Inhibition of the Escherichia coli pyruvate dehydrogenase complex E1 subunit and its tyrosine 177 variants by thiamin 2-thiazolone and thiamin 2-thiothiazolone diphosphates. Evidence for reversible tight-binding inhibition.

Variants of the pyruvate dehydrogenase subunit (E1; EC ) of the Escherichia coli pyruvate dehydrogenase multienzyme complex with Y177A and Y177F substitutions were created. Both variants displayed pyruvate dehydrogenase multienzyme complex activity at levels of 11% (Y177A E1) and 7% (Y177F E1) of the parental enzyme. The K(m) values for thiamin diphosphate (ThDP) were 1.58 microm (parental E1) and 6.65 microm (Y177A E1), whereas the Y177F E1 variant was not saturated at 200 microm. According to fluorescence studies, binding of ThDP was unaffected by the Tyr(177) substitutions. The ThDP analogs thiamin 2-thiazolone diphosphate (ThTDP) and thiamin 2-thiothiazolone diphosphate (ThTTDP) behaved as tight-binding inhibitors of parental E1 (K(i) = 0.003 microm for ThTDP and K(i) = 0.064 microm for ThTTDP) and the Y177A and Y177F variants. This analysis revealed that ThTDP and ThTTDP bound to parental E1 via a two-step mechanism, but that ThTDP bound to the Y177A variant via a one-step mechanism. Binding of ThTDP was affected and that of ThTTDP was unaffected by substitutions at Tyr(177). Addition of ThDP or ThTDP to parental E1 resulted in similar CD spectral changes in the near-UV region. In contrast, binding of ThTTDP to either parental E1 or the Y177A and Y177F variants was accompanied by the appearance of a positive band at 330 nm, indicating that ThTTDP was bound in a chiral environment. In combination with x-ray structural evidence on the location of Tyr(177), the kinetic and spectroscopic data suggest that Tyr(177) has a role in stabilization of some transition state(s) in the reaction pathway, starting with the free enzyme and culminating with the first irreversible step (decarboxylation), as well as in reductive acetylation of the dihydrolipoamide acetyltransferase component.

Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) - evidence for tertiary and quaternary interactions.

The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvic acid with C221 located on the beta domain at >20 A from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitutions were made at H92 on the alpha domain, across the domain divide from C221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 together appear to have a critical impact on the Hill coefficient, providing a pathway for information transfer. To study the activation by ThDP.Mg(II), variants at G231 (of the conserved GDG triplet) and at N258 and C259 (all three being part of the putative ThDP fold) of the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg(II) ligands are very important to activation of the enzymes by cofactors.

Systematic study of the six cysteines of the E1 subunit of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: none is essential for activity.

Variants of the Escherichia coli 1-lip pyruvate dehydrogenase multienzyme complex (1-lip PDHc) with the C259N and C259S substitutions in the putative thiamin diphosphate-(ThDP-) binding motif of the pyruvate dehydrogenase component (E1, EC 1.2.4.1) were characterized. Single substitutions were made at the five remaining cysteines of the E1 component, creating the C120A, C575A, C610A, C654A, and C770S variants to test the hypothesis that the activity loss that accompanies exposure of the enzyme to fluoropyruvate, bromopyruvate, and 2-oxo-3-butynoic acid is the result of the modification of approximately one cysteine residue per E1 monomer. Surprisingly, all single cysteine E1 variants could be reconstituted with E2-E3 subcomplex and showed PDHc activity ranging from 74% to 96% that of the parental enzyme. The specific activities of C259N and C259S variants of 1-lip PDHc were 58% and 27% relative to that of the parental 1-lip PDHc. All five single cysteine E1 variants, along with the C259N and C259S variants of 1-lip PDHc, could also (1) be inactivated with fluoropyruvate and 2-oxo-3-butynoic acid, (2) were subject to inactivation by the monoclonal antibody 18A9 reported from one of our laboratories, and (3) were subject to regulation by pyruvate and acetyl-CoA. It was therefore concluded that none of the six cysteine residues is essential for the activity of the E1 component or of the complex. When tested with the putative transition-state analogue, thiamin 2-thiothiazolone diphosphate, all but the C259S and C259N variants were very potently inhibited, the stoichiometry for parental E1 being about 1.6 mol of inhibitor/mol of E1 subunit. The C259S and C259N E1 variants required at least 25-fold greater inhibitor concentration to achieve the same level of inhibition. C259 is located in the putative thiamin diphosphate-binding motif of the enzyme [more exactly, it is adjacent to a ligand to the Mg(II) ion]. It is therefore concluded that thiamin 2-thiothiazolone diphosphate is not a transition-state analogue; rather, it is a potent inhibitor of the complex because of a specific interaction with the C259 residue.

2-Oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex.

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.

Effect of substitutions in the thiamin diphosphate-magnesium fold on the activation of the pyruvate dehydrogenase complex from Escherichia coli by cofactors and substrate.

