The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase.
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ABSTRACT:
SUBMITTER: Wilton DC
PROVIDER: S-EPMC1186462 | biostudies-other | 1979 Mar
REPOSITORIES: biostudies-other
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Project description:1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2. In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.
| S-EPMC1172421 | biostudies-other
Project description:1. Pig heart pyruvate dehydrogenase phosphate complex in which all three sites of phosphorylation were completely phosphorylated was re-activated at a slower rate by phosphatase than complex predominantly phosphorylated in site 1. The ratio of initial rates of re-activation was approx. 1:5 with a comparatively crude preparation of phosphatase and with phosphatase purified by gel filtration and ion-exchange chromatography. 2. The ratio of apparent first-order rate constants during dephosphorylation of fully phosphorylated complex averaged 1/3.8/1.3 for site 1/site 2/site 3. Only site-1 dephosphorylation was linearly correlated with re-activation of the complex throughout dephosphorylation. Dephosphorylation of site 3 was linearly correlated with re-activation after an initial burst of dephosphorylation. 3. Because dephosphorylation of site 1 was always associated with dephosphorylation of site 2, it is concluded that dephosphorylation cannot be purely random. 4. The ratio of apparent first-order rate constants for dephosphorylation of site 1 (partially/fully phosphorylated complexes) averaged 1.72. This ratio is smaller than the ratio of approx. 5 for the initial rates of re-activation. Possible mechanisms involved in the diminished rate of re-activation of fully phosphorylated complex are discussed.
| S-EPMC1162854 | biostudies-other
Project description:The crystal structure of D-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 A. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus D-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a ;closed' conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.
| S-EPMC2802865 | biostudies-literature
Project description: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.
| S-EPMC1152782 | biostudies-other
Project description:Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F420)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated Mycobacterium tuberculosis var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (ndh) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type ndh Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.
| S-EPMC7179590 | biostudies-literature
Project description:We have reinvestigated the conformation of NAD+ bound to dogfish lactate dehydrogenase (LDH) by using an NMR experiment that allows one to exploit nuclear Overhauser effects to determine internuclear distances between pairs of protons, without perturbation of spin-diffusion effects from other protons belonging either to the cofactor or to the binding pocket of the enzyme. The analysis indicates that the structure of bound NAD+ is in accord with the conformation determined in the solid state by x-ray diffraction for the adenosine moiety, but deviates significantly from that of the nicotinamide. The NMR data indicate conformational averaging about the glycosidic bond of the nicotinamide nucleotide. In view of the strict stereospecificity of catalysis by LDH and the conformational averaging of bound NAD+ that we infer from solution-state NMR, we suggest that LDH binds the cofactor in both syn and anti conformations, but that binding interactions in the syn conformation are not catalytically productive.
| S-EPMC20731 | biostudies-literature
Project description:The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.
| S-EPMC1142596 | biostudies-literature
Project description:1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.
| S-EPMC1161174 | biostudies-other
Project description:BACKGROUND & AIMS:Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. METHODS:Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, ?-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. RESULTS:Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. CONCLUSION:PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. LAY SUMMARY:Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.
| S-EPMC6057136 | biostudies-literature
Project description:BACKGROUND: Various Pseudomonas strains can use L-lactate as their sole carbon source for growth. However, the L-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied. METHODOLOGY/PRINCIPAL FINDINGS: An NAD-independent L-lactate dehydrogenase (L-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of L-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), L-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on L-lactate, but retained the ability to grow on pyruvate. CONCLUSIONS/SIGNIFICANCE: It is proposed that L-iLDH plays an indispensable function in Pseudomonas L-lactate utilization by catalyzing the conversion of L-lactate into pyruvate.
| S-EPMC3344892 | biostudies-literature