Asp > Cys (Fig. 1). In all instances water is an additional zinc ligand and can be activated to be ionized, polarized, or displaced. The catalytic zinc site of alcohol dehydrogenase is unique, the only one so far comprising only one histidine and two cysteines (Cys-46 and Cys-174) (11). The amino acid ligands, their spacing in the protein sequence, and the vicinal properties of the active center created by protein folding are critical for the mechanism of action of each particular enzyme. The nature of the ligands and the potential of the ionization of the bound water affect the possible net charge of the resultant complex, which ranges

impressions. The enzymatic roles of zinc are expressed in catalytic, structural, and cocatalytic (or coactive) motifs (1). Catalytic zinc atoms participate directly in the bond-making or -breaking step, and their removal by chelating or other agents completely abolishes activity. Changes in local and/or overall conformation may or may not accompany this loss. In multizinc enzymes the cocatalytic zinc (or other metal) atoms operate in concert to affect catalysis and perhaps stabilization of active-site conformation directly and/or indirectly (1). Structural zinc atoms can maintain the tertiary structure of an enzyme in a manner analogous to that of disulfide bridges and may serve as conduits of local conformational changes. Thus far, the dimeric alcohol dehydrogenases are the only enzymes known to contain both a catalytic and a structural zinc atom. Although a large number of zinc enzymes are now known, x-ray crystallographic analysis has identified the amino acid ligands and the resultant structural motifs of catalytic, cocatalytic, and structural zinc coordination sites in relatively few of these (1-4). However, their number is now sufficient to draw conclusions regarding the structural identities of the zinc binding sites in the relevant families. Imidazole nitrogen, carboxylate oxygen, and cysteine thiol predominate as ligands to the catalytic, cocatalytic, and structural sites, respectively. Here we will specifically detail the character-

from a dication for the lyase carbonic anhydrase, to a monocation for many of the hydrolases, to a neutral complex *In three of these enzymes a fourth protein ligand with a relatively longer bonding distance has been noted. All of them are encountered in three histidine-ligated catalytic zinc sites. In the cadmiumsubstituted 3-lactamase Cys-168 has been thought to be a weak ligand at 4.5 A (7). In astacin Tyr-149 is believed to be a ligand with a bond length of 2.6 A (8), and in the inhibitor-complexed form of adenosine deaminase Asp-295 ligates the zinc; the bond length is 2.5

A (9, lo). tZnl refers to catalytic zinc atoms in all instances. Zn2 and Zn3 are

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cocatalytic. 2715

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Proc. Natl. Acad. Sci. USA 90 (1993)

H20 I Zn LL1 SHORT

CAI, II -L AD DD-CPD A TL, NP AE CPD A, B PL C N P1 AP ADH

L2

H-i-H H I1-H HA 1 -H H- 2 H H 3 -H H- 3-H H H- 3 H- 2 -E E- 3 ------ H D 3 -H D- 3-H C-20-- H

--

L LON LONG 22 121 196 40 5 19 19 123 13 12 80 106

H H H H H E E H H H H C

FIG. 1. Ligand and spacer relationships of catalytic zinc sites in carbonic anhydrases I and II (CA I, II), P-lactamase (P-L), adenosine deaminase (AD), DD carboxypeptidase (DD-CPD), astacin (A), thermolysin (TL), neutral protease (NP), aeruginosa elastase (AE), carboxypeptidases A and B (CPD A, B), phospholipase C (PL C), nuclease P1 (N P1), alkaline phosphatase (AP), and alcohol dehydrogenase (ADH). Amino acid ligands L1, L2, and L3 and the length of the short and long spacers (in amino acid residues) are indicated.

for the oxidase alcohol dehydrogenase. The systematic spacing between the coordinating ligands is striking (Fig. 1). A short spacer (1-3 amino acids) initially enables formation of a bidentate zinc complex, whereas the long spacer (5-196 amino acids) provides other catalytic and/or substratebinding groups that form the active center and the ligand that completes the coordination sphere which poises zinc for catalysis. Three histidines are typical of the lyases human carbonic anhydrase I and 11 (12, 13) and the hydrolases Bacillus cereus ,3-lactamase (7), DD carboxypeptidase of Streptomyces albus G (14), adenosine deaminase (10), and astacin (8). Two histidines are characteristic of bovine carboxypeptidases A and B (15, 16); thermolysin, the neutral protease of Bacillus thermoproteolyticus (17); B. cereus neutral protease (18); Pseudomonas aeruginosa elastase (19); B. cereus phospholipase C (20); Escherichia coli alkaline phosphatase (21); and Penicillium citrinum nuclease P1 (22). Structural Zinc Sites. The structural zinc sites are compact domains whose construction is based solely on cysteine ligands arranged in tetrahedral coordination. In point of fact, aspartate carbamoyltransferase is the only enzyme containing only a structural zinc atom located in the regulatory unit (23). Alcohol dehydrogenase, in addition to its catalytic zinc atom, also has a second, structural zinc atom (11). Cocatalytic Zinc Sites. These occur only in multimetal zinc enzymes where-prior to the x-ray structure determination the function of these metal atoms was dubbed "modulating" or "regulatory" (24, 25). X-ray structures of these enzymes in numbers sufficient to allow the systematization of their zinc binding sites have become available only in the course of the last 2 years. All contain two or more zinc and/or another metal atom (usually magnesium). Since the structures of alkaline phosphatase (21) and leucine aminopeptidase of lens (26) were solved we have referred to the motifs of these zinc binding sites as "cocatalytic" or "coactive" (1). Cocatalytic zinc binding sites feature two or more metal atoms, one, two, or all of which can be zinc. They are physically close to one another and operate as a functional unit. One amino acid, either aspartate or glutamate, simultaneously binds two metal atoms. Thus, it is characteristic for the three-metal group of multizinc enzymes that aspartate provides a bridge between Zn2 and Zn3 atoms or between Zn2 and a magnesium atom. Three enzymes in the three-metal cocatalytic site group whose structures are now known metabolize phosphate. B.

