Hsocitrate Lyase and Adenosine Triphosphate Malate Eyase as Key. Enzymes for the Methylotrophic Growth of Bacterium 5W2. ROGER B. COX and L. J. ...
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Hsocitrate Lyase and Adenosine Triphosphate Malate Eyase as Key Enzymes for the Methylotrophic Growth of Bacterium 5W2 ROGER B. COX and L. J. ZATMAN Department of Microbiology, University of Reading, Reading RGI 5 A Q, U.K. During an investigation of the carbon-assimilation pathway of C1 compounds by the facultative methylotroph bacterium 5H2 (Hampton & Zatman, 1973) isocitrate lyase (EC 4.1.3.1) activities in cell-free extracts of organisms grown on a variety of substrates were measured by the method of Dixon & Kornberg (1959). The method of Kramer et a/.(1959) was used to confirm that a product of the reaction was glyoxylate, the phenylhydrazone of the latter producing the intensely red-coloured 1,Sdiphenylformazancarboxylic acid on treatment with ferricyanide. The isocitrate lyase specific activities of bacterium 5H2 grown on tetramethylammonium chloride, trimethylamine, dimethylamine, methylamine, and on acetate, were respectively, 72, 84, 103, 95 and 150munits/mg of protein, whilst the values for extracts of cells grown on fructose, glutamate and on glycerol were respectively 1.9, I .5 and 2.0munits/mg of protein. These results suggest that isocitrate lyase is important for the inethylotrophic growth of bacterium 5H2 and, not unexpectedly, for growth on acetate. High specific activities of malate synthase (EC 4.1.3.2), measured by method of Dixon & Kornberg (1959), were found in cell-free extracts of bacterium 5H2 grown on CI as well as non-C1 substrates. The results of the following induction experiment confirmed the importance of isocitrate lyase for the growth of bacterium 5H2 on trimethylamine. Glycerol-grown cells, shaken at 30°C in a mineral medium to which 0.2% trimethylarnine had been added, showed no significant growth for the first 6h but the specific activity of isocitrate lyase increased from 2.0 to approximately 45 munits/mg of protein during this period. The specific activity of isocitrate lyase reached a maximum value of approximately lOOmunits/mg of protein about 3 h later, by which time the bacteria were growing exponentially. In a simultaneous experiment in which glycerol-grown cells were shaken at 30°C in the mineral medium containing 0.2% glycerol, exponential growth occurred immediately and there was no increase in the specific activity of the isocitrate lyase throughout the experiment. Present evidence (Salem et al., 1972; Dunstan el a[., 1972; Quayle, 1972) suggests that, in organisms which use the serine pathway of C1 assimilation, regeneration of the glycine acceptor for C1 units arises via the cleavage of a C4 metabolite. During a search for such a C, cleavage reaction, it was observed that crude cell-free extracts of trimethylamine-grown bacterium 5H2 (French press, 35000g supernatant) catalyse, in the presence of hydroxylamine, an ATP-, CoA- and Mgz+-dependent formation of acetylhydroxamate from L-malate. Further investigation indicated that the reaction is mediated by a malate cleavage enzyme (ATP malate lyase) which splits L-malate to acetylCoA and glyoxylate and which is similar to the malate lyase cleavage activity described by Tuboi & Kikuchi (1963) in Rhodopseirclomorias spheroicks grown phototrophically with malate. Our reaction mixture ( I ml total volume) contained (pniol): Tris-HC1 buffer pH7.8, 200; ATP, 10; CoA, 