Manipulating pyruvate decarboxylase by addition of enzyme regulators during fermentation of Rhizopus oryzae to enhance lactic acid production.

Ethanol was found as the major by-product in lactate fermentation by Rhizopus oryzae. Several methods have been conducted in order to limit ethanol formation, thus increasing the lactate yield. The direct way to suppress ethanol production can be done by inhibition of the responsible enzymes in the related pathway. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are responsible for ethanol production in R. oryzae. Shunting the ethanol production pathway by targeting at PDC was attempted in this study. Three compounds including 4-methylpyrazole, glyoxylic acid, and 3-hydroxypyruvate with the in vitro reversible inhibitory effect on PDC were selected from the literature and were used to regulate the living cell of R. oryzae during the fermentation. The results show that 0.1 mM 4-methylpyrazole of which the structure resembled a thiazolium ring in thiamine diphosphate, PDC cofactor, and 1.0 µm 3-hydroxypyruvate, pyruvate analog, effectively hampered ethanol production. Further observation on the enzyme expression indicated that these two regulators not only targeted PDC but also caused changes in ADH and lactate dehydrogenase (LDH) activities. This was perhaps due to the living cell of R. oryzae that responded to the presence of the regulators to balance the pyruvate flux and subsequently maintain its metabolic activities.

The microRNA miR-696 regulates PGC-1{alpha} in mouse skeletal muscle in response to physical activity.

MicroRNAs (miRNAs) are small noncoding RNAs involved in posttranscriptional gene regulation that have been shown to be involved in growth, development, function, and stress responses of various organs. The purpose of this study was to identify the miRNA response to physical activity, which was related to functions such as nutrient metabolism, although the miRNAs involved are currently unknown. C57BL/6 mice were divided into exercise and control groups. The exercise group performed running exercise, with a gradual increase of the load over 4 wk. On the other hand, to examine the effect of muscle inactivity, the unilateral hindlimbs of other mice were fixed in a cast for 5 days. Microarray analysis for miRNA in gastrocnemius revealed that miR-696 was markedly affected by both exercise and immobilization, showing opposite responses to these two interventions. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which was increased by exercise and decreased by immobilization in the protein level, was predicted as a target regulated by miR-696. In cultured myocytes, intracellular miR-696 variation led to negative regulation of PGC-1alpha protein along with the expression of mRNAs for downstream genes. In addition, we found decreases in the biogenesis of mitochondria and fatty acid oxidation in miR-696-overexpressing myocytes compared with normal control myocytes. These observations demonstrate that miR-696 is a physical activity-dependent miRNA involved in the translational regulation of PGC-1alpha and skeletal muscle metabolism in mice.

Synthesis and biological evaluation of pyrophosphate mimics of thiamine pyrophosphate based on a triazole scaffold.

Novel triazole-based pyrophosphate analogues of thiamine pyrophosphate (TPP) have been synthesised and tested for inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis. The thiazolium ring of thiamine was replaced by a triazole in an efficient two-step procedure. Pyrophosphorylation then gave extremely potent triazole inhibitors with K(I) values down to 20 pM, compared to a K(D) value of 0.35 microM for TPP. This triazole scaffold was used for further investigation and six analogues containing mimics of the pyrophosphate group were synthesised and tested for inhibition of PDC. Several effective analogues were found with K(I) values down to around 1 nM.

Inhibition of pyruvate decarboxylase from Z. mobilis by novel analogues of thiamine pyrophosphate: investigating pyrophosphate mimics.

Replacement of the thiazolium ring of thiamine pyrophosphate with a triazole gives extremely potent inhibitors of pyruvate decarboxylase from Z. mobilis, with K(I) values down to 20 pM; this system was used to explore pyrophosphate mimics and several effective analogues were discovered.

Inhibition of thiamin diphosphate dependent enzymes by 3-deazathiamin diphosphate.

3-Deazathiamin diphosphate (deazaTPP) and a second thiamin diphosphate (TPP) analogue having a benzene ring in place of the thiazolium ring have been synthesised. These compounds are both extremely potent inhibitors of pyruvate decarboxylase from Zymomonas mobilis; binding is competitive with TPP and is essentially irreversible even though no covalent linkage is formed. DeazaTPP binds approximately seven-fold faster than TPP and at least 25,000-fold more tightly (K(i) less than 14 pM). DeazaTPP is also a potent inhibitor of the E1 subunit of alpha-ketoglutarate dehydrogenase from E. coli and binds more than 70-fold faster than TPP. In this case slow reversal of the inhibition could be observed and a K(i) value of about 5 nM was calculated (ca. 500-fold tighter binding than TPP).

Catalytic acid-base groups in yeast pyruvate decarboxylase. 1. Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions.

