Mutagen Stability of Alkylation-Sensitive Mutants of Bacillus subtilis

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.     

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

beta-Ketoadipate pathway in Trichosporon cutaneum modified for methyl-substituted metabolites.

Trichosporon cutaneum, when grown with p-cresol, catalyzed intradiol fission of the benzene nucleus of 4-methylcatechol before the complete catabolism of these two substrates. Steps in their conversion to pyruvate and acetyl coenzyme A were investigated by using cell extracts, and some properties of various new microbial catabolites are also described. These included (-)-2,5-dihydro-3-methyl-5-oxofuran-2-acetic acid (beta-methylmuconolactone) and (-)-3-keto-4-methyladipic acid and its coenzyme A ester; the latter was degraded by an enzymatic reaction sequence that included the coenzyme A esters of methylsuccinic, itaconic, and citramalic acids. A notable feature of this sequence is the formation of beta-methylmuconolactone which can be readily metabolized, in contrast to the analogous reaction in bacteria that gives the "dead-end" compound gamma-methylmuconolactone; this compound cannot be enzymatically degraded and so renders the beta-ketoadipate pathway unavailable for methyl-substituted bacterial sources of carbon that are catabolized by way of 4-methylcatechol.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

Amino acid and lactate catabolism in trimethylamine oxide respiration of Alteromonas putrefaciens NCMB 1735.

The nonfermentative Alteromonas putrefaciens NCMB 1735 grew anaerobically in defined media with trimethylamine oxide as external electron acceptor. All amino acids tested, except taurine and those with a cyclic or aromatic side chain, were utilized during trimethylamine oxide-dependent anaerobic growth. Lactate, serine, and cysteine (which are easily converted to pyruvate) and glutamate and aspartate (which are easily converted to tricarboxylic acid cycle intermediates) were metabolized at the fastest rate. Growth with lactate as growth-limiting substrate gave rise to the formation of 40 mol% acetate, whereas serine and cysteine were nearly completely oxidized to CO2. Molar growth yields with the latter substrates were the same and were 50% higher than with lactate. This showed that more ATP was formed when acetyl coenzyme A entered the tricarboxylic acid cycle than when it was converted via acetyl phosphate to acetate. Also, growth with formate as substrate indicated that the reduction of trimethylamine oxide to trimethylamine was coupled with energy conservation by a respiratory mechanism.     

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS I. : General Properties of the Formate-Exchange Reaction.

McCormick, N. G. (University of Washington, Seattle), E. J. Ordal, and H. R. Whiteley. Degradation of pyruvate by Micrococcus lactilyticus. I. General properties of the formate-exchange reaction (J. Bacteriol. 83:887-898. 1962.-At an alkaline pH, extracts of Micrococcus lactilyticus(2) catalyze the phosphoroclastic degradation of pyruvate to formate and acetyl phosphate and the rapid exchange of formate into the carboxyl group of pyruvate. At an acid pH, hydrogen, carbon dioxide, and acetyl phosphate are produced, and carbon dioxide is exchanged into the carboxyl group of pyruvate. A concentration of approximately 1 m phosphate is required for the phosphoroclastic reaction and formate exchange; the production of carbon dioxide and hydrogen is greatly inhibited by high concentrations of phosphate. Formate exchange requires a divalent metal ion and is stimulated by reducing agents and an atmosphere of hydrogen. Inhibition by p-chloromercuribenzoate, Zn(++), Cd(++), and arsenite indicates that sulfhydryl groups on the enzyme are involved in the reaction; the inhibition by arsenite and Cd(++) may be relieved by 2,3-dimercaptopropanol, suggesting that vicinal dithiols may be required. Inhibition by hypophosphite may reflect a competition with formate for a site on the enzyme.At an alkaline pH, alpha-ketobutyrate is degraded to propionate and formate, whereas alpha-ketoglutarate is fermented to succinate, propionate, carbon dioxide, hydrogen, and formate. Formate is exchanged into the carboxyl groups of alpha-ketobutyrate and alpha-ketoglutarate under these conditions. Only traces of alpha-ketovalerate and alpha-ketoisovalerate are fermented at an alkaline pH and the exchange of formate into these compounds is very low.The addition of viologen dyes under the conditions used for formate exchange causes a reduction of pyruvate, alpha-ketobutyrate, alpha-ketovalerate, and alpha-ketoisovalerate to the corresponding alpha-hydroxy acids.     

