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PubMed2001

Impairment of pyruvate dehydrogenase activity by acetaldehyde.

The facial features that are characteristic of fetal alcohol syndrome (FAS) are strikingly similar to those seen in pyruvate dehydrogenase (PDH) deficiency. Furthermore, alcohol-induced central nervous system insult results in midline anomalies such as agenesis of the corpus callosum, which has also been described in several metabolic diseases, including PDH deficiency. The purpose of this work was to examine the effect of acetaldehyde on PDH in vitro. The activity of PDH was measured in the presence of acetaldehyde (10 microM-1 mM) by measuring the formation of the reduced form of nicotinamide-adenine dinucleotide at 340 nm. Pyruvate dehydrogenase was separated by using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique after incubation with [1,2-(14)C]-acetaldehyde to detect the formation of covalent adducts autoradiographically. The effect of acetaldehyde on the phosphorylation of the complex was also determined autoradiographically after incubating of PDH with (32)P-adenosine triphosphate. The results of this study show that acetaldehyde impairs PDH activity by a mixed inhibition type mechanism (Kic=62.4+/-25.7 microM, Kiu=225+/-68 microM), which is not a result of the formation of covalent adducts with PDH, nor of a stimulation of phosphorylation or inactivation of the complex. Because PDH levels are low throughout development and that the competition between pyruvate and acetaldehyde may be enhanced due to ethanol-induced lowering of ambient pyruvate concentrations, we conclude that impairment of PDH may have a significant effect on the developing fetus.

Acetaldehyde↗
PubMed2003

Sodium pyruvate protects against H(2)O(2) mediated apoptosis in human neuroblastoma cell line-SK-N-MC.

Free radicals are involved in neuronal damage. The present study was aimed to investigate the protective effect of sodium pyruvate-a free radical scavenger against hydrogen peroxide (H(2)O(2)) induced apoptosis in human neuroblastoma cell line-SK-N-MC. On exposure to H(2)O(2) (0.025 mM) cells exhibited apoptosis within 24 h, demonstrating a high caspase 3 activity by 3 h followed by cleavage of PARP that was maximum at 24 h. A break down in the mitochondrial membrane potential was observed 3 h onwards. Sodium pyruvate protected cells significantly (P<0.05) against apoptosis in a dose dependent manner as assessed for cell viability by dye exclusion method and apoptosis by TUNEL. Sodium pyruvate significantly inhibited caspase 3 activity, cleavage of PARP and breakdown of mitochondrial membrane potential. These data suggest that sodium pyruvate protects neuronal damage caused by H(2)O(2).

Antioxidants↗
PubMed1997

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide.

Instability of hydrogen peroxide solutions was noted during the experimental exposure of human cells in culture to hydrogen peroxide in experiments designed to study the production and repair of DNA single-strand breaks. A hydrogen peroxide concentrate was diluted into culture medium, which was then added to experimental cell cultures at various times, with all cultures assessed for DNA damage at 2 h. Only cells treated by the first addition had observable DNA damage. This result was unexpected since these cells had had the maximum repair time. It was determined that the hydrogen peroxide had been eliminated by the culture medium. To determine the mechanism of this elimination, 200 microM hydrogen peroxide was added to various cell culture components, and the solutions were assayed for hydrogen peroxide after 1 h at 37 degrees C. Although most components (except the balanced salts) showed some hydrogen peroxide degradation, it was found that sodium pyruvate was most effective, by a wide margin, in eliminating hydrogen peroxide and its toxic effects. This was confirmed by addition of pyruvate to balanced salt solutions or buffers, and observing the same elimination of hydrogen peroxide. We subsequently found a few earlier reports describing the decarboxylation reaction between hydrogen peroxide and pyruvate, but no kinetic measurements have been published and there seems to be no general appreciation for the very high efficiency of this reaction. The present work presents a preliminary assessment of the importance of pyruvate in the study of hydrogen peroxide and other reactive oxygen species in mammalian cell culture.

Azides↗
PubMed1999

The linkage of catalysis and regulation in enzyme action: oxidative diversion in the hysteretically regulated yeast pyruvate decarboxylase.

