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PubMed1993

Characterization of the covalent enzyme intermediates formed during pyruvate phosphate dikinase catalysis.

The intermediacy of a pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) in the Clostridium symbiosum pyruvate phosphate dikinase catalyzed interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PPi), and phosphoenolpyruvate (PEP) was examined using transient kinetic techniques. Single-turnover experiments with [gamma-32P]ATP or [14C]ATP and PPDK were carried out in the presence and absence of Pi to test for pyrophosphorylenzyme and AMP formation, respectively. Formation of the E-PP.AMP complex was found to be followed by Pi binding and the formation of the E-P.AMP.PPi complex. The level of pyrophosphorylenzyme accumulated during a single turnover was found to be dependent on the divalent metal cofactor used (Mn2+ > Co2+ > Mg2+). Single-turnover experiments with [32P]PEP and PPDK were carried out in the presence and absence of PPi and pyruvate to test for phosphorylenzyme formation in the reverse, ATP-forming direction of the reaction. Phosphorylenzyme formed from the reaction of the E.PEP complex was converted in the presence of AMP and PPi to free enzyme at a rate exceeding the steady-state turnover rate. The reaction sequence for pyruvate phosphate dikinase was determined to be [formula see text] 31P NMR analysis of the phosphorylenzyme in the native (-4.0 ppm) and denatured form (-3.9 ppm) revealed a 3-N-phosphohistidine residue. Complexation of Mg2+ resulted in a 0.3 ppm upfield shift of the phosphorus resonance from native phosphorylenzyme while Mn2+ complexation lead to extensive line broadening, indicative of metal cofactor binding in close vicinity to the phosphoryl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1991

A rate-determining proton relay in the pyruvate kinase reaction.

This study ascribes the large steady-state D2O isotope effect on kcat of pyruvate kinase (PEP + ADP----pyruvate + ATP) to the reprotonation of the product form of the enzyme for use in forming pyruvate. Previous tritium trapping experiments [Rose, I. A., & Kuo, D. J. (1989) Biochemistry 28, 9579-9585] with muscle pyruvate kinase showed that the proton used for ketonization of enolpyruvate is derived from an enzyme "pool" that contains three kinetically equivalent hydrogens that could be trapped in a nontritiated "chase" medium by high levels of ADP and PEP. The exchange of this pool with the medium was rapid in the free enzyme (approximately 1400 s-1), prior to addition of PEP, and apparently much less in the completed complex. The dissociation rate constant was determined by using the dissociation-competition equation koffT = K1/2kcat/Km, where kcat/Km is the steady-state parameter for PEP and K1/2 is the concentration of PEP required to trap 50% of the isotope that could be trapped. The present study shows that the competition constant, K1/2, is decreased by approximately 5-fold in D2O, the same effect see on kcat under conditions where kcat/Km, measured in the steady state, is not changed. The common effect of D2O on kcat in the steady state and koffT in pulse/chase suggests that the forward reaction rate is determined by hydrogen transfer to the enzyme. Further evidence indicates that the kinetically important proton in question is the proton used for ketonization of enolpyruvate, the substrate proton.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

The conversion of alanine into glutamine in guinea-pig renal cortex. Essential role of pyruvate carboxylase.

1. The metabolism of L-alanine was studied in isolated guinea-pig kidney-cortex tubules. 2. In contrast with previous conclusions of Krebs [(1935) Biochem. J. 29, 1951-1969], glutamine was found to be the main carbon and nitrogenous product of the metabolism of alanine (at 1 and 5 mM). Glutamate and ammonia were only minor products. 3. At neither concentration of alanine was there accumulation of glucose, glycogen, pyruvate, lactate, aspartate or tricarboxylic acid-cycle intermediates. 4. Carbon-balance calculations and the release of 14CO2 from [U-14C]alanine indicate that oxidation of the alanine carbon skeleton occurred at both substrate concentrations. 5. A pathway involving alanine aminotransferase, glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, pyruvate carboxylase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of alanine into glutamine. 6. Strong evidence for this pathway was obtained by: (i) suppressing alanine removal by amino-oxyacetate, and inhibitor of transaminases, (ii) measuring the release of 14CO2 from [1-14C]alanine, (iii) the use of L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from alanine, and (iv) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from alanine. 7. In this pathway, the central role of pyruvate carboxylase, which explains the discrepancy between our results and those of Krebs (1935), was also demonstrated.