The Utilization of Choline and Acetyl Coenzyme A for the Synthesis of Acetylcholine

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high-affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high-affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high-affinity transport and acetylation of choline are kinetically coupled.     

Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.     

Pyruvate oxidase activity dependent on thiamine pyrophosphate, flavin adenine dinucleotide and orthophosphate in Streptococcus sanguis

Abstract Oxygen uptake by Streptococcus sanguis ATCC10556 in the presence of pyruvate was studied. In permeabilized cells pyruvate oxidase activity dependent on thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and orthophosphate was demonstrated. The activity was ten times higher in cells grown aerobically than in cells grown anaerobically. Acetyl phosphate was a product, and 1.1 mol of acetyl phosphate was formed per mol of oxygen taken up. No pyruvate dehydrogenase activity dependent on NAD, coenzyme A (CoA) and TPP was detected.     

Lipoic acid and diabetes—Part III: Metabolic role of acetyl dihydrolipoic acid

Rat liver lipoyl transacetylase catalyzes the formation of acetyl dihydrolipoic acid from acetyl coenzyme A and dihydrolipoic acid. In an earlier paper the formation of acetyl dihydrolipoic from pyruvate and dihydrolipoic acid catalyzed by pyruvate dehydrogenase has been reported. Acetyl dihydrolipoic acid is a substrate for citrate synthase, acetyl coenzyme A carboxylase and fatty acid synthetase. The V_max. for citrate synthase with acetyl dihydrolipoic acid was identical to acetyl coenzyme A (approximately 1 μmol citrate formed/min/mg protein) while the apparent K_m was approximately 4 times higher with acetyl dihydrolipoic acid as the substrate. This may be due to the fact that synthetic acetyl dihydrolipoic acid is a mixture of 4 possible isomers and only one of them may be the substrate for the enzymatic reaction. While dihydrolipoic acid can replace coenzyme A in the activation of succinate catalyzed by succinyl coenzyme A synthetase, the transfer of coenzyme A between succinate and acetoacetyl dihydrolipoic acid catalyzed by succinyl coenzyme A: 3 oxo-acid coenzyme A transferase does not occur.     

Metabolic Pathways in Methanococcus jannaschii and Other Methanogenic Bacteria

Eleven strains of methanogenic bacteria were divided into two groups on the basis of the directionality (oxidative or reductive) of their citric acid pathways. These pathways were readily identified for most methanogens from the patterns of carbon atom labeling in glutamate, following growth in the presence of [2-¹³C]acetate. All used noncyclic pathways, but members of the family Methanosarcinaceae were the only methanogens found to use the oxidative direction. Methanococcus jannaschii failed to incorporate carbon from acetate despite transmembrane equilibration comparable to other weak acids. This organism was devoid of detectable activities of the acetate-incorporating enzymes acetyl coenzyme A synthetase, acetate kinase, and phosphotransacetylase. However, incorporation of [1-¹³C]-, [2-¹³C]-, or [3-¹³C]pyruvate during the growth of M. jannaschii was possible and resulted in labeling patterns indicative of a noncyclic citric acid pathway operating in the reductive direction to synthesize amino acids. Carbohydrates were labeled consistent with glucogenesis from pyruvate. Leucine, isoleucine, phenylalanine, lysine, formate, glycerol, and mevalonate were incorporated when supplied to the growth medium. Lysine was preferentially incorporated into the lipid fraction, suggesting a role as a phytanyl chain precursor.     

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS II. : Studies of Cofactors in the Formate-Exchange Reaction.