The homotropic regulation of the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) by its coenzyme thiamin diphosphate and its substrate pyruvate was re-examined with complexes containing three and one lipoyl domains per E2 chain, and several variants of the latter, containing substitutions in the putative thiamin diphosphate fold of E1 (G231A, G231S, C259S, C259N, and N258Q). It was found that all of the E1 variants had significantly reduced specific activities, as reported elsewhere (Russell, G. C., Machado, R. S., and Guest, J. R. (1992) Biochem. J. 287, 611-619). In addition, extensive kinetic studies were performed in an attempt to determine the effects of the amino acid substitutions on the Hill coefficients with respect to thiamin diphosphate and pyruvate. All but one of the variants were incapable of being saturated with thiamin diphosphate, even at concentrations > 5 mM. Most importantly, the striking activation lag phase lasting for many seconds in the parental complexes containing three and one lipoyl domains per E2 chain was totally eliminated in the variants. Furthermore, activation by the coenzyme was localized to the E1 subunit, because resolved E1 exhibits virtually the same behavior during the activation lag phase as does the complex. In the parental complexes two distinct lag phases could be resolved, the duration of both decreases with increasing ThDP concentration. A mechanism that is consistent with all of the kinetic data on the parental complexes involves rapid equilibration of the first ThDP with the E1 dimer, followed by a slow conformational equilibration, that in turn is followed by slow addition of the second ThDP to form the fully activated dimer. When the diphosphate site is badly impaired, the binding affinity is very much reduced, this perhaps eliminates the slow step leading to the activated dimer form of the E1.

[Activity of the pyruvate dehydrogenase complex in Walker-256 carcinosarcoma]. / Aktivnost' piruvatdegidrogenaznogo kompleksa kartsinosarkomy Uoker-256.

Pyruvate dehydrogenase complex activity in the coarsely purified enzyme fractions (supernatant and mitochondrial extract) isolated from Walker-256 carcinosarcoma is shown to be much lower than the complex activity in the rat brain. Acetoin was not found in the incubation medium containing pyruvate dehydrogenase tumour complex. The rate of the reaction of nonoxidative formation of acetaldehyde by pyruvate dehydrogenase complex of the tumour was approximately five times lower than the rate of reaction catalyzed by the complex from the rat brain. It is shown that in the presence of adenine nucleotides AMP, ADP or ATP (0.5 mM) in the medium pyruvate: NAD+ oxidoreductase activity of the tumour complex is inhibited by 40-56% as compared to the control and reaction of nonoxidative decarboxylation of pyruvate is approximately 2 times activated. It is supposed that the accumulation of nonoxidative reaction products may be an essential factor for development and functioning of the tumour.

[Coenzyme-binding sites of the brain pyruvate dehydrogenase complex]. / Izuchenie kofermentsviazyvaiushchikh tsentrov piruvatdegidrogenaznogo kompleksa mozga.

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.

[The effect of phosphorylation on oxidative and CoA- and NAD+-independent turnover of pyruvate by pyruvate dehydrogenase complex in the brain]. / Vliianie fosforilirovaniia na okislitel'noe i CoA- i NAD+- nezavisimoe prevrashchenie piruvata piruvatdegidrogenzanym kompleksom mozga.

It was shown that in the presence of ATP and Mg2+ the phosphorylation of the partially purified pyruvate dehydrogenase complex and the enzyme in isolated brain mitochondria inhibited the oxidative activity of the pyruvate dehydrogenase complex. The phosphorylation did no affect essentially the nonoxidative decarboxylation of pyruvate to form CO2 and acetaldehyde. In native mitochondria from the bovine brain the nonoxidative activity of the pyruvate dehydrogenase complex reached about 10% as compared to the oxidative activity of enzyme.

[Pyruvate dehydrogenase from pigeon breast muscle. Chemical modification of the enzyme associated with conformation changes in the protein molecule]. / Piruvatdegidrogenaza grudnoi myshtsy golubia. Khimicheskaia modifikatsiia fermenta, soprovozhdaiushchaiasia konformatsionnym perekhodom belka.

The two-phase character of essential histidine residues modification of pyruvate dehydrogenase component of pyruvate dehydrogenase complex from pigeon breast muscle by diethylpyrocarbonate has been demonstrated. The relative amplitude of the fast phase increases with increasing the modificator concentration. The model of chemical modification of dimeric enzyme where the modification of the residue in one subunit leads to the change of reactivity of corresponding residue in the other subunit is used for the description of inactivation kinetics. The expression for the diminishing of enzyme activity in the course of chemical modification and the methods of kinetic parameters estimation have been proposed. The following values of kinetic parameters for the modification of pyruvate dehydrogenase component by diethylpyrocarbonate were obtained (pH 6.0; 20 degrees C): k1 = 6400 +/- 400 M-1 min-1 (the microscopic rate constant for the modification of histidine residue in the intact dimer), k2 = 890 +/- 200 M-1 min-1 (the rate constant for the modification of histidine residue in the intact subunit in the dimer which contains one modified subunit) and kt = 0.9 +/- 0.2 min-1 (the rate constant for conformational transition of the dimer induced by modification of histidine residue in one of the subunits in the dimeric molecule).