Alanine↗
PubMed1986

The Mr-50 000 polypeptide of mammalian pyruvate dehydrogenase complex participates in the acetylation reactions.

The mammalian pyruvate dehydrogenase complex, Mr 8.5 X 10(6), contains an additional tightly bound 50 000-Mr polypeptide, component X, which copurifies with the intact assembly. Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One-dimensional peptide mapping studies with 125I-labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2-14C]pyruvate and N-ethyl-[2,3-14C]maleimide confirm that distinct 14C-labelled peptides are generated from these two species. These protein-chemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase (Mr 70 000) subunit. Incubation of the native PDC in the presence of [2-14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (E1) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetylation of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N-ethylmaleimide.

Acetylation↗
PubMed1990

Purification and partial characterization of pyruvate decarboxylase from Oryza sativa L.

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 mu kat/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC, the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxia and would be activated by small changes in pyruvate concentration.

Chlorides↗
PubMed1993

Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes.

Metformin (dimethylbiguanide) has been used for more than 30 years as an antihyperglycemic agent in the treatment of diabetes mellitus, but its effect on gluconeogenesis is still controversial. In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/pyruvate (10:1, mol/mol), fructose, alanine or glutamine, following metformin addition, is observed. Moreover, in hepatocytes perifused with dihydroxyacetone as the gluconeogenic substrate and treated with 0.5 mM metformin, an inhibition of the glucose flux and a simultaneous stimulation of the lactate/pyruvate flux were observed. This enhancement of lactate/pyruvate formation appears to be due to an effect on the pyruvate-kinase enzyme. A direct effect of metformin on pyruvate kinase cannot explain this result, since pyruvate-kinase activity was not affected by metformin at this concentration. In contrast, the addition of metformin caused a significant decrease in the cellular ATP concentration, a known allosteric inhibitor of this enzyme. This could explain the stimulation of pyruvate-kinase activity following metformin addition and thus the inhibition of gluconeogenesis.

Adenosine Diphosphate↗
PubMed1998

The RAG3 gene of Kluyveromyces lactis is involved in the transcriptional regulation of genes coding for enzymes implicated in pyruvate utilization and genes of the biosynthesis of thiamine pyrophosphate.

The RAG3 gene of Kluyveromyces lactis, a homolog of PDC2 of Saccharomyces cerevisiae, is known to be a regulator of the pyruvate decarboxylase gene KlPDC1. We have identified new target genes for Rag3p. The RAG3 gene product was found to be required for the transcription of two genes of the biosynthetic pathway of thiamine (a cofactor of pyruvate decarboxylase). Conversely, the RAG3 gene product partially repressed the expression of the pyruvate dehydrogenase gene KlPDA1. Therefore, RAG3 may act as a general regulator in the balance of the two alternative pathways of pyruvate metabolism in yeast.

Blotting, Northern↗
PubMed1975

Absence of pyruvate decarboxylase activity in man: a cause of congenital lactic acidosis.

A complete deficiency in the pyruvate dehydrogenase system activity contributed to the death of a 6-month-old infant with congenital lactic acidosis. The enzymatic block could be isolated to the first component, pyruvate decarboxylase (E1) of the pyruvate dehydrogenase complex. This enzymatic deficiency allowed a demonstration of an "intercomplex" exchange of the components of the mammalian pyruvate dehydrogenase system and indicated that the first component is normally present in an apparent excess.