The reaction catalyzed by the thiamin-diphosphate-dependent yeast pyruvate decarboxylase, which is hysteretically regulated by pyruvate, undergoes paracatalytic oxidative diversion by 2,6-dichlorophenolindophenol, which traps a carbanionic intermediate and diverts the product from acetaldehyde to acetate (Christen, P. Meth. Enzymol. 1977, 46, 48). This reaction is now shown to exhibit an oxidant on-rate constant somewhat faster than that for pyruvate in the normal catalytic cycle and a product off-rate constant about 60-fold smaller than that for acetaldehyde. Both on-rates and off-rates exhibit an inverse solvent isotope effect of 1.5-2, observed in normal catalysis as a signal of sulfhydryl addition to the keto group of pyruvate at the allosteric regulatory site. The findings are consistent with a model for regulation in which the sulfhydryl-addition process mediates access to a fully catalytically competent active site, the oxidative-diversion reaction being forced to make use of the normal entry exit machinery.

2,6-Dichloroindophenol↗
PubMed1976

Pyruvate metabolism in Friedreich's ataxia.

Friedreich's ataxia patients show evidence of an abnormally elevated and prolonged response of pyruvate and lactate to a glucose load, with normal fasting levels. However, ther is a bimodal distribution of this response with high and low pyruvate responders. This trait appears to be determined genetically, However, although in vivo tests suggest low oxidation of pyruvate, we were unable to confirm any in vitro impairment of each of the components of the pyruvate dehydrogenase (PDH) complex. We conclude that the defect is in the metabolic regulation of PDH, probably at the E3 (lipoamide dehydrogenase) step.

Alanine Transaminase↗
PubMed1993

Electron nuclear double resonance study of the Mn2+ environs in the oxalate-ATP complex of pyruvate kinase.

Electron nuclear double resonance (ENDOR) and the related pulse technique of pulse field sweep EPR (PFSEPR) were used to probe the site I environment of Mn2+ in the oxalate-ATP complex of pyruvate kinase. Assignment of features and an estimate of hyperfine couplings have shown proximity of protons to the metal ions through their dipolar interaction and proximity of 31P and 17O because of a contact interaction from direct Mn(2+)-ligand covalent spin transfer. Since Mn2+ is a spin5/2 ion whose Ms = +/- 1/2, +/- 3/2, and +/- 5/2 electron spin states can all contribute to EPR and ENDOR, we have developed experimental and theoretical strategies for elucidating hyperfine couplings to the Mn2+ electron spin states. Solvent-exchangeable proton ENDOR features were evident with couplings very similar to the hyperfine couplings of H2O in [Mn(H2O)6]2+. ENDOR of exchangeable, more distant protons originated from a dipolar coupling such as could be expected from protons residing 5.5 A from Mn2+ and hydrogen-bonded to a nonliganding oxygen or nitrogen. Nonexchangeable proton ENDOR features indicated dipolar coupling to proton(s) from the protein residing at approximately 4.5 A from the Mn2+. The approximately 4-MHz 31P phosphate hyperfine couplings in Mn(II)-nucleotide models and in pyruvate kinase were similar, but a detailed ENDOR and PFSEPR comparison revealed that the hyperfine coupling to the ATP gamma-phosphate in pyruvate kinase was approximately 10% less than coupling to phosphates of Mn(II)-nucleotides. [In pyruvate kinase only the gamma-phosphate has been shown to bind to Mn2+ at site I (Lodato & Reed, 1987).](ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1991

Minimum requirements for protease activation of flavin pyruvate oxidase.

Previous investigations have shown that the catalytic efficiency (kcat/KM) of pyruvate oxidase can be enhanced 450-fold by chymotryptic cleavage of a 23-residue peptide (alpha-peptide) from the carboxy terminus of the enzyme. The minimum requirement for proteolytic activation has been investigated by exposing pyruvate oxidase to a variety of carboxypeptidases, either singly or in combination. The extent of carboxypeptidase hydrolysis was followed by analyzing the release of amino acids and by mass spectral analysis of the truncated alpha-peptides which were derived from the carboxypeptidase-treated preparations. The results indicate that the removal of 7 carboxy-terminal residues does not activate the enzyme whereas the removal of 10 or 11 residues produces activated pyruvate oxidase. Activation of pyruvate oxidase by endoproteinase Glu-C confirms the carboxypeptidase results. Endoproteinase Glu-C specificity predicts hydrolytic cleavage of the peptide bond between Glu-561 and Val-562 with the removal of 11 residues from the carboxy terminus of the enzyme.

