Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate.

Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.     

Catalytic acid-base groups in yeast pyruvate decarboxylase. 2. Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products.

Yeast pyruvate decarboxylase (YPDC), in addition to forming its metabolic product acetaldehyde, can also carry out carboligase reactions in which the central enamine intermediate reacts with acetaldehyde or pyruvate (instead of the usual proton electrophile), resulting in the formation of acetoin and acetolactate, respectively (typically, 1% of the total reaction). Due to the common mechanism shared by the acetaldehyde-forming and carboligase reactions through decarboxylation, a detailed analysis of the rates and stereochemistry of the carboligase products formed by the E477Q, D28A, and D28N active center YPDC variants was undertaken. While substitution at either position led to an approximately 2-3 orders of magnitude lower catalytic efficiency in acetaldehyde formation, the rate of acetoin formation by the E477Q and D28N variants was higher than that by wild-type enzyme. Comparison of the steady-state data for acetaldehyde and acetoin formation revealed that the rate-limiting step for acetaldehyde formation by the D28A, H114F, H115F, and E477Q variants is a step post-decarboxylation. In contrast to the wild-type YPDC and the E477Q variant, the D28A and D28N variants could synthesize acetolactate as a major product. The lower overall rate of side-product formation by the D28A variant than wild-type enzyme attests to participation of D28 in steps leading up to and including decarboxylation. The results also provide insight into the state of ionization of the side chains examined. (R)-Acetoin is produced by the variants with greater enantiomeric excess than by wild-type YPDC. (S)-Acetolactate is the predominant enantiomer produced by the D28-substituted variants, the same configuration as produced by the related plant acetolactate synthase.     

Paracoccus denitrificans PD1222 Utilizes Hypotaurine via Transamination Followed by Spontaneous Desulfination To Yield Acetaldehyde and,Finally, Acetate for Growth

Hypotaurine (HT; 2-aminoethane-sulfinate) is known to be utilized by bacteria as a sole source of carbon, nitrogen, and energy for growth, as is taurine (2-aminoethane-sulfonate); however, the corresponding HT degradation pathway has remained undefined. Genome-sequenced Paracoccus denitrificans PD1222 utilized HT (and taurine) quantitatively for heterotrophic growth and released the HT sulfur as sulfite (and sulfate) and HT nitrogen as ammonium. Enzyme assays with cell extracts suggested that an HT-inducible HT:pyruvate aminotransferase (Hpa) catalyzes the deamination of HT in an initial reaction step. Partial purification of the Hpa activity and peptide fingerprinting-mass spectrometry (PF-MS) identified the Hpa candidate gene; it encoded an archetypal taurine:pyruvate aminotransferase (Tpa). The same gene product was identified via differential PAGE and PF-MS, as was the gene of a strongly HT-inducible aldehyde dehydrogenase (Adh). Both genes were overexpressed in Escherichia coli. The overexpressed, purified Hpa/Tpa showed HT:pyruvate-aminotransferase activity. Alanine, acetaldehyde, and sulfite were identified as the reaction products but not sulfinoacetaldehyde; the reaction of Hpa/Tpa with taurine yielded sulfoacetaldehyde, which is stable. The overexpressed, purified Adh oxidized the acetaldehyde generated during the Hpa reaction to acetate in an NAD⁺-dependent reaction. Based on these results, the following degradation pathway for HT in strain PD1222 can be depicted. The identified aminotransferase converts HT to sulfinoacetaldehyde, which desulfinates spontaneously to acetaldehyde and sulfite; the inducible aldehyde dehydrogenase oxidizes acetaldehyde to yield acetate, which is metabolized, and sulfite, which is excreted.     

Mutagenesis at asp27 of pyruvate decarboxylase from Zymomonas mobilis. Effect on its ability to form acetoin and acetolactate.

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three-dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by-product from the hydroxyethyl-ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by-product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild-type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30-fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild-type. The time-courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild-type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild-type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild-type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.     

Redirection of the Reaction Specificity of a Thermophilic Acetolactate Synthase toward Acetaldehyde Formation

Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H⁺ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme.     

Coenzyme-binding sites of the brain pyruvate dehydrogenase complex]

It is shown that the relative amount of the holoenzyme in the highly purified pyruvate dehydrogenase complex from the bovine brain is higher when the enzyme activity is assayed in the reaction of nonoxidative formation of acetaldehyde as compared to the pyruvate: NAD+ reductase reaction. The S0.5 values for thiamine pyrophosphate are as following: (TPP) (0.314 +/- 0.22) x 10(-7) M with reaction of nonoxidative formation of acetaldehyde, (0.188 +/- 0.08) x 10(-6) M and (1.65 +/- 1.16) x 10(-6) M in case of the pyruvate: NAD+ reductase reaction. TPP in the concentration of (0.5-6.0) x 10(-7) M completely protects the sites of nonoxidative formation of acetaldehyde from modification by the coenzyme analogs, 4'-oxythiamine pyrophosphate and tetrahydrothiamine pyrophosphate. However, the pyruvate: NAD+ reductase activity of the pyruvate dehydrogenase complex is inhibited in this case by 30-34%. The data obtained suggest that in contrast to the pyruvate: NAD+ reductase reaction the conversion of pyruvate to acetaldehyde occurs by the sites which tightly bound TPP.     