Acidosis↗
PubMed1982

Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis.

Six different pyruvate decarboxylase mutants of Saccharomyces cerevisiae were isolated. They belong to two unlinked complementation groups. Evidence is presented that one group is affected in a structural gene. The fact that five of the six mutants had residual pyruvate decarboxylase activity provided the opportunity for an intensive physiological characterization. It was shown that the loss of enzyme activity in vitro is reflected in a lower fermentation rate, an increased pyruvate secretion, and slower growth on a 2% glucose medium. The different effects of antimycin A on leaky mutants grown on ethanol versus the same mutants grown on glucose support the view that glucose induces some of the glycolytic enzymes, especially pyruvate decarboxylase.

Antimycin A↗
PubMed1992

Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis.

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.

Acetolactate Synthase↗
PubMed1963

Degradation of pyruvate by Micrococcus lactilyticus. III. Properties and cofactor requirements of the carbon dioxide-exchange reaction.

Whiteley, H. R. (University of Washington, Seattle) and N. G. McCormick. Degradation of pyruvate by Micrococcus lactilyticus. III. Properties and cofactor requirements of the carbon dioxide-exchange reaction. J. Bacteriol. 85:382-393. 1963.-At an acid pH, extracts of Micrococcus lactilyticus (Veillonella alcalescens) catalyze the oxidative decarboxylation of pyruvate to carbon dioxide, hydrogen, and acetyl phosphate, and the rapid exchange of carbon dioxide into the carboxyl group of pyruvate. These reactions take place only under anaerobic conditions and require phosphate (or arsenate), a reducing agent, diphosphothiamine, coenzyme A, an electron acceptor (ferredoxin, flavins, dyes, or certain inorganic anions), and a divalent cation (Co(++)> Mn(++) > Mg(++) > Fe(++)). High concentrations of coenzyme A and electron acceptors stimulate pyruvate breakdown but inhibit CO(2) exchange. Exchange is also inhibited by p-chloromercuribenzoate but not by arsenite. Extracts rapidly lose the ability to mediate the exchange reaction after passage through diethylaminoethyl- or triethylaminoethyl-cellulose or Dowex-1; this loss in activity may be prevented by adding a reducing agent and the above cofactors. The exchange of CO(2) and formate by M. lactilyticus is compared.

Carbon Dioxide↗
PubMed1992

Postnatal development of pyruvate oxidation in quadriceps muscle of the rat.

In order to evaluate the age dependency of enzymes involved in the energy-generating system, skeletal muscle specimens from rats of different ages were investigated for several mitochondrial enzymes. [1-14C]pyruvate (+/- ADP) oxidation rates and pyruvate dehydrogenase complex (PDHC) activity increased significantly from low early values during the neonatal period to nearly adult values at the end of the suckling period. Other enzymes of the pyruvate oxidation route such as citrate synthase and cytochrome c oxidase showed similar patterns of development. Immunoblot studies of PDHC detected a clear increase in the intensity of the bands of the alpha subunits of E1 (pyruvate dehydrogenase) and E2 (dihydrolipoyl transacetylase) within the first 3 weeks of life. The ratio between the individual PDHC proteins indicated that E1 alpha, the regulatory subunit of the multienzyme complex, is the most rapidly increasing protein with age.

Acetyltransferases↗
PubMed1995

Palmitate-induced beta-cell insensitivity to glucose is coupled to decreased pyruvate dehydrogenase activity and enhanced kinase activity in rat pancreatic islets.