cereus phospholipase C (20) and P. citrinum nuclease P1 (22) contain three zinc atoms. Alkaline phosphatase of E. coli (21) contains two zinc atoms and one magnesium atom. In all these one zinc atom is catalytic (Znl) while the remaining pair of metal atoms (Zn2 and Zn3 or Mg) are linked by a bridging amino acid so that all three metal atoms are in close physical proximity to one another. The interatomic Znl-Zn2 distance of alkaline phosphatase is 3.94 A, whereas the Zn2-Mg distance is 4.88 A (Fig. 2). The catalytic Znl site comprises two ligands, Asp-327 and His331, separated by a 3-amino acid short spacer and a third ligand, His-412, separated from His-331 by an 80-amino acid long spacer. The second zinc atom, Zn2, is coordinated to Asp-369, His-370, Ser-102, and 081 of Asp-51 and bridged to magnesium by coordinating with 0o2 of Asp-51, one of the E oxygens of Glu-322, the hydroxyl of Thr-155, and three water molecules in a slightly distorted octahedron (21). In phospholipase C, His-128, His-142, and Glu-146 are the ligands to Znl, the catalytic zinc site. Aspartate bridges Zn2 and Zn3. Zn2 is bound to Q81 of the bridging ligand Asp-122 and to His-118, His-69, and Asp-55. Zn3 is bound to O02 ofthe bridging ligand Asp-122 and to the amino and carbonyl groups of Trp-1, the nitrogen of His-14, and an oxygen of a water molecule which is an additional bridge for the two metal sites (20). The interatomic distances are 4.4 and 3.3 A for Znl-Zn2 and Zn2-Zn3, respectively. In nuclease P1 the interatomic distances between Znl and Zn2 and between Zn2 and Zn3 are 3.2 A and 5.8 A, respectively (22). The catalytic Znl site comprises His-149 and Asp-153 separated by a 3-amino acid short spacer and a third ligand, His-126, separated from His-149 by a long spacer of 12 amino acids. Asp-120 is the bridging ligand for Zn2 and Zn3. In addition, a water molecule or hydroxide ion bridges the two zinc atoms in a fashion similar to that seen in phospholipase C. His-45, His-65, and His-116 coordinate with Zn2 while the amino and carbonyl groups of Trp-1 and His-6 are coordinated to Zn3. The nature of these cocatalytic metal sites shows that the ligands are dissimilar from the details of the catalytic and structural sites. Thus, the identity of the ligands and the coordination number and geometries of the complexes, as well as the existence of a bridging ligand(s) in these motifs, clearly set cocatalytic sites apart from other enzymatic zinc sites. The interatomic distances between the component metal atoms are their most obvious diagnostic physicalchemical features. We shall here single out the two-metal

FIG. 2. Schematic of cocatalytic zinc site for E. coli alkaline phosphatase. Amino acid ligands are shown in the one-letter code. Distances are in angstroms.

