Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.2 (PDK1)
2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.
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PMID:Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate. 221 97

The effects of various metabolites on pyruvate dehydrogenase (PDH) kinase-catalyzed inactivation of the pyruvate dehydrogenase complex (PDC) were studied in extracts of mitochondria purified from green leaf tissue of Pisum sativum L. Pyruvate was an uncompetitive inhibitor of PDH kinase with respect to ATP whereas ADP was a competitive inhibitor. In the absence of pyruvate a fivefold excess of ADP over ATP was required to inhibit PDH kinase, however, in the presence of pyruvate much lower ADP concentrations were required. Inhibition of PDH kinase by pyruvate and ADP was synergistic and the addition of ADP changed pyruvate from an uncompetitive inhibitor to a noncompetitive inhibitor. This result indicates that pyruvate acts as a "dead-end" inhibitor, binding to the PDH kinase-ADP reaction intermediate. Evidence is also presented that inhibition by pyruvate in the presence of thiamine pyrophosphate is due to the formation of hydroxyethyl thiamine pyrophosphate. The results are discussed in terms of the regulation of PDC activity by pyruvate and ADP during periods of increased demand for carbon skeleton biosynthesis by way of the tricarboxylic acid (TCA) cycle despite constraints imposed on TCA cycle flux by a high ATP/ADP ratio.
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PMID:Mechanism of pyruvate inhibition of plant pyruvate dehydrogenase kinase and synergism with ADP. 232 60

The effects of monovalent ions on endogenous pyruvate dehydrogenase (PDH) kinase activity in purified bovine heart pyruvate dehydrogenase complex were investigated. Activity of PDH kinase was stimulated 1.9-, 1.95-, 1.65-, and 1.4-fold by 10 mM K+, Rb+, NH+4, and Cs+, respectively, whereas Na+ and Li+ had no effect on PDH kinase activity. The crystal radii of stimulatory ions were in the range of 1.33 to 1.69 A while the crystal radii of nonstimulatory ions were in the range of 0.6 to 0.94 A. Stimulation of PDH kinase by monovalent ions was not pH dependent. Protein dilution studies showed that monovalent ion stimulation was measurable within 10 s after protein addition to PDH kinase assays. Furthermore, stimulation occurred at all protein concentrations tested. At ATP concentrations from 12.5 to 25 microM, K+ and NH+4 stimulation was constant from 0 to 110 and 0 to 30 mM, respectively. At higher ATP concentrations, from 50 to 500 microM, K+ and NH+4 stimulation peaked at approximately 30 and 3 mM, respectively, and thereafter declined as the ion concentration increased. Maximal PDH kinase stimulation by K+ or NH+4 also declined as Na+ was increased from 0 to 120 mM, but at a fixed salt concentration of 120 mM, both K+ and NH+4 stimulated PDH kinase activity. Phosphopeptide analysis demonstrated that K+ and NH+4 stimulated phosphorylation at sites 1 and 2, but that site 3 phosphorylation was relatively constant under all conditions. Thiamin pyrophosphate and 5,5'-dithiobis-(2-nitrobenzoate) blocked monovalent ion stimulation half-maximally at 4 and 6 microM, respectively. However, neither thiamin pyrophosphate nor 5,5'-dithiobis-(2-nitrobenzoate) significantly inhibited PDH kinase activity in the absence of monovalent ions. The results indicate that heart PDH kinase stimulation by monovalent ions does not occur by changing the binding equilibrium between PDH and dihydrolipoyl transacetylase core. Instead, monovalent ions bind and exert their regulatory effects at or near the active site of PDH kinase.
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PMID:Bovine heart pyruvate dehydrogenase kinase stimulation by monovalent ions. 274 10

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.
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PMID:[Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex]. 282 86