Transient releases of acetaldehyde from tree leaves − products of a pyruvate overflow mechanism?

Emissions of acetaldehyde from tree leaves were investigated by proton‐transfer‐reaction mass spectrometry (PTR‐MS), a technique that allows simultaneous monitoring of different leaf volatiles, and confirmed by derivatization and high‐performance liquid chromatography analysis. Bursts of acetaldehyde were released by sycamore, aspen, cottonwood and maple leaves following light–dark transitions; isoprene emission served as a measure of chloroplastic processes. Acetaldehyde bursts were not accompanied by ethanol, but exposure of leaves to inhibitors of pyruvate transport or respiration, or anoxia, led to much larger releases of acetaldehyde, accompanied by ethanol under anoxic conditions. These same leaves have an oxidative pathway for ethanol present in the transpiration stream, resulting in acetaldehyde emissions that are inhibited in vivo by 4‐methylpyrazole, an alcohol dehydrogenase (Adh) inhibitor. Labelling of leaf volatiles with ¹³CO₂ suggested that the pools of cytosolic pyruvate, the proposed precursor of acetaldehyde bursts, were derived from both recent photosynthesis and cytosolic carbon sources. We hypothesize that releases of acetaldehyde during light–dark transitions result from a pyruvate overflow mechanism controlled by cytosolic pyruvate levels and pyruvate decarboxylase activity. These results suggest that leaves of woody plants contribute reactive acetaldehyde to the atmosphere under different conditions: (1) metabolic states that promote the accumulation of cytosolic pyruvate, triggering the pyruvate decarboxylase reaction; and (2) leaf ethanol oxidation resulting from ethanol transported from anoxic tissues.     

乳酸菌风味代谢物质的基因调控

乳酸菌的主要风味代谢物质包括丁二酮,乙醛以及各种氨基酸。利用基因工程和代谢工程的相关技术提高乙醛和丁二酮产量,是当前乳酸菌研究的热点之一。乙醛的代谢调控主要是针对丝氨酸羟甲基转移酶的表达进行调控,或是针对丙酮酸脱羧酶和NADH氧化酶的表达采用联合调控策略;而丁二酮的代谢调控则主要集中于乳酸脱氢酶、NADH氧化酶、α-乙酰乳酸合成酶和α-乙酰乳酸脱羧酶中任意两种关键酶基因间的联合调控,并且存在进行乳酸脱氢酶,α-乙酰乳酸合成酶和α-乙酰乳酸脱羧酶3种关键酶基因联合调控的可行性。     

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.     

Probing the molecular basis of substrate specificity, stereospecificity, and catalysis in the class II pyruvate aldolase, BphI

BphI, a pyruvate-specific class II aldolase found in the polychlorinated biphenyls (PCBs) degradation pathway, catalyzes the reversible C-C bond cleavage of (4S)-hydroxy-2-oxoacids to form pyruvate and an aldehyde. Mutations were introduced into bphI to probe the contribution of active site residues to substrate recognition and catalysis. In contrast to the wild-type enzyme that has similar specificities for acetaldehyde and propionaldehyde, the L87A variant exhibited a 40-fold preference for propionaldehyde over acetaldehyde. The specificity constant of the L89A variant in the aldol addition reaction using pentaldehyde is increased ～50-fold, making it more catalytically efficient for pentaldehyde utilization compared to the wild-type utilization of the natural substrate, acetaldehyde. Replacement of Tyr-290 with phenylalanine or serine resulted in a loss of stereochemical control as the variants were able to utilize substrates with both R and S configurations at C4 with similar kinetic parameters. Aldol cleavage and pyruvate α-proton exchange activity were undetectable in the R16A variant, supporting the role of Arg-16 in stabilizing a pyruvate enolate intermediate. The pH dependence of the enzyme is consistent with a single deprotonation by a catalytic base with pK(a) values of approximately 7. In H20A and H20S variants, pH profiles show the dependence of enzyme activity on hydroxide concentration. On the basis of these results, a catalytic mechanism is proposed.     

Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde.

The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K(m) with no effect on k(cat), and rate-limiting product release has been proposed to account for the lack of an isotope effect on k(cat) (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k(cat)/K(m) for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ((H)(k(cat)/K(m))/(D)(k(cat)/K(m)) = 4.5, and (D)k(cat) = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k(cat) is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K(d) values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic control of individual reaction steps and by loose binding of all ligands.     