The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods. The steady-state kinetic constants were determined in the pH range of activity for the enzyme. The substitutions result in large changes in k(cat) and k(cat)/S(0.5) (and related terms), indicating that all four groups have a role in transition state stabilization. Furthermore, these results also imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release). With the exception of some modest effects, the shapes of neither the bell-shaped k(cat)/S(0.5)-pH (and related functions) plots nor the k(cat)-pH plots are changed by the substitutions. Yet, the fractional activity still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing the C2H. There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions. These results strongly imply that the base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction. The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.

Catalytic acid-base groups in yeast pyruvate decarboxylase. 3. A steady-state kinetic model consistent with the behavior of both wild-type and variant enzymes at all relevant pH values.

The widely quoted kinetic model for the mechanism of yeast pyruvate decarboxylase (YPDC, EC 4.1.1.1), an enzyme subject to substrate activation, is based on data for the wild-type enzyme under optimal experimental conditions. The major feature of the model is the obligatory binding of substrate in the regulatory site prior to substrate binding at the catalytic site. The activated monomer would complete the cycle by irreversible decarboxylation of the substrate and product (acetaldehyde) release. Our recent kinetic studies of YPDC variants substituted at positions D28 and E477 at the active center necessitate some modification of the mechanism. It was found that enzyme without substrate activation apparently is still catalytically competent. Further, substrate-dependent inhibition of D28-substituted variants leads to an enzyme form with nonzero activity at full saturation, requiring a second major branch point in the mechanism. Kinetic data for the E477Q variant suggest that three consecutive substrate binding steps may be needed to release product acetaldehyde, unlikely if YPDC monomer is the minimal catalytic unit with only two binding sites for substrate. A model to account for all kinetic observations involves a functional dimer operating through alternation of active sites. In the context of this mechanism, roles are suggested for the active center acid-base groups D28, E477, H114, and H115. The results underline once more the enormous importance that both aromatic rings of the thiamin diphosphate, rather than only the thiazolium ring, have in catalysis, a fact little appreciated prior to the availability of the 3-dimensional structure of these enzymes.

Is a hydrophobic amino acid required to maintain the reactive V conformation of thiamin at the active center of thiamin diphosphate-requiring enzymes? Experimental and computational studies of isoleucine 415 of yeast pyruvate decarboxylase.

The residue I415 in pyruvate decarboxylase from Saccharomyces cerevisiae was substituted with a variety of uncharged side chains of varying steric requirements to test the hypothesis that this residue is responsible for supporting the V coenzyme conformation reported for this enzyme [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. Changing the isoleucine to valine and threonine decreased the kcat value and shifted the kcat-pH profile to more alkaline values progressively, indicating that the residue at position 415 not only is important for providing the optimal transition state stabilization but also ensures correct alignment of the ionizable groups participating in catalysis. Substitutions to methionine (the residue used in pyruvate oxidase for this purpose) or leucine (the corresponding residue in transketolase) led to greatly diminished kcat values, showing that for each thiamin diphosphate-dependent enzyme an optimal hydrophobic side chain evolved to occupy this key position. Computational studies were carried out on the wild-type enzyme and the I415V, I415G, and I415A variants in both the absence and the presence of pyruvate covalently bound to C2 of the thiazolium ring (the latter is a model for the decarboxylation transition state) to determine whether the size of the side chain is critically required to maintain the V conformation. Briefly, there are sufficient conformational constraints from the binding of the diphosphate side chain and three conserved hydrogen bonds to the 4'-aminopyrimidine ring to enforce the V conformation, even in the absence of a large side chain at position 415. There appears to be increased coenzyme flexibility on substitution of Ile415 to Gly in the absence compared with the presence of bound pyruvate, suggesting that entropy contributes to the rate acceleration. The additional CH3 group in Ile compared to Val also provides increased hydrophobicity at the active center, likely contributing to the rate acceleration. The computational studies suggest that direct proton transfer to the 4'-imino nitrogen from the thiazolium C2H is eminently plausible.

Reactivity at the substrate activation site of yeast pyruvate decarboxylase: inhibition by distortion of domain interactions.

The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered [Baburina et al. (1994) Biochemistry 33, 5630-5635] and is located on the beta domain [Arjunan et al. (1996) J. Mol. Biol. 256, 590], while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains. The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors. Protein chemical methods, in conjunction with [1-14C] and [3-3H] analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14. However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme. Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol. Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes. Other potential nucleophilic sites for the inhibitor [C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site] were also substituted by a nonnucleophilic side chain. All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process. It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound [1-14C]-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site. Upon combining the tritiated inhibitor with [2-14C]-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein. It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol. Among other known activators, ketomalonate was found to react at C221 only. Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center. The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme [forming a Cys221S(-) + HHis92 ion pair; see Baburina et al. (1996) Biochemistry 35, 10249-10255, and Baburina et al. (1998) Biochemistry 37, 1235-1244]. Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone. All three compounds reduced the Hill coefficient and hence appear to react at C221. It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation.

Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess.

In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.

[Inactivation of yeast pyruvate decarboxylase in the presence of substrate and quinone electron acceptors]. / Inaktivatsiia drozhzhevoi piruvatdekarboksilazy v prisutstvii substrata i khinonovykh aktseptorov élektronov.

The rate constants of paracatalytic inactivation of pyruvate decarboxylase in the presence of 1,4-naphthoquinones and 1,4-benzoquinones are determined by redox potentials of the oxidant. The logarithm of k2 depends hyperbolically on the redox potential of quinone E0(Q/Q-.) with the coefficient of proportionality which approximates 8.4, The absence of considerable deviations in this correlation for the oxidants of different structures with closed values Eo(Q/Q-.) indicates that the enzyme produces no additional steric barrier.

[The interaction of pyruvate decarboxylase with the quinone-ferricyanide oxidative system]. / Vzaimodeistvie piruvatdekarboksilazy s okislitel'noi sistemoi khinon--ferritsianid.

Inactivation of yeast pyruvate decarboxylase in the presence of substrate and oxidative system containing substituted quinone and ferricyanide has been investigated. It was established that ferricyanide at pH 5.2-6.4 can prevent irreversible inactivation of the pyruvate decarboxylase caused by the concerted action of pyruvate and substituted quinone. The influence of ferricyanide which depends on the redox potential of the substituted quinone is decreasing in a series tetramethyl-p-benzoquinone, trimethyl-p-benzoquinone, 2-methyl-5-isopropyl-p-benzoquinone. It is supposed that the effect of the oxidative system partially converting the nonoxidative to oxidative function of pyruvate decarboxylase is attributed to the oxidation of active acetaldehyde by substituted quinone and reaction of resultant semiquinone radical with ferricyanide.

A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. Competitive epitope mapping studies.

Presented here are competitive epitope mapping studies on a monoclonal antibody library to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC1.2.4.1) subunit (E1). Several of the monoclonal antibodies had been found to inhibit PDHc from 0 to 98%. Of the 10 monoclonal antibodies that showed the greatest inhibition of PDHc, 4 were elicited by PDHc and 6 by E1. Surface plasmon resonance was used for competitive epitope mapping and revealed that these 10 monoclonal antibodies had at least 6 separate binding regions on the PDHc. The three monoclonal antibodies that demonstrated the strongest inhibition appeared to bind the same region on the PDHc. Mapping studies with the E1 antigen using an additional five monoclonal antibodies demonstrated that the two strongest inhibitory monoclonal antibodies (18A9 and 21C3) shared the same binding region on E1, whereas the third strongest inhibitor (15A9) displayed an epitope region that overlapped the previous two on the E1 subunit. Antibody 15A9 had been shown to counteract GTP regulation of PDHc. Simultaneous multiple site binding experiments confirmed that the defined epitope regions were indeed independent. Limited competitive epitope binding experiments using radiolabeled E1 confirmed the surface plasmon resonance results.

A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. A biochemical analysis and their ability to inhibit the enzyme complex.

A library of monoclonal antibodies to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC 1.2.4.1; E1) subunit is reported. 21 monoclonal antibodies were generated, and 20 were investigated, of which 9 were elicited to PDHc and 11 to pure E1 subunit; 19 were of the IgG1 isotype and one of the IgG3 isotype. According to an enzyme immunoassay, all 20 of the monoclonal antibodies bound the PDHc, and 17 bound the E1 subunit. According to Western blot analysis, 14 of the 19 monoclonal antibodies bound to the E1 subunit. The monoclonal antibodies inhibited PDHc from 0 to > 98%. The six monoclonal antibodies that displayed greater than 30% inhibition of E. coli PDHc were unable to inhibit porcine heart PDHc nor did they bind porcine heart PDHc according to dot blot analysis. Radiolabeling gave binding constants ranging from 5 to 10 x 10(8) M-1 on these six monoclonal antibodies, with greater than 80% of maximal inhibition achieved in less than 1 min. One of the six, 18A9, gave > 98% inhibition, required two antibodies/E1 subunit for maximum inhibition, and was shown to be a non-competitive inhibitor. Monoclonal antibody 15A9 was shown to counteract GTP-induced inhibition, while 1F2 influenced the conformation of E1, allowing two antibodies, which did not previously bind E1, to bind to it. A new mechanism-based kinetic assay is presented that is specific for the E1 component of 2-keto acid dehydrogenases. This assay confirmed that the three most strongly inhibitory monoclonal antibodies specifically inhibited the E1 function while antibody 1F2 led to enhanced activity, suggesting an induced conformational change in PDHc or in E1.

Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.

Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

[Paracatalytic inactivation of pyruvate decarboxylase in the presence of quinones]. / Parakataliticheskaia inaktivatsiia piruvatdekarboksilazy v prisutstvii khinonov.

Pyruvate promotes the yeast pyruvate decarboxylase inactivation under the influence of substituted p-benzoquinones. Pyruvate decarboxylase activity is not renewed after the removal of low-molecular impurities by gel filtration and subsequent addition of dithiothreitol, thiamine diphosphate, magnesium chloride. The inactivation rate under joint action of 2-methyl-5-isopropyl-p-benzoquinone and pyruvate is regulated by the pseudo-first-order equation. The relationship between pseudo-first-order rate constant and pyruvate concentration takes the shape of hyperbola. The inactivation order with respect to quinone is determined by oxidant concentration and pH value. Maximum pseudo-first-order rate constant values in the presence of the excess substrate and 2-methyl-5-isopropyl-p-benzoquinone are observed at pH 5.9-6.0. The data obtained evidence for the fact that during inactivation quinone interacts with "active acetaldehyde" being the intermediate in the process of catalysis with pyruvate decarboxylase.

[Mechanism of interaction between quinones and 2alpha-carbanion in the active center of pyruvate decarboxylase]. / Mekhanizm vzaimodeistviia khinonov s 2alpha-karbanionom v aktivnom tsentre piruvatdekarboksilazy.

The kinetics of paracatalytic inactivation of pyruvate decarboxylase by joint action of the substrate and exogenous oxidant p-benzoquinone, methyl-p-benzoquinone, 2-methyl-5-isopropyl-p-benzoquinone, trimethyl-p-benzoquinone, tetramethyl-p-benzoquinone has been investigated. Nonlinear correlation between the observed second-order rate constants and redox potentials of quinones has been found. It is supposed that negative deviations from linear dependence was caused by changing the rate determining step by the interaction of quinone with 2 alpha-carbanion in the active centre of pyruvate decarboxylase. According to structure of the oxidant the inactivation rate is limited by one electron transfer or the following protonating of the formed anion radical of quinone.

Inhibition of transketolase and pyruvate decarboxylase by omeprazole.

Omeprazole inhibited two thiamin diphosphate-dependent enzymes, pyruvate decarboxylase (EC 4.1.1.1, PDC) from Zymomonas mobilis and transketolase (EC 2.2.1.1, TK) from human erythrocytes. Inhibition of PDC was competitive with the coenzyme with a Ki value of 42 +/- 3 microM, whereas inhibition of TK was complex.

[Inhibition of yeast pyruvate decarboxylase by alkyl phosphates]. / Ingibirovanie drozhzhevoi piruvatdekarboksilazy alkilfosfatami.

It is found that yeast pyruvate decarboxylase is inhibited by alkyl phosphates. Inhibition is competitive with respect to a substrate. The inhibition constants with n-butyl and n-heptyl esters of phosphoric acid are the values of the same order of magnitude. With an increase in the length of the alkyl phosphates hydrocarbon chain from 7 to 10 carbon atoms inhibition constants change drastically. For n-heptyl phosphate and n-decyl phosphate values KI are equal to 1.6 x 10(-4) M and 1.7 x 10(-6) M, respectively. A further increase in the number of carbon atoms in the alkyl substituent of phosphoric acid ester induces no reduction of the inhibition constant. Multiple-inhibitor experiments of pyruvate decarboxylase show that inorganic phosphate and n-decyl ester of phosphoric acid are mutually exclusive. It is suggested that the inhibition mechanism with alkyl phosphates includes the competition of the phosphoric acid residue with alpha-ketocarboxyl group of pyruvate as well as the interaction between a hydrocarbon radical and hydrophobic parts on the enzyme surface, one of them being outside the substrate binding site.

Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi.

Pyruvate decarboxylase from the obligate anaerobe Sarcina ventriculi was purified eightfold. The subunit Mr was 57,000 +/- 3000 as estimated from SDS-PAGE, and the native Mr estimated by gel filtration on a Superose 6 column was 240,000, indicating that the enzyme is a tetramer. The Mr values are comparable to those for pyruvate decarboxylase from Zymomonas mobilis and Saccharomyces cerevisiae, which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6.3-6.7. It displayed sigmoidal kinetics for pyruvate, with a S0.5 of 13 mM, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from Z. mobilis. No activators were found. p-Chloromercuribenzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of S. cerevisiae than Z. mobilis pyruvate decarboxylase.