We previously found that long-term exposure to fatty acids impairs glucose-induced insulin release. In the present study, we investigated whether impairment is related to decreased pyruvate dehydrogenase (PDH) and increased PDH kinase activity. Rat pancreatic islets were cultured for 48 h in RPMI-1640 medium with or without 0.125 mmol/l palmitate. Potentiation of insulin responses to succinic acid monomethylester (SAM) by 10 mmol/l acetate and pyruvate were subsequently compared in order to assess whether generation of acetyl-coenzyme A (CoA) from pyruvate was deficient in the intact beta-cell. Potentiation by acetate was similar in control and palmitate-preexposed islets. In contrast, pyruvate potentiated SAM-induced response by 122% in control but by only 39% in palmitate-exposed islets (P < 0.001). In extracts of palmitate-exposed islets, the active (unphosphorylated) form of PDH was decreased by 50% and total PDH activity (assessed after phosphatase treatment) by 25%. The proportion of active form to total PDH activity was also reduced (42.7 +/- 2.6% after palmitate vs. 66.6 +/- 4.3% in control islets, P < 0.01). In the same preparations, PDH kinase activity was enhanced 1.7-fold by palmitate in terms of the rate constant of ATP-dependent inactivation of PDH (P < 0.05). To test for a role of free (not PDH-bound) kinase, a PDH-free mitochondrial fraction was prepared, and its kinase activity was tested against a pig heart PDH preparation. Free kinase activity was increased 1.9-fold in palmitate-treated islets (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1992

The catalytic power of pyruvate decarboxylase. A stochastic model for the molecular evolution of enzymes.

Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate anion by a factor of around 10(12), compared with the non-enzymic decarboxylation by thiamine, under standard state conditions of 1 mM pyruvate and thiamine diphosphate (TDP), pH 6.2. Free-energy diagrams constructed on the basis of earlier measurements for the enzymic and non-enzymic reactions give some information on catalysis by PDC. PDC stabilizes the reactant state preceding TDP addition to pyruvate by 76 kJ mol-1 and the transition state for the addition by 83 kJ mol-1. PDC stabilizes the reactant state preceding decarboxylation (presumably alpha-lactyl-TDP) by 27 kJ mol-1 and the transition state for decarboxylation by 68 kJ mol-1. In addition, the free-energy diagrams reveal a leveling of reactant-state free energies in the enzymic reaction compared with the non-enzymic reaction, in that the former are nearly equal to each other. The enzyme-bound transition-state energies are similarly leveled. The energetic leveling of reactant states has been noted by Albery, Knowles and their coworkers in many enzymic reactions and termed 'matched internal thermodynamics.' They showed that the result would arise naturally (and inevitably) in the 'evolution to perfection' of enzymes, when the evolutionary process was treated by a deterministic model. The critical assumption of this model was the validity of a Marcus-type or Brønsted-type linear free-energy relationship between rate and equilibrium constants for reactions occurring wholly within enzyme complexes. Here a completely stochastic simulation of molecular evolution, with no deterministic assumptions, is shown to reproduce both 'matched internal thermodynamics' and the 'matched internal kinetics' or leveling of transition-state energies noted here. The Albery-Knowles result is thus more general than might have been supposed.

Biological Evolution↗
PubMed2002

Therapeutic potential of the mammalian pyruvate dehydrogenase kinases in the prevention of hyperglycaemia.