Binding in vitro to rat liver receptors does not correlate with activities in vivo of bovine somatotropin. Use of chemically modified derivatives as probes.

The mitochondrial fraction from the free-living nematode Panagrellus redivivus decarboxylates pyruvate to produce significant amounts of acetaldehyde. Acetaldehyde formation is stimulated by thiamin pyrophosphate, shows a sharp optimum at pH 6.8 and is greater under anaerobic than aerobic conditions. The pyruvate decarboxylase activity cofractionates with, and is probably a partial reaction of, the pyruvate dehydrogenase complex. Acetaldehyde production is modulated by NAD+, ATP and acetyl-CoA and is greatly stimulated by lipoic acid. The pyruvate decarboxylase system is extremely sensitive to thiol-group inhibitors and is inhibited by oxygen in the presence of pyruvate.     

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases.

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 mm pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 mm, than of higher concentrations (1.0 mm) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K_m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.     

Studies on the continuous production of (R)-(−)-phenylacetylcarbinol in an enzyme-membrane reactor

The optimization of a continuous enzymatic reaction yielding (R)-(−)-phenylacetylcarbinol ((R)-PAC), a key intermediate of the (1R,2S)-(−)-ephedrine synthesis, is presented. We compare the suitability of different mutants of the pyruvate decarboxylase (PDC) from Zymomonas mobilis with respect to their application in biotransformation using pyruvate or acetaldehyde and benzaldehyde as substrates, respectively. Starting from 90 mM pyruvate and 30 mM benzaldehyde, (R)-PAC was obtained with a space time yield of 27.4 g/(L·day) using purified PDCW392I in an enzyme-membrane reactor. Due to the high stability of the mutant enzymes PDCW392I and PDCW392M towards acetaldehyde, a continuous procedure using acetaldehyde instead of pyruvate was developed. The kinetic results of the enzymatic synthesis starting from acetaldehyde and benzaldehyde demonstrate that the carboligation to (R)-PAC is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Starting from an inlet concentration of 50 mM of both aldehydes, (R)-PAC was obtained with a space-time yield of 81 g/(L·day) using the mutant enzyme PDCW392M. The new reaction strategy allows the enzymatic synthesis of (R)-PAC from cheap substrates free of unwanted by-products with potent mutants of PDC from Z. mobilis in an aqueous reaction system.     

Kinetic analysis and modelling of enzymatic (R)-phenylacetylcarbinol batch biotransformation process

Initial rate and biotransformation studies were applied to refine and validate a mathematical model for enzymatic (R)-phenylacetylcarbinol (PAC) production from pyruvate and benzaldehyde using Candida utilis pyruvate decarboxylase (PDC). The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U ml-1 carboligase. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde, and acetoin. These values were determined from data for three batch biotransformations performed over a range of initial concentrations (viz. 50-150 mM benzaldehyde, 60-180 mM pyruvate, 1.1-3.4 U ml-1 enzyme activity). The finalized model was then used to predict a batch biotransformation profile at 120/100 mM initial pyruvate/benzaldehyde (initial enzyme activity 3.0 U ml-1). The simulated kinetics gave acceptable fitting (R2 = 0.9963) to the time courses of these latter experimental data for substrates pyruvate and benzaldehyde, product PAC, by-products acetaldehyde and acetoin, as well as enzyme activity level.     

The use of steady-state treatment in the rapid kinetics of horse liver alcohol dehydrogenase. The effect of addition of product on the "burst" reaction.

The effect of addition of product on the amplitude of the “burst” reaction of horse liver alcohol dehydrogenase was studied using a stopped-flow spectrophotofluorimeter. The amplitude of the “burst” formation of enzyme-bound NADH fluorescence was completely diminished by the addition of excess acetaldehyde or benzaldehyde in the reaction with NAD⁺ and ethanol or NAD⁺ and benzylalcohol, respectively. The results indicate that a significant concentration of the ternary enzyme-coenzyme-substrate complex was formed during the steady-state in the presence of product, and this ternary complex did not exhibit NADH fluorescence. The dissociation constants for the ternary complex were determined from the amplitudes of the “burst” reactions. The “active site” titration of the enzyme with NAD⁺ in the presence of ethanol and iso-butyramide is also described.     

A high affinity pyruvate decarboxylase is present in cottonwood leaf veins and petioles: A second source of leaf acetaldehyde emission?

Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the range of 2 mM. The leaf vein form of the enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the K_m value for pyruvate was 42 μM. This K_m is lower than the typical millimolar range seen for PDCs from other sources. The purified leaf PDC also decarboxylates 2-ketobutyric acid (K_m = 2.2 mM). We conclude that there are several possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles, and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabolically active vascular bundle cells from the effects of oxygen deprivation.     

Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol

We have found that acetohydroxyacid synthase (AHAS) is an efficient catalyst for the enantiospecific (> or =98% enantiomeric excess) synthesis of (R)-phenylacetylcarbinol (R-PAC) from pyruvate and benzaldehyde, despite the fact that its normal physiological role is synthesis of (S)-acetohydroxyacids from pyruvate and a second ketoacid. (R)-phenylacetylcarbinol is the precursor of important drugs having alpha and beta adrenergic properties, such as L-ephedrine, pseudoephedrine, and norephedrin. It is currently produced by whole-cell fermentations, but the use of the isolated enzyme pyruvate decarboxylase (PDC) for this purpose is the subject of active research and development efforts. Some of the AHAS isozymes of Escherichia coli have important advantages compared to PDC, including negligible acetaldehyde formation and high conversion of substrates (both pyruvate and benzaldehyde) to PAC. Acetohydroxyacid synthase isozyme I is particularly efficient. The reaction is not limited to condensation of pyruvate with benzaldehyde and other aromatic aldehydes may be used.     

New model for activation of yeast pyruvate decarboxylase by substrate consistent with the alternating sites mechanism: demonstration of the existence of two active forms of the enzyme

Pyruvate decarboxylase from yeast (YPDC, EC 4.1.1.1) exhibits a marked lag phase in the progress curves of product (acetaldehyde) formation. The currently accepted kinetic model for YPDC predicts that, only upon binding of substrate in a regulatory site, a slow activation step converts inactive enzyme into the active form. This allosteric behavior gives rise to sigmoidal steady-state kinetics. The E477Q active site variant of YPDC exhibited hyperbolic initial rate curves at low pH, not consistent with the model. Progress curves of product formation by this variant were S-shaped, consistent with the presence of three interconverting conformations with distinct steady-state rates. Surprisingly, wild-type YPDC at pH < or =5.0 also possessed S-shaped progress curves, with the conformation corresponding to the middle steady state being the most active one. Reexamination of the activation by substrate of wild-type YPDC in the pH range of 4.5-6.5 revealed two characteristic transitions at all pH values. The values of steady-state rates are functions of both pH and substrate concentration, affecting whether the progress curve appears "normal" or S-shaped with an inflection point. The substrate dependence of the apparent rate constants suggested that the first transition corresponded to substrate binding in an active site and a subsequent step responsible for conversion to an asymmetric conformation. Consequently, the second enzyme state may report on "unregulated" enzyme, since the regulatory site does not participate in its generation. This enzyme state utilizes the alternating sites mechanism, resulting in the hyperbolic substrate dependence of initial rate. The second transition corresponds to binding a substrate molecule in the regulatory site and subsequent minor conformational adjustments. The third enzyme state corresponds to the allosterically regulated conformation, previously referred to as activated enzyme. The pH dependence of the Hill coefficient suggests a random binding of pyruvate in a regulatory and an active site of wild-type YPDC. Addition of pyruvamide or acetaldehyde to YPDC results in the appearance of additional conformations of the enzyme.     

Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria.

1. Evidence is given for three sites of phosphorylation in the alpha-chains of the decarboxylase component of purified rat heart pyruvate dehydrogenase complex, analogous to those established for procine and bovine complexes. Inactivation of rat heart complex was correlated with phosphorylation of site 1. Relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. 2. Methods are described for measurement of incorporation of 32Pi into the complex in rat heart mitochondria oxidizing 2-oxoglutarate + L-malate (total, sites 1, 2 and 3). Inactivation of the complex was related linearly to phosphorylation of site 1 in mitochondria of normal or diabetic rats. The relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. Rates of site-2 and site-3 phosphorylation may have been closer to that of site 1 in mitochondria of diabetic rats than in mitochondria of normal rats. 3. The concentration of inactive (phosphorylated) complex was varied in mitochondria from normal rats by inhibiting the kinase reaction with pyruvate at concentrations ranging from 0.15 to 0.4 mM. The results showed that the concentration of inactive complex is related linearly to incorporation of 32Pi into site 1. Inhibition of 32Pi incorporations with pyruvate at all concentrations over this range was site 3 greater than site 2 greater than site 1. 4. With mitochondria from diabetic rats, pyruvate (0.15-0.4 mM) inhibited incorporation of 32Pi into site 3, but it had no effect on the concentration of inactive complex or on incorporations of 32Pi into site 1 or site 2. It is concluded that site-3 phosphorylation is not required for inactivation of the complex in rat heart mitochondria. 5. Evidence is given that phosphorylation of sites 2 and 3 may inhibit reactivation of the complex by dephosphorylation in rat heart mitochondria.