The branched-chain alpha-ketoacid dehydrogenase complex, like the pyruvate dehydrogenase complex, is an intramitochondrial enzyme subject to regulation by covalent modification. Phosphorylation causes inactivation and dephosphorylation causes activation of both complexes. The branched-chain alpha-ketoacid dehydrogenase kinase, believed distinct from pyruvate dehydrogenase kinase, is an integral component of the branched-chain alpha-ketoacid dehydrogenase complex and is sensitive to inhibition by branched-chain alpha-ketoacids, alpha-chloroisocaproate, phenylpyruvate, clofibric acid, octanoate and dichloroacetate. Phosphorylation of branched-chain alpha-ketoacid dehydrogenase occurs at two closely-linked serine residues (sites 1 and 2) of the alpha-subunit of the decarboxylase. HPLC and sequence data suggest homology of the amino acid sequence adjacent to phosphorylation sites 1 and 2 of complexes isolated from several different tissues. Stoichiometry for phosphorylation of all of the complexes studies was about 1 mol P/mol alpha-subunit for 95% inactivation and 1.5 mol P/mol alpha-subunit for maximally phosphorylated complex. Site 1 and site 2 were phosphorylated at similar rates until total phosphorylation exceeded 1 mol P/mol alpha-subunit. The complexes from rabbit kidney, rabbit heart, and rat heart showed 30-40% additional phosphorylation of the alpha-subunit beyond 95% inactivation. Site specificity studies carried out with the kinase partially inhibited with alpha-chloroisocaproate suggest that phosphorylation of site 1 is primarily responsible for regulation of the complex. The capacity of the branched-chain alpha-ketoacid dehydrogenase to oxidize pyruvate (Km = 0.8 mM, Vmax = 20% of that of alpha-ketoisovalerate) interferes with the estimation of activity state of the hepatic pyruvate dehydrogenase complex. The disparity between the activity states of the two complexes in most physiologic states contributes to this interference. An inhibitory antibody for branched-chain alpha-ketoacid dehydrogenase can be used to prevent interference with the pyruvate dehydrogenase assay. Almost all of the hepatic branched-chain alpha-ketoacid dehydrogenase in chow-fed rats is active (greater than 90% dephosphorylated). In contrast, almost all of the hepatic enzyme of rats fed a low-protein (8%) diet is inactive (greater than 85% phosphorylated). Fasting of chow-fed rats has no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e. greater than 90% of the enzyme remains in the active state. However, fasting of rats maintained on low-protein diets greatly activates the hepatic enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Regulation of branched-chain alpha-ketoacid dehydrogenase complex by covalent modification. 302 49

Tryptic digestion of the fully phosphorylated Ascaris suum pyruvate dehydrogenase complex yielded a single tetradecapeptide containing 2 phosphorylated serine residues. Its amino acid sequence was Tyr-Ser-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Ser(P)-Tyr-Arg and was very similar to one of the tryptic phosphopeptides isolated from mammalian and yeast pyruvate dehydrogenases. At partial phosphorylation, three peptides were isolated which corresponded to the monophosphorylated (sites 1 and 2) and diphosphorylated tetradecapeptides. In contrast to results reported from mammalian complexes, phosphorylation of the ascarid complex paralleled inactivation, and no additional phosphorylation occurred after inactivation was complete. Complete inactivation of the complex was associated with the incorporation of 1.7-1.9 mol of phosphoryl groups/mol of alpha-pyruvate dehydrogenase subunit, and the strict preference of the pyruvate dehydrogenase kinase for site 1 was not observed. Whereas site 1 was initially phosphorylated more rapidly than site 2, at 50% inactivation, 41% of the incorporated phosphoryl groups were incorporated into site 2. In addition, substantial amounts of peptide monophosphorylated at site 2 also accumulated, suggesting that prior phosphorylation at site 1 was not necessary for phosphorylation at site 2. Phosphorylation also caused a marked decrease in the mobility of the alpha-pyruvate dehydrogenase subunit on sodium dodecyl sulfate-polyacrylamide gels and the apparent separation of mono- and diphosphorylated forms of the enzyme. The significance of these observations in the regulation of the unique anaerobic mitochondrial metabolism of A. suum is discussed.
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PMID:Phosphorylation and inactivation of the pyruvate dehydrogenase from the anaerobic parasitic nematode, Ascaris suum. Stoichiometry and amino acid sequence around the phosphorylation sites. 319 13