Amino Acid Sequence↗
PubMed1983

Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate.

(E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.

Binding Sites↗
PubMed1984

Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss.

A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.

Acetaldehyde↗
PubMed1986

Identification of the high-affinity lipid binding site in Escherichia coli pyruvate oxidase.

Pyruvate oxidase from Escherichia coli is a peripheral membrane associated enzyme which is activated by lipids. We have investigated the high-affinity lipid binding site associated with lipid activation of pyruvate oxidase by covalent attachment of [14C]lauric acid to the enzyme. Lauric acid is bound stoichiometrically (1 mol/mol of active sites), and the enzyme is essentially irreversibly activated. Mild tryptic digestion of the modified enzyme shows that the lauric acid is bound within the last 100 residues of the 572-residue monomer. Digestion with thermolysin releases two closely related peptides, A and B, in approximately equal amounts. Comparison of the amino acid composition of peptide A with the entire sequence of the protein shows that peptide A corresponds to the sequence from Ala-543 to Ile-554. The analysis of peptide B is very similar to that of A. Limited sequence analysis of peptide B shows that residue 1 is Ala and residue 2 is labeled. These results support the assignment of residue 1 in peptide B as Ala-543 and indicate that lauric acid is bound to Lys-544. Previous work in this laboratory has shown that pyruvate oxidase may be activated independently of lipids by mild protease digestion. Proteolytic activation is accompanied by the release of a small peptide (residues 550-572) from the carboxyl terminus of the protein. The present work locates the lipid binding site very close to this peptide. The significance of these results for the mechanism of activation of pyruvate oxidase and other lipid-activated systems is discussed.

Amino Acid Sequence↗
PubMed1986

Carbon-13 and deuterium isotope effects on oxalacetate decarboxylation by pyruvate carboxylase.

Deuterium and 13C isotope effects for the enzymic decarboxylation of oxalacetate showed that both deuterium- and 13C-sensitive steps in the reaction are partially rate limiting. A normal alpha-secondary effect of 1.2 per deuterium was calculated for the reaction in which pyruvate-d3 was the substrate, suggesting that the enolate of pyruvate was an intermediate in the reaction. The large normal alpha-secondary deuterium isotope effect of 1.7 when oxalacetate-d2 was the substrate suggests that the motions of the secondary hydrogens are coupled to that of the primary hydrogen during the protonation of the enolate of pyruvate. The reduction in the magnitude of the 13C isotope effect for the oxamate-dependent decarboxylation of oxalacetate from 1.0238 to 1.0155 when the reaction was performed in D2O (primary deuterum isotope effect = 2.1) clearly indicates that the transfer of the proton and carboxyl group between biotin and pyruvate does not occur via a single concerted reaction. Mechanisms in which biotin is activated to react with CO2 (prior to transfer of the proton on N-1) by bond formation between the sulfur and the ureido carbon, or in which the sequence of events is decarboxylation of oxalacetate, proton transfer from biotin to enolpyruvate, and carboxylation of enolbiotin, predict that the 13C isotope effect in D2O should be substantially lower than the observed value. A stepwise mechanism that does fit the data is one in which a proton is removed from biotin by a sulfhydryl group on the enzyme prior to carboxyl transfer, as long as the sulfhydryl group has an abnormally low pK.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Modification of two essential cysteines in rabbit muscle pyruvate kinase by the guanine nucleotide analogue 5'[p-(fluorosulfonyl) benzoyl] guanosine.

