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)

Propionate inhibited the metabolic flux through the pyruvate dehydrogenase reaction in the perfused rat liver when the perfusate concentration of propionate was below 10 mM and the perfusate pyruvate concentration was held within the physiological range. At higher propionate concentrations (e.g., 20 mM) the inhibition of pyruvate dehydrogenase was alleviated and the activation state of the pyruvate dehydrogenase complex was nearly doubled. In livers perfused with a high pyruvate concentration (e.g., 5 mM), propionate coinfusion at all concentrations inhibited the rate of pyruvate decarboxylation. Additional studies were performed in liver mitochondria maintained in State 3 where the ATP/ADP and the NADH/NAD+ ratios were held constant. Low propionate concentrations (e.g., 0.5 mM) inactivated the mitochondrial pyruvate dehydrogenase complex, whereas propionate levels in excess of 1 mM activated the enzyme complex. CoA distribution analyses of the mitochondrial incubations indicated that the presence of either 0.5 or 10 mM propionate caused a substantial accumulation of propionyl-CoA and methylmalonyl-CoA at the expense of free CoASH. Experiments were performed in which the ratios of various acyl-CoA derivatives to CoASH were varied by sequentially increasing the L-carnitine concentrations in the incubation. An inverse relationship between the propionyl-CoA/CoASH and methylmalonyl-CoA/CoASH ratios and the activity of the pyruvate dehydrogenase complex was observed. Experiments using freeze-thawed liver mitochondrial membranes indicated that propionate protected the pyruvate dehydrogenase complex from ATP-mediated inactivation by the pyruvate dehydrogenase kinase. It is our contention that the inactivation of pyruvate dehydrogenase complex at low propionate levels may be due to an increase in the mitochondrial acyl-CoA/CoASH ratios, whereas the activation of the enzyme complex demonstrated at high propionate levels is due to the inhibition of the pyruvate dehydrogenase kinase in a manner similar to that caused by pyruvate or dichloroacetic acid.
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PMID:The effect of propionate on the regulation of the pyruvate dehydrogenase complex in the rat liver. 682 32

The effects of myocardial ischemia and reperfusion on pyruvate dehydrogenase (PDH) activity were studied in isolated rat hearts. PDH remained largely (80%) in the active form during 10 min of whole heart ischemia in hearts receiving 11 mM glucose as substrate. With reperfusion, PDH was converted to the inactive form (45% by 2 min) and then returned slowly to control levels. Addition of pyruvate (10 mM) to the glucose containing perfusate during reperfusion prevent the reperfusion inactivation of PDH (96% active). The maintenance of a high percent of PDH in the active form during ischemia occurred in spite of high mitochondrial ratios of NADH/NAD and acetyl CoA/CoA and was related to a very low mitochondrial ATP/ADP ratio. The low ATP and high ADP would restrict PDH kinase phosphorylation and inactivation of PDH during ischemia. Reperfusion resulted in a rapid increase in mitochondrial ATP/ADP ratio and the increased availability of ATP as substrate for the kinase coupled with continued high levels of NADH and acetyl CoA which stimulate kinase activity may have accounted for the early inactivation of PDH with reperfusion. Addition of pyruvate to the perfusate probably inhibited the PDH kinase and prevent the reperfusion inactivation of PDH.
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PMID:Effects of ischemia and reperfusion on pyruvate dehydrogenase activity in isolated rat hearts. 687 85

Four mitochondrial protein kinases have been cloned. These proteins represent a new family of protein kinases, related by sequence to the bacterial protein kinases but by function to the eukaryotic serine protein kinases. Arg288 is required for recognition by BCKDK of the phosphorylation site on the E1alpha subunit of the BCKDH complex. BCKDK inhibits the dehydrogenase activity of the BCKDH complex by introducing a negative charge into the active-site pocket of the E1 component. Protein starvation of rats induces an increase in the amount of BCKDK bound to the BCKDH complex. This causes inactivation of the BCKDH complex and conserves branched-chain amino acids for protein synthesis in the protein-starved state. Expression of the different PDK isoenzymes is tissue specific, and the different PDK isoenzymes are unique with respect to kinetic parameters for ATP and ADP and sensitivity to allosteric effectors (NADH, NAD+, coenzyme A, acetyl-CoA, pyruvate, and dichloroacetate). Preliminary experiments indicate that an increased amount of PDK2 protein partly explains the increase in PDK activity that occurs in rat liver in response to chemically induced diabetes.
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PMID:Mitochondrial alpha-ketoacid dehydrogenase kinases: a new family of protein kinases. 934 45