McCormick, N. G. (University of Washington, Seattle), E. J. Ordal, and H. R. Whiteley. Degradation of pyruvate by Micrococcus lactilyticus. II. Studies of cofactors in the formate-exchange reaction. J. Bacteriol. 83:899-906. 1962.-Enzyme preparations from Micrococcus lactilyticus(2) are rendered inactive with respect to formate exchange by treatment with charcoal or Dowex-50, by dialysis, or by fractionation with ammonium sulfate. The activity may be completely restored by a "kochsaft" preparation (BES) obtained from M. lactilyticus and partially restored by similar BES preparations from Escherichia coli and Clostridium butyricum. Diphosphothiamine is required for formate exchange but full activity cannot be restored by known cofactors. Brief exposure to increased temperatures, air, extremes of pH, and absorption with charcoal and Dowex-50 decrease the cofactor activity of BES preparations. The addition of BES preparations from E. coli and Streptococcus faecalis causes a shift in the degradation of pyruvate by extracts of M. lactilyticus from the phosphoroclastic cleavage (to acetyl phosphate and formate) to the dismutation of pyruvate (to lactate, acetate, and carbon dioxide).C. cylindrosporum was found to mediate the formate-exchange reaction; the activity of crude extracts was stimulated by M. lactilyticus and C. butyricum BES preparations. M. lactilyticus BES also increased the formate-exchange activity of extracts of E. coli.     

Disruption of the operon encoding Ehb hydrogenase limits anabolic CO2 assimilation in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO2 assimilation in methanococci.     

Effect of dilution rate and carbon availability on Bifidobacterium breve fermentation

Bifidobacterium breve NCFB 2257 was grown in glucose-limited and nitrogen (N)-limited chemostats at dilution rates (D) from 0.04 to 0.60 h^–1, to study the effect of nutrient availability on carbohydrate metabolism. The results showed that D had little effect on fermentation product formation, irrespective of the form of nutrient limitation. However, marked differeces were observed in the distribution of fermentation products, that were attributable to glucose availability. In glucose-limited cultures, formate and acetate were the principal end-products of metabolism. Lactate was never detected under these growth conditions. In contrast, lactate and acetate were mainly formed when glucose was in excess, and formate was not produced. These results are explained by the metabolic fate of pyruvate, which can be dissimilated by either phosphoroclastic cleavage to acetyl phosphate and formate, or alternatively, it may be reduced to lactate. Enzymic studies were made to establish the mechanisms that regulated pyruvate metabolism. The data demonstrated that control was not exercised through regulation of the synthesis and activity of lactate dehydrogenase (LDH), phosphofructokinase or alcohol dehydrogenase. It is possible however, that there was competition for pyruvate by LDH and the phosphoroclastic enzyme, which would determine the levels of lactate and formate produced respectively. These results demonstrate the metabolic flexibility of B. breve, which preferentially uses lactate as an electron sink during N-limited growth, whereas under energy-limitation, carbon flow is directed towards acetyl phosphate to maximise ATP synthesis. Correspondence to: B. A. Degnan     

Pyruvate Metabolism in Sarcina maxima

The mechanisms of pyruvate cleavage and hydrogen production by Sarcina maxima were studied. It was found that a phosphoroclastic system for pyruvate oxidation, similar to that occurring in saccharolytic clostridia, is present in S. maxima. Cleavage of pyruvate by extracts of the latter organism resulted in the formation of acetyl phosphate, CO(2), and electrons which were transferred to ferredoxin. Formate was not an intermediate in this system. Pyruvate oxidation was coupled with ferredoxin-dependent nicotinamide adenine dinucleotide phosphate (NADP) reduction. A hydrogenase, active in particulate extracts of S. maxima, did not accept electrons from reduced ferredoxin. Formate was detected as a fermentation product when S. maxima was grown in media buffered with CaCO(3). Whole cells and extracts degraded formate to H(2) and CO(2). The evidence suggests that electrons generated by ferredoxin-linked pyruvate oxidation by S. maxima are not used for H(2) production, but that they serve for the reduction of NADP. Reduced NADP may be utilized by the organisms for synthesis of cell material. Production of H(2) by S. maxima may occur through a pyruvate clastic system similar to that present in coliform bacteria.     