Reaction of rabbit muscle pyruvate kinase with the affinity label 5'-[p-(fluorosulfonyl) benzoyl] guanosine (5'-FSBG), at pH 7.65 and 7.93, leads to a loss in enzyme activity. The inactivation is characterized by a biphasic kinetic profile, with the initial phase accounting for approximately 55% of the reduction in enzymatic activity. For both the rapid and slow phases, at pH 7.93, the inactivation rate constants are linearly proportional to the reagent concentration (from 0.48 to 3.0 mM), yielding second-order rate constants of 195 min-1 M-1 and 19 min-1 m-1, respectively. The effect of ligands was tested on the two phases of inactivation. For both, a decrease in the inactivation rate was produced by Mg2+ alone, but the best protection was provided by Mg2+ plus either ADP or GDP, suggesting that the reaction occurs in the region of the metal-nucleotide binding site. Modified pyruvate kinase is completely reactivated by incubation with 20 mM dithiothreitol, indicating the involvement of cysteine in the inactivation, indicating the involvement of cysteine in the inactivation process. Reaction with [5'=3H]-5'-FSBG leads to the incorporation of up to 1.3 mol of radioactive reagent per mol of enzyme subunit; however, identical radiolabel incorporation is observed before or after dithiothreitol reactivation of modified enzyme. This result implies that the labeled amino acid residue, measured by means of incorporation, is not directly involved in the inactivation process. In contrast, inactivation was found to correlate well with the loss of two free sulfhydryl groups per enzyme subunit and the restoration of activity to correlate with the regeneration of two free sulfhydryls after treatment of modified enzyme with dithiothreitol. It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide. A negative cooperatively in the interaction of pyruvate kinase subunits with 5'-[p-(fluorosulfonyl)-benzoyl] guanosine might best account for the biphasic inactivation kinetics.

Adenosine Diphosphate↗
PubMed1980

Elementary steps in the reaction mechanism of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: kinetics of acetylation and deacetylation.

The kinetics of the acetylation of the pyruvate dehydrogenase complex from Escherichia coli by [3-14C]pyruvate and of the deacetylation of the complex by coenzyme A have been studied by using rapid mixing-quench techniques. The time course for acetylation in 4 mM thiamin pyrophosphate, 2 mM MgSO4, and 0.02 M potassium phosphate (pH 7.0) at 4 degrees C can be analyzed in terms of two kinetic processes. At long times 10 nmol of acetyl groups is incorporated per mg of enzyme complex (48 sites per complex of molecular weight 4.8 X 10(6)). The slower process is much too slow to be of catalytic significance. The rate constant for the faster process is not dependent on enzyme concentration and reaches a limiting value of 40--65 s-1 at high pyruvate concentrations; the exact value is dependent on the detailed acetylation mechanism assumed. The minimum molar turnover number of the enzyme complex is 420 s-1 (17.5 s-1 per pyruvate decarboxylase). The acetylated lipoic acids are deacetylated by coenzyme A at a rate much faster than that of acetylation. Complete deacetylation is obtained only if the deacetylation is carried out within seconds of the acetylation, apparently because dead-end intramolecular transfers of acetyl groups from the lipoic acids to other functional groups on the enzyme not essential for catalytic activity can occur. The results obtained suggest only about half of the acetylation reactions are on the main catalytic pathway.

Acetylation↗
PubMed1975

Stereochemistry of propionyl-coenzyme A and pyruvate carboxylations catalyzed by transcarboxylase.

The stereochemistry of the two half-reactions catalyzed by the biotin-containing enzyme, transcarboxy-lase from Propionobacteria shermanii, has been determined. The pro-R hydrogen at C-2 of propionyl-coenzyme A is replaced by CO2 in formation of the S isomer of methylmalonyl-CoA, defining the process as retention of configuration. This C-2 hydrogen is abstracted at a rate identical with product formation. For the other half-reaction, pyruvate to oxalacetate, the chiral methyl group methodology of Rose (I. A. Rose (1970), J. Biol. Chem. 245, 6052) was employed. First, it was determined with [3-2-He]pyruvate that a kinetic deuterium isotope effect of 2.1 occurs at Vmax in this carboxyl transfer, indicating that the necessary requirement for discrimination against heavy isotopes of hydrogen existed. Then, 3(S)-[3-2-H,3-H]pyruvate, generated from 3(S)-]E-2-H,3-H]phosphoglycerate, was carboxylated and the oxalacetate trapped as [3030H]malate using malate dehydrogenase. Exhaustive incubation of the tritiated malate (3-H/14-C = 1.95) with fumarase to labilize the pro-R hydrogen at C-3 resulted in release of 65% of the tritium into water. Reisolation of the malate after fumarase action yielded a 30H/14-C ration of 0.67, indicating 34% retention as expected. The theoretical enantiotopic distribution for the observed k1H/k2H of 2.1 is 68:32. Selective enrichment of tritium in the pro-R position at C-3 of malate indicates enzymatic carboxylation of pyruvate with retention of configuration in this half-reaction also.

