Cooperative interaction of pyruvate dehydrogenase from pigeon breast muscle]

Cooperative interaction of pyruvate with the pyruvate dehydrogenase (PD) complex from pigeon breast muscle was shown. The sigmoidal dependence of the reaction rate on pyruvate concentration was observed for the PD complex. The Hill coefficient is equal to 1,5; no inhibition by the substrate (up to 2.2.10(-3) M) was found. The kinetic behaviour of the isolated pyruvate dehydrogenase component (PDH) analyzed under similar conditions, is more complex; this may be probably due to the presence of oligomeric forms with different molecular weights and specific activities. The competitive inhibitor of the PD complex--an amide of pyruvic acid (PA) (Ki=6.3-10(-6) M) activates the enzyme at low concentrations (less than 2,10(-6) M). When PA is present, the dependence of the reaction rate on pyruvate concentration gives a usual hyperbolic curve, v of [S]o. It is concluded that pyruvate may have a regulatory effect on the activity of muscle PD complex.     

Half-of-the-site reactivity of the decarboxylating component of the pyruvate dehydrogenase complex from pigeon breast muscle with respect to 2-hydroxyethyl thiamine pyrophosphate

The holopyruvate dehydrogenase is characterized by the charge transfer complex formation between tryptophan residue and thiamine pyrophosphate in each of two active centres. Interaction of apoenzyme with one mole of 2-hydroxyethyl thiamine pyrophosphate results in appearance of the same spectral band which does not change in intensity with further increase in ligand concentration. 2-hydroxyethyl thiamine pyrophosphate: acceptor oxidoreductase activity abolishes after oxidation of only one tryptophan residue per mole of the protein or blocking of one of the active centres with inactive analogue of the coenzyme. In the latter case the charge transfer complex band induced by interaction of apoenzyme with 2-hydroxyethyl thiamine pyrophosphate was not shown at all. These facts testify to half-of-the-site reactivity of pyruvate dehydrogenase with respect to 2-hydroxyethyl thiamine pyrophosphate.     

Turnover of pegeon breast muscle pyruvate dehydrogenase complex.

The pigeon breast muscle pyruvate dehydrogenase complex was resolved into three component enzymes: lipoate acetyltransferase, pyruvate dehydrogenase, and lipoamide dehydrogenase. The antibodies against each component enzyme were prepared. All of the antibodies against component enzymes precipitated the pyruvate dehydrogenase complex. The enzyme complex was recovered as the immunoprecipitate from the extract of breast muscle of a pigeon that had received a single injection of L-[4,5-3H]leucine. The immunoprecipitate was separated into each component enzyme by SDS-polyacrylamide gel electrophoresis. The relative isotopic leucine incorporations per mg of protein into each component enzyme 4 h after the injection were 1.0 : 0.9 : 1.4 : 2.7 for lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase, respectively. The half-lives of lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase were 7.7, 2.5, 2.6, and 1.8 days, respectively. These results indicate that the component enzymes of the pyruvate dehydrogenase complex were synthesized and degraded at different rates.     

Regulation of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle]

The activity of alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle is controlled by ADP and the reaction products, i. e. succinyl-CoA and NADH. ADP activates the alpha-ketoglutarate dehydrogenase component of the complex, whereas NADH inhibits alpha-ketoglutarate dehydrogenase and lipoyl dehydrogenase. In the presence of NADH the kinetic curve of the complex with respect to alpha-ketoglutarate and NAD and the dependence of upsilon versus [NAD] and upsilon versus [Lip (SH)2] in the lipoyl dehydrogenase reaction are S-shaped. In the absence of inhibitor ADP had no activating effect on lipoyl dehydrogenase; however, in the presence of NADH ADP decreases the cooperativity for NAD. The cooperative kinetics of the constituent enzymes of the complex are indicative of its allosteric properties. Isolation of the alpha-ketoglutarate dehydrogenase complex and its lipoyl dehydrogenase and alpha-ketoglutarate dehydrogenase components in a desensitized state confirms their allosteric nature. It is assumed that NADH effects of isolated alpha-ketoglutarate dehydrogenase is due to a shift in the equilibrium between different oligomeric forms of the enzyme.     

Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.

One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.     

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.     

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.     

Pyruvate dehydrogenase kinase activity of pig heart pyruvate dehydrogenase (E1 component of pyruvate dehydrogenase complex).

The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.     

The pyruvate dehydrogenase multienzyme complex

During the review period, several structures of component enzymes and domains of enzymes of this multienzyme complex were determined. Three structures of the flavoprotein component, dihydrolipoamide dehydrogenase, became available. The structure of the core component, dihydrolipoyl acetyltransferase, can in principle be constructed from the known structures of its modules: the lipoyl, the peripheral subunit-binding and the catalytic domain. Dynamic aspects, such as the structure and function of the inter-domain linkers in dihydrolipoyl acetyltransferase and the conformational changes invlved in the mechanism of electron transfer in dihydrolipoamide dehydrogenase, remain to be clarified. Although several questions concerning the structure of the individual components of the complex have been solved, there is still much to learn about the assembly pathway. In mammalian complexes, the structure and function of protein X remains something of a riddle.     

Substrate-dependent inactivation of muscle pyruvate dehydrogenase: identification of the acetyl-substituted enzyme form

The properties of the pyruvate dehydrogenase component isolated from the pigeon breast muscle pyruvate dehydrogenase complex were studied upon inactivation of the enzyme in an incomplete reaction mixture: in the presence of cofactors and pyruvate, and in the absence of electron acceptors. The substrate-dependent inactivation was shown to result in the modification of two sulfhydryl groups per mole of the enzyme, in the appearance of a maximum at 235 nm in the protein absorption spectrum, and in the involvement of 1.5 moles of the [2-14C]-pyruvate fragment per mole of the pyruvate dehydrogenase. The fragment-protein bond is acid-stable, labile in alkali, and breaks up in the presence of performic acid, neutral hydroxylamine and dithiothreitol. An acetyl-substituted form of pyruvate dehydrogenase appearing with the participation of sulfhydryl enzyme groups is suggested.     

Immunological identification of a new component of Ascaris pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex was purified from Ascaris muscle both with and without MgCl2 treatment at the first stage of purification. The specific activity of complex purified with MgCl2 treatment was about 2-fold as high as that purified without it. In addition to three component enzymes, two unknown polypeptides of 46 and 41 kDa were found in the complex purified by the two procedures. The quantity of unknown polypeptide of 41 kDa was increased in the complex purified with MgCl2 treatment as compared with that without it. Antibodies against the three component enzymes were prepared. All the antibodies precipitated the two unknown polypeptides in addition to the three component enzymes in immunoprecipitation experiments. Antibody against the alpha-subunit of pyruvate dehydrogenase reacted with the 41 kDa polypeptide as well as the alpha-subunit in the immunoblotting method. The unknown polypeptide of 46 kDa did not react with any antibody. These results suggest that the unknown 41 kDa polypeptide is a derivative of the alpha-subunit and that the unknown 46 kDa polypeptide is not a proteolytic-degradative product of component enzymes but is a component of the Ascaris pyruvate dehydrogenase complex. When the Ascaris complex was incubated with [2-14C]pyruvate in the absence of CoASH, only lipoate acetyltransferase was acetylated. In rat heart pyruvate dehydrogenase complex, lipoate acetyltransferase and another protein (referred to as component x or protein x) were acetylated. These results indicate that the unknown polypeptide of 46 kDa is a new component.