Kinetic characterization of the [3'-32P]coenzyme A/acetyl coenzyme A exchange catalyzed by a three-subunit form of the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum

The ability of acetyl coenzyme A synthesizing carbon monoxide dehydrogenase isolated from Clostridium thermoaceticum to catalyze the exchange of [3'-32P]coenzyme A with acetyl coenzyme A is studied. This exchange is found to have a rate exceeding that of the acetyl coenzyme A carbonyl exchange also catalyzed by CO dehydrogenase ([1-14C]acetyl coenzyme A + CO in equilibrium acetyl coenzyme A + 14CO). These two exchanges are diagnostic of the ability of CO dehydrogenase to synthesize acetyl coenzyme A from a methyl group, coenzyme A, and carbon monoxide. The kinetic parameters for the coenzyme A exchange have been determined: Km(acetyl coenzyme A) = 1500 microM, Km(coenzyme A) = 50 microM, and Vmax = 2.5 mumol min-1 mg-1. Propionyl coenzyme A is shown to be a substrate (Km approximately 5 mM) for the coenzyme A exchange, with a rate 1/15 that of acetyl coenzyme A, but is not a substrate for the carbonyl exchange. CO dehydrogenase capable of catalyzing both these two exchanges, and the oxidation of CO to CO2, is isolated as a complex of molecular weight 410,000 consisting of three proteins in an alpha 2 beta 2 gamma 2 stoichiometry. The proposed gamma subunit, not previously reported as part of CO dehydrogenase, copurifies with the enzyme and has the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the disulfide reductase previously separated from CO dehydrogenase in a final chromatographic step.     

Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage

Recently, Knappe and co-workers [Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., & Ganzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332] have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using [2H2]hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from [2H]formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.     

Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate)

Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.     

Pyruvate dehydrogenase and 3-fluoropyruvate: chemical competence of 2-acetylthiamin pyrophosphate as an acetyl group donor to dihydrolipoamide

The pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex catalyzes the decomposition of 3-fluoropyruvate to CO2, fluoride anion, and acetate. Acetylthiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with 3-fluoro[1,2-14C]pyruvate, TPP, coenzyme A (CoASH), and either NADH or pyruvate as reducing systems leads to the formation of [14C]acetyl-CoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH (87 +/- 2%) and water (13 +/- 2%). When the E1 component is incubated with 3-fluoro[1,2-14C]pyruvate, TPP, and dihydrolipoamide, [14C]acetyldihydrolipoamide is produced. The formation of [14C]acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration (0.25-16 mM). A plot of the extent of acetyl group partitioning to dihydrolipoamide as a function of 1/[dihydrolipoamide] showed 95 +/- 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration was extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate.     

Properties of oxaloacetate decarboxylase from Veillonella parvula.

Oxaloacetate decarboxylase was purified to 136-fold from the oral anaerobe Veillonella parvula. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity of the enzyme was exhibited at pH 7.0 for both carboxylation and decarboxylation. At this pH, the Km values for oxaloacetate and Mg2+ were at 0.06 and 0.17 mM, respectively, whereas the Km values for pyruvate, CO2, and Mg2+ were 3.3, 1.74, and 1.85 mM, respectively. Hyperbolic kinetics were observed with all of the aforementioned compounds. The Keq' was 2.13 X 10(-3) mM-1 favoring the decarboxylation of oxaloacetate. In the carboxylation step, avidin, acetyl coenzyme A, biotin, and coenzyme A were not required. ADP and NADH had no effect on either the carboxylation or decarboxylation step, but ATP inhibited the carboxylation step competitively and the decarboxylation step noncompetitively. These types of inhibition fitted well with the overall lactate metabolism of the non-carbohydrate-fermenting anaerobe.