Biochemistry: Vallee and Auld cocatalytic site of leucine aminopeptidase to illustrate this further.t In lens aminopeptidase, Zn2 is bound less tightly than Znl, being coordinated to the E-amino group of Lys-250, the carboxylate oxygens of Asp-273, and to one carboxylate oxygen of the bridging ligand Glu-334 (26, 29). The other carboxylate oxygen of the bridging Glu-334 is bound to the Znl, 2.9 A away. The carboxylate oxygens of Asp-255 and Asp-332, as well as the carbonyl oxygen of the latter, complete the coordination of the first zinc site. A weakly bound water molecule at 3.2 A and Zn2 at 2.9 A are also believed to be involved in this coordination site. We have previously referred to superoxide dismutase in the context of zinc enzyme motifs (1) although functionally it is a copper enzyme (ref. 30 and references therein). However, its enzymatically active copper atom is bound to His-61 (31), the same residue which also ligates the sole zinc atom, whose role remains conjectural (Fig. 3). Cocatalytic zinc atoms are distinguished by yet other characteristic coordination features (see below). Superoxide dismutase is part of this presentation largely so that it becomes inclusive regarding the possible roles of zinc. We realize, of course, that superoxide dismutase might represent the only example so far of yet another, different constellation and/or motif. Before such a decision is made the potential identities and differences of its characteristics deserve further study. The existence of a bridging ligand is the most dramatic feature of the cocatalytic site and is distinctive in differentiating the cocatalytic site from catalytic and structural zinc sites (Fig. 3). The interatomic distances between the constituent metals are diagnostic of cocatalytic sites, reflecting their close proximity. Moreover, cocatalytic zinc sites differ from all others in regard to the type of ligands and the existence of 5- or even 6-coordinate zinc complexes in the absence of substrate. The structures of the carboxylate and imidazole side chains of aspartate, glutamate, and histidine are all suitable to serve as bridging ligands, since they feature two electronegative oxygen atoms or a nitrogen attached to a central carbon. The x-ray structures so far available show that aspartate is the bridging amino acid in three enzymes which metabolize phosphate; but lens leucine aminopeptidase employs glutamate (Fig. 3). Aspartate may well be the bridging ligand of choice when the closest proximity between two metal atoms is desired. It is less flexible than glutamate and would most likely make a better conduit for conformational changes in protein zinc sites on interaction with substrate. In this regard it should be noted that in 19 of the phosphatases, glycine abuts both the C- and N-terminal sides of Asp-51 and the C-terminal side in all the phospholipases C and P1-like nucleases (32). The lack of steric hindrance in a glycine residue, together with its capacity to allow for sharp turns in the protein chain, might assist the structural roles of such bridging aspartates. Understanding the interactive relationship between zinc and its associated bridging ligands will be crucial in revealing the mechanistic pathways of the group of enzymes containing cocatalytic zinc sites. The coordination number of cocatalytic zinc sites in the resting enzyme is frequently 5 or 6, which contrasts with the tetracoordination encountered in catalytic and structural zinc sites of monozinc enzymes. In all Zn2 sites of the phosphatemetabolizing enzymes, zinc is coordinated to four different amino acid ligands. Surprisingly, only three amino acid ligands have been identified in either zinc site of lens leucine aminopeptidase. However, the extreme proximity of the two

tZinc and magnesium have also been implicated in the 3'-5' exonuclease activity of E. coli DNA polymerase 1 (27, 28). In that case zinc binds to Asp-355, Glu-357, and Asp-501. Asp-355 bridges magnesium to this site.

Proc. Natl. Acad. Sci. USA 90 (1993)

AP

Zn2

Mg

2717

51

GGM G D

L L I G 122

PL C

Zn2 Zn3

VV N QP

H YL G 120

N P1 APEP

SOD

M T Q P

H F I G

Zn3 Zn2 N TD A Zn3

Cu

334

G R L I 61

S AG P

F N P

L

FIG. 3. Cocatalytic bridging ligands and their adjoining amino acid sequences in E. coli alkaline phosphatase (AP), B. cereus phospholipase C (PL C), P. citrinum nuclease P1 (N P1), bovine lens aminopeptidase (APEP), and bovine superoxide dismutase (SOD). The only copper enzyme in this group, SOD, is set apart from the rest.

zinc atoms (2.9 A) has led to the somewhat unorthodox suggestion that Znl might be a ligand for Zn2 and presumably vice versa (29). While histidine and cysteine predominate in catalytic and structural sites, aspartate appears to be the preferred ligand in cocatalytic sites. The participation of ligands other than histidine, glutamate, aspartate, and cysteine is yet an additional feature of the cocatalytic sites. Thus, a lysine residue binds to Zn2 of lens aminopeptidase, an N-terminal tryptophan is a bidentate ligand to both the Zn3 sites of phospholipase C and nuclease P1, the catalytic serine binds to the Zn2 of the phosphatases, and serine or threonine ligates the magnesium site of the phosphatases. These arrangements obviously differ significantly from those encountered in monozinc enzymes, where the implication is strong that the displacement, ionization, or polarization of water is the primary objective of the motif of the catalytic site. Amino acid ligands other than histidine, glutamate, aspartate, and cysteine have not been recognized thus far in catalytic or structural sites of any monozinc enzymes or DNA-binding zinc proteins. Metal coordination in multizinc enzymes clearly is distinct from that in monozinc enzymes. While a water molecule involved in catalysis is the fourth ligand in catalytic zinc sites, it may be a fourth, fifth, or sixth ligand in cocatalytic sites. Further, in cocatalytic sites lysine, serine, threonine, or tryptophan residues may participate as ligands, generating greater diversity of the zinc coordination sphere and, hence, creating increased potential for catalytic polarizability. In alkaline phosphatase, for example, the catalytic serine coordinated to zinc is released in the process of substrate interaction to become phosphorylated. The phosphorylation of this residue, long known to result in an intermediate of phosphatase action, in this enzyme seems to be an objective of the cocatalytic motif in concert with the activation of water. This work was supported by the Endowment for Research in Human Biology, Inc. 1. Vallee, B. L. & Auld, D. S. (1992) Faraday Discuss. Chem. Soc. 93, 47-65. 2. Vallee, B. L. & Auld, D. S. (1989) FEBS Lett. 257, 138-140. 3. Vallee, B. L. & Auld, D. S. (1990) Proc. Natl. Acad. Sci. USA 87, 220-224.

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