The mitochondrial pyruvate dehydrogenase complex (PDC) catalyses the oxidative decarboxylation of pyruvate, and links glycolysis to the tricarboxylic acid cycle and ATP production. Adequate flux through PDC is important in tissues with a high ATP requirement, in lipogenic tissues (since it provides cytosolic acetyl-CoA for fatty acid (FA) synthesis), and in generating cytosolic malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase (CPT I). Conversely, suppression of PDC activity is crucial for glucose conservation when glucose is scarce. This review describes recent advances relating to the control of mammalian PDC activity by phosphorylation (inactivation) and dephosphorylation (activation, reactivation), in particular regulation of PDC by pyruvate dehydrogenase kinase (PDK) which phosphorylates and inactivates PDC. PDK activity is that of a family of four proteins (PDK1-4). PDK2 and PDK4 appear to be expressed in most major tissues and organs of the body, PDK1 appears to be limited to the heart and pancreatic islets, and PDK3 is limited to the kidney, brain and testis. PDK4 is selectively upregulated in the longer term in most tissues and organs in response to starvation and hormonal imbalances such as insulin resistance, diabetes mellitus and hyperthyroidism. Parallel increases in PDK2 and PDK4 expression appear to be restricted to gluconceogenesic tissues, liver and kidney, which take up as well as generate pyruvate. Factors that regulate PDK4 expression include FA oxidation and adequate insulin action. PDK4 is also either a direct or indirect target of peroxisome proliferator-activated receptor (PPAR) alpha. PPAR alpha deficiency in liver and kidney restricts starvation-induced upregulation of PDK4; however, the role of PPAR alpha in heart and skeletal muscle appears to be more complex. These observations may have important implications for the pharmacological modulation of PDK activity (e.g. use of PPAR alpha activators) for the control of whole-body glucose, lipid and lactate homeostasis in disease states and suggest that therapeutic interventions must be tissue targeted so that whole-body fuel homeostasis is not adversely perturbed.

Animals↗
PubMed1976

Lactic acidosis in three sibs due to defects in both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes.

A Canadian Indian family is described in which three of the children were mentally retarded, and had seizures and other neurological abnormalities. They had chronic metabolic acidosis associated with elevated blood levels of lactate, pyruvate, and alanine. Two of the children excreted large amounts of pyruvic and alpha-ketoglutaric acids in the urine and had elevated plasma levels of glutamic acid and proline. Hypoglycemia occurred with fasting in two of the children. Treatment with pharmacological doses of thiamine, lipoic acid, biotin, riboflavin, and various dietary regimes was without effect. One child died at 3 1/2 months and another at 4 1/2 months; the third is still alive at 23 months of age. Enzyme assays revealed a low level of activity of both the pyruvate and alpha-ketoglutarate dehydrogenase complexes in cultured fibroblasts of one of the sibs. These patients appeared to have partial defects in the oxidation of pyruvate, as well as of alpha-ketoglutarate within the tricarboxylic acid cycle.

Acidosis↗
PubMed1987

The catalytic requirements for reduction and acetylation of protein X and the related regulation of various forms of resolved pyruvate dehydrogenase kinase.

The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.

Acetylation↗
PubMed1978

Regulation of pyruvate oxidation and the conservation of glucose.

In animals the pyruvate dehydrogenase reaction is mainly responsible for the irreversible loss of glucose carbon by oxidation. Regulation of this reaction is shown to be a major determinant of glucose conservation in starvation and diabetes. Estimates of conservation in man in starvation and diabetes are reviewed. The pyruvate dehydrogenase complex is inhibited by products of its reactions; it is also regulated by a phosphorylation-dephosphorylation cycle catalysed by a kinase intrinsic to the complex and by a more loosely associated phosphatase. Inactivation is largely accomplished by phosphorylation of the tetrameric decarboxylase component (alpha2beta2) to alpha2Pbeta2. Complete phosphorylation produces the (alpha2P3)beta2 form. Both forms are completely reactivated by phosphatase action but the initial rate of reactivation of a complex containing alpha2Pbeta2 is approximately three times that of (alpha2P3)beta2. The proportion of active (dephosphorylated) complex is decreased in rat tissues by starvation and diabetes and in perfused rat heart by oxidation of fatty acids and ketone bodies. In adipose tissue in vitro, insulin increases the proportion of active complex and lipolytic hormones may decrease this proportion. It is suggested that rates of oxidation of lipid fuels may be a major determinant of the activity of pyruvate dehydrogenase in tissues in relation to the actions of insulin and lipolytic hormones and the effects of diabetes and starvation. Phosphorylation and inactivation of the complex are enhanced by high mitochondrial ratios of [acetyl-CoA]/[CoA], [ATP]/[ADP], [NADH]/[NAD+] and low concentrations of pyruvate, Mg2+ and Ca2+, and vice versa.

Animals↗