Carboxy-Lyases↗
PubMed1997

Refolding and reconstitution studies on the transacetylase-protein X (E2/X) subcomplex of the mammalian pyruvate dehydrogenase complex: evidence for specific binding of the dihydrolipoamide dehydrogenase component to sites on reassembled E2.

Reconstitution studies have been conducted on the dihydrolipoamide acetyltransferase-protein X core subcomplex of the mammalian pyruvate dehydrogenase complex. GdnHCl-induced dissociation of this core is an ordered cooperative event involving formation of specific lower-Mr intermediates corresponding to dihydrolipoamide acetyltransferase trimers and monomers. Recovery profiles of the dihydrolipoamide acetyltransferase-protein X core, unfolded in 6 M GdnHCl prior to the removal of denaturant by either (a) slow dialysis or (b) rapid dilution, demonstrated rapid initial reappearance of substantial levels of dihydrolipoamide acetyltransferase activity with complete recovery occurring in 4-6 h. Immunological analysis of reconstituted cores revealed reduced levels of protein X (approximately 30-35%) after slow dialysis and the total absence of this component following rapid dilution. The dihydrolipoamide acetyltransferase core, devoid of protein X, was unable to sustain overall complex activity when reconstituted with stoichiometric amounts of its companion pyruvate decarboxylase and dihydrolipoamide deydrogenase components, whereas the protein X-depleted core could sustain 30-35% of control activity. Further reconstitution analyses of overall complex function with these two types of reassembled core structures in the presence of excess dihydrolipoamide dehydrogenase (100-fold) demonstrated significant additional stimulation of pyruvate dehydrogenase complex activity (25-30%) which was dependent on the source of exogenous dihydrolipoamide dehydrogenase. Thus, this constituent enzyme can interact directly with the dihydrolipoamide acetyltransferase oligomer with low affinity in addition to its normal high-affinity binding to the protein X subunit. These results provide definitive in vitro evidence in support of recent clinical observations reporting residual pyruvate dehydrogenase activity (10-20%) in cell lines derived from patients lacking protein X.

Acetyltransferases↗
PubMed1997

Sulfhydryl chemistry detects three conformations of the lipid binding region of Escherichia coli pyruvate oxidase.

Site-specific disulfide cross-linking experiments detected a conformational change within the C-terminal segment of Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, upon substrate binding [Chang, Y.-Y., & Cronan, J. E., Jr. (1995) J. Biol. Chem. 270, 7896-7901]. The C-terminal lipid binding regions were cross-linked only in the presence of the substrate, pyruvate, and the thiamine pyrophosphate cofactor, indicating close proximity of a pair of C termini. We have now systematically substituted cysteine at 18 additional amino acid positions within the C-terminal region to obtain a panel of 21 proteins each having a single residue changed to cysteine. These proteins have been studied by disulfide cross-linking and by accessibility of the cysteine side chain to a variety of sulfhydryl agents. In the absence of pyruvate, the cysteine residues of the modified PoxB proteins failed to form disulfide bonds, generally failed to react with a large and rigid hydrophilic sulfhydryl reagent, 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid (IASD), and in some cases reacted weakly with a smaller more hydrophobic reagent, N-ethylmaleimide. Therefore, in this conformation, the C termini appear fixed in a rigid environment having limited exposure to solvent. In the presence of pyruvate, all of the C-terminal cysteine residues (except the two most distal from the C terminus) reacted with both sulfhydryl reagents and readily formed disulfide cross-linked species, indicating conversion to a structure having a high degree of conformational freedom. In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacted with the membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelles. For most of the proteins, similar extents of disulfide formation were seen upon addition of an oxidizing agent in the presence or absence of lipid activators. An exception was PoxB D560C which was much more readily cross-linked in the presence of lipid. Moreover, a subset of PoxB proteins that cross-linked to lower extents in the presence of lipids was found. The behavior of these proteins provides strong support for the model in which two C termini associate to form the functional lipid binding domain. These data are discussed in terms of three distinct PoxB conformers and the known crystal structure of a highly related protein.

Animals↗
PubMed1998

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.

Amino Acid Substitution↗