The work investigated the mechanisms for modulation of renal and hepatic pyruvate dehydrogenase complex (PDH) activities after carbohydrate re-feeding of 48 h-starved rats, and identified a regulatory role for tri-iodothyronine. Glucose re-feeding decreased blood concentrations of lipid fuels in both euthyroid and hyperthyroid rats. This treatment was not associated with re-activation of hepatic PDH in either group of rats, or of renal PDH in hyperthyroid rats (where activity was already high), but it increased renal PDH in euthyroid rats. Dichloroacetate (DCA), an activator of PDH kinase, increased renal PDH activities in euthyroid rats, but not hyperthyroid rats, and effects of glucose re-feeding or hyperthyroidism were no longer apparent. These treatments therefore exert their effects on renal PDH through changes in PDH kinase. DCA re-activation of hepatic PDH was more marked in hyperthyroid than in euthyroid rats, suggesting that, under conditions of inhibited kinase activity, PDH phosphatase is more active in livers of hyperthyroid rats. The limited effect of DCA on hepatic PDH in euthyroid rats was potentiated by glucose re-feeding or insulin, but not by inhibition of lipolysis, demonstrating a direct effect of insulin to increase hepatic PDH phosphatase. Glucose re-feeding, inhibition of lipolysis or insulin administration did not increase hepatic PDH in DCA-treated hyperthyroid rats, indicating that effects of hyperthyroidism and of insulin on PDH phosphatase are not additive.
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PMID:Regulation of renal and hepatic pyruvate dehydrogenase complex on carbohydrate re-feeding after starvation. Possible mechanisms and a regulatory role for thyroid hormone. 329 32

The effect of chronic sepsis on the concentration of active pyruvate dehydrogenase complex has been investigated in liver and skeletal muscle of normal, sterile inflammatory, and chronic septic (small and large abscess) animals. Hyperdynamic sepsis was induced by the intraperitoneal introduction of a rat fecal-agar pellet of known size and bacterial composition (Escherichia coli + Bacteroides fragilis). Total pyruvate dehydrogenase complex activity was not altered in either liver or skeletal muscle in any of the conditions studied. In hepatic tissue, sterile inflammation increased the proportion of active complex 2.5-fold compared with control. The same increase in the concentration of active complex was observed in animals with a small abscess. When the abscess size was increased (large abscess), the concentration of active complex was decreased relative to sterile inflammatory or small abscess septic animals. In contrast to liver, sterile inflammation did not alter the proportion of active complex in skeletal muscle. Sepsis (either small or large septic abscess) resulted in threefold decrease in the concentration of active complex relative to control or sterile inflammatory animals. Changes in the concentration of active complex did not appear to be dependent on the ATP/ADP concentration ratio or tissue pyruvate levels but were consistent with changes in the acetyl-coenzyme A-to-coenzyme A concentration ratio. The mechanism responsible for altered concentration of active complex may be mediated through changes in the activity of the pyruvate dehydrogenase kinase, secondary to alterations in the effector concentration ratios.
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PMID:Effect of sepsis on activity of pyruvate dehydrogenase complex in skeletal muscle and liver. 352 10

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.
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PMID:Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast. 353 83

In contrast to the pyruvate dehydrogenase complex (PDC) from animal mitochondria, our in situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings. Pyruvate was a competitive inhibitor of ATP-dependent inactivation (Ki = 59 microM), while the PDC had a Km for pyruvate of microM. Thiamine pyrophosphate, the coenzyme for the pyruvate dehydrogenase (PDH) component of the complex, did not inhibit ATP-dependent inactivation when used alone but it enhanced inhibition by pyruvate. As such, thiamine pyrophosphate was a competitive inhibitor (Ki = 130 nM) of ATP-dependent inactivation. A model is proposed for the pyruvate plus thiamine pyrophosphate inhibition of ATP-dependent inactivation of the pyruvate dehydrogenase complex in which pyruvate exerts its inhibition of inactivation by altering or protecting the protein substrate from phosphorylation and not by directly inhibiting PDH kinase.
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PMID:Regulation of pea mitochondrial pyruvate dehydrogenase complex activity: inhibition of ATP-dependent inactivation. 367 88


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