0.1; MgCI2, 5 ; L-malate, 100; NH20H,HC1 freshly neutralized, 500; crude cell-free extract (I-2mg of protein). This was incubated without shaking at 30°C, usually for 30min, the reaction being stopped by heating at 100°C for 2min, and a sample of the supernatant being chromatographed on paper by the method of Tuboi & Kikuchi (1962) to identify the hydroxarnates. Acetyl-, succinyl- and malyl-hydroxamic acids for marker spots on the chromatograms were prepared from their respective anhydrides by the method of Lipmann & Tuttle (1945). A measure of the total hydroxamic acids in the reaction mixture was obtained by the FeCI, method of Lipmann & Tuttle (1945). The major FeCI,-positive spot on the chromatogranis was identified as acetylhydroxamic acid, and a minor spot accounting for approximately 5 % of the total hydroxamate, was identified as malylhydroxamic acid. Glyoxylate production was determined i n reaction mixtures in which the hydroxylamine was
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BIOCHEMICAL SOCIETY TRANSACTIONS ,Glycine
Serine
Hydroxypyruvate ---+ Glycerate ---+ 3-Phosphoglycerate
Glyoxylate
Malate
-
I Acetyl-CoA
1
l’hosphoenolpyruvate
Oxaloacetate
Succina te Glyoxylate cycle
% CELL MATERIALS
Scheme 1. Proposed pathway of C,assimilation in hacteriitm 5H2
replaced by 35 pmol of neutralized phenylhydrazine and measured continuously by following the increase in absorbance at 324nm due to glyoxylate phenylhydrazone formation and by the formazan method of Kramer et al. (1959). Determination of the stoicheiometry of the reaction indicated that approximately equimolecular amounts of acetyl-CoA and glyoxylate were produced from L-rnalate. A hydroxaniate was produced from D-malate which was identified by chromatography as D-malylhydroxamate; no acetyl-hydroxamate or glyoxylate phenylhydrazone was detected using this substrate. It is concluded that whereas L-malate is activated and cleaved, D-malate is activated but not cleaved. ATP malate lyase in trimethylanline-grown extracts of bacterium 5H2 has a specific activity of approx. 50munits/mg of protein ; our preliminary results indicate that the specific activity of the enzyme in organisms grown on glutamate or glycerol is less than 2 % of that found in trimethylamine-grown organisms. It is concluded that ATP malate lyase is important for the methylotrophic growth of bacterium 5H2. Methylotrophically grown cells contain hydroxypyruvate reductase but no hexose phosphate synthase, indicating that assimilation of C1 compounds occurs via the serine pathway; Harder & Quayle (1971) have presented evidence that glyoxylate is the immediate precursor of glycine in this pathway. We therefore propose that assimilation in bacterium 5H2 occurs as shown in Scheme 1 , in which the serine pathway is linked to the glyoxylate cycle. The overall net reaction is thus the formation of 1 molecule of succinate from 2 C , units 2 CO,; succinate thus becomes the starting point for the biosynthesis of cell materials. Hersh & Bellion (1972) have also recently demonstrated the presence of a malate lyase in methylamine-grown Pseudomonas MA and Hyphomicrobiirm vulgare and, in agreement with our results for bacterium 5H2 conclude that it plays a key role in the metabolism of C, compounds in Pseudomonas MA. It is of interest that we have been unable to detect any malate cleavage activity in methylamine-grown Pseudomonas AM1 ; the source of the glyoxylate for the regeneration of glycine in this organism thus remains obscure.
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Dixon, G. H. & Kornberg, H. L. (1959) Biochem. J . 72, 3~ Dunstan, P. M., Anthony, C. & Drabble, W. T. (1972) Biochem. J. 128, 107-115 Hampton, D. & Zatman, L. J. (1973) Biochem. SOC.Trans. 1, 667-668 Harder, W. & Quayle, J. R. (1971) Biochem. J . 121, 763-769 Hersh, L. B. & Bellion, E. (1972) Biochem. Biophys. Res. Commun. 48, 712-719 Kramer, D. N., Klein, N. & Baselice, R. A. (1959) Anal. Chem. 31, 250-251 Lipmann, F. & Tuttle, L. C . (1945) J . Biol. Chem. 159, 21-28
1973
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Quayle, J. R. (1972) Adunrr. Microbiul Physiol. 7, 119-203 Salem, A . R., Large, P. J. RL Quaylc, J. R. (1972) Biuchem. J. 128, 1203-1211 Tuboi, S. & Kikuchi, G . (1962) Bioclrim. Biuphys. Actn 62, 188-190 Tuboi, S. & Kikuchi, G. (1963) J . Biochem. (Tokyo) 53,364373
Om the Specificity off the Tramsferr Ribonucleic Acid AUIdmOa@yhtiC9U?lRQactiQm J. P. EBEL, R. GIEGfi, J. BONNET, D. KERN, N. BEFORT, C. BOLLACK, F. FASLOLO, J. GANGLOFF and G. DIRHEIMER Laboratoire de Chimie Biologique, Uiiiversite‘Louis Pasterrr, Rue Descartes, Esplanade, 67000 Strasbozrrg, France
It is generally believed that the specificity of tRNA aminoacylation results solely from a specific recognition between the aniinoacyl-tRNA synthetase and the cognate tRNA. In fact, in this report we demonstrate that this specificity is not absolute. This assumption is supported by the following observations from our laboratory : (1) The existence of tRNA mischarging (Giege et al., 1971, 1972; Kern et al., 1972) implies that interactions take place between a tRNA and an aminoacyl-tRNA synthetase that does not correspond to this tRNA. With total tRNA, these mischarging reactions are more easily detected under special experimental conditions and in heterologous systems (Giege et al., 1971, 1972; Kern et al., 1972), but if isolated tRNA species are used, these incorrect aminoacylations can also be detected under standard conditions and in homologous systems (e.g. mischarging of yeast tRNAAlaand tRNAPhcby yeast valyl-tRNA synthetase and of yeast tRNAAspby yeast arginyl-tRNA synthetase). (2) The existence of non-specific interactions between an aminoacyl-tRNA synthetase and ‘non-cognate’ tRNA species is confirmed by the competitive inhibitions produced by ‘non-cognate’ tRNA species (e.g. yeast tRNATy‘, tRNAASPand tRNAPhe)in a correct aminoacylation reaction (e.g. the aminoacylation of yeast tRNAValby yeast valyl-tRNA synthetase). (3) The aminoacyl-tRNA synthetase-catalysed deacylation of aminoacyl-tRNA species (AMP- and PPi-independent) possesses a decreased specificity (Bonnet et al., 1972) (e.g. yeast alanyl-tRNAAIa, phenylalanyl- and valyl-tRNAPhe,seryl-tRNASer, tryptophyl-tRNATrP can be deacylated by yeast valyl-tRNA synthetase). We have evidence that during this deacy!ation reaction, the aniinoacyl-tRNA occupies the same site on the enzyme that the tRNA does during the aminoacylation reaction. Therefore it can be assumed that various aminoacyl-tRNA species which do not correspond to a given aminoacyl-tRNA synthetase are recognized by this enzyme. (4) Finally, non-specific complexes between tRNA species and aminoacyl-tRNA synthetases have been isolated (e.g. between Escherichia coli valyl-tRNA”*’ and yeast phenylalanyl-tRNA synthetase and between yeast tRNAAsPand yeast arginyl-tRNA synthetase), thus demonstrating directly the existence of non-specific interactions. All these results lead us to explain the high specificity of the aminoacylation reaction, which obviously exists, other than by an absolute specificity o f recognition between the tRNA and the aminoacyl-tRNA synthetase. We propose that tRNA aminoacylation depends more on the velocity of aminoacylation than on the recognition between aminoacyl-tRNA synthetase and tRNA. This concept is supported by kinetic measurements in which the V,,,. values for mischarging are diminished by three or four orders of magnitude as compared with that of the normal aminoacylation, whereas the K,,, values are only increased by one or two orders of magnitude. The strong decrease of the V,,,,,. values could probably result from a non-optimal positioning of the 3’-terminal adenosine of the tRNA on the amino acids of the catalytic site of the enzyme. Several experimental arguments lead us to think that the region near the 3‘-terminus of the VOl.
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