The purpose of the study was to examine the roles of active pyruvate dehydrogenase (PDH(a)), glycogen phosphorylase (Phos), and their regulators in lactate (Lac(-)) metabolism during incremental exercise after ingestion of 0.3 g/kg of either NaHCO(3) [metabolic alkalosis (ALK)] or CaCO(3) [control (CON)]. Subjects (n = 8) were studied at rest, rest postingestion, and during constant rate cycling at three stages (15 min each): 30, 60, 75% of maximal O(2) uptake (VO(2 max)). Radial artery and femoral venous blood samples, leg blood flow, and biopsies of the vastus lateralis were obtained during each power output. ALK resulted in significantly (P < 0.05) higher intramuscular Lac(-) concentration ([Lac(-)]; ALK 72.8 vs. CON 65.2 mmol/kg dry wt), arterial whole blood [Lac(-)] (ALK 8.7 vs. CON 7.0 mmol/l), and leg Lac(-) efflux (ALK 10.0 vs. CON 4.2 mmol/min) at 75% VO(2 max). The increased intramuscular [Lac(-)] resulted from increased pyruvate production due to stimulation of glycogenolysis at the level of Phos a and phosphofructokinase due to allosteric regulation mediated by increased free ADP (ADP(f)), free AMP (AMP(f)), and free P(i) concentrations. PDH(a) increased with ALK at 60% VO(2 max) but was similar to CON at 75% VO(2 max). The increased PDH(a) may have resulted from alterations in the acetyl-CoA, ADP(f), pyruvate, NADH, and H(+) concentrations leading to a lower relative activity of PDH kinase, whereas the similar values at 75% VO(2 max) may have reflected maximal activation. The results demonstrate that imposed metabolic alkalosis in skeletal muscle results in acceleration of glycogenolysis at the level of Phos relative to maximal PDH activation, resulting in a mismatch between the rates of pyruvate production and oxidation resulting in an increase in Lac(-) production.
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PMID:Effect of induced metabolic alkalosis on human skeletal muscle metabolism during exercise. 1066 17

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.
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PMID:Marked differences between two isoforms of human pyruvate dehydrogenase kinase. 1074 34

The mechanism of action of structurally distinct pyruvate dehydrogenase kinase (PDK) inhibitors was examined in assays with experimental contexts ranging from an intact pyruvate dehydrogenase complex (PDC) with and without supplemental ATP or ADP to a synthetic peptide substrate to PDK autophosphorylation. Some compounds directly inhibited the catalytic activity of PDKs. Some of the inhibitor classes tested inhibited autophosphorylation of recombinant PDK1 and PDK2. During these studies, PDC was shown to be directly inhibited by a novel mechanism; the addition of supplemental recombinant PDKs, an effect that is ADP-dependent and partly alleviated by members of each of the compound classes tested. Overall, these data demonstrate that small molecules acting at diverse sites can inhibit PDK activity.
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PMID:Diverse mechanisms of inhibition of pyruvate dehydrogenase kinase by structurally distinct inhibitors. 1100 68

The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd approximately 175 microM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd approximately 3 microM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in approximately 8-fold tighter binding of PDK2 to GST-L2red, which was approximately 300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound approximately 18 PDK2 dimers with a Kd similar to GST-L2. E2.E1 bound more PDK2 (approximately 27.6) than E2 with approximately 2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2.E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5'-(beta,gamma-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2.E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed "hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate.
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PMID:Facilitated interaction between the pyruvate dehydrogenase kinase isoform 2 and the dihydrolipoyl acetyltransferase. 1281 49

The transacetylase component (E2) of PDC (pyruvate dehydrogenase complex) plays a critical role in the regulation of PDHK (pyruvate dehydrogenase kinase) activity. The present study was undertaken to investigate further the molecular mechanism by which E2 modulates the activity of PDHK. In agreement with the earlier results, it was found that the inner L2 (lipoyl-bearing domain 2) of E2 expressed with or without the C-terminal hinge region had little, if any, effect on the kinase activity, indicating a lack of direct allosteric effect of L2 on PDHK. In marked contrast, significant activation of PDHK was observed with the construct consisting of L2 and the E1BD (E1-binding domain) of E2 (L2-E1BD didomain) suggesting that co-localization and/or mutual orientation of PDHK and E1, facilitated by E2 binding, largely account for the activation of PDHK by the transacetylase component. Isothermal titration calorimetry and glutathione S-transferase pull-down assays established that binding of adenyl nucleotides to the PDHK molecule facilitated the release of L2 domain. In contrast, binding of the L2 domain caused a significant decrease in the affinity of PDHK for ATP. The cross-talk in binding of adenyl nucleotides and the L2 domain to PDHK may indicate the existence of a highly integrated mechanism whereby the exchange of lipoyl-bearing domains presented to PDHK by E2 is coupled with ADP/ATP exchange.
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PMID:Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity. 1550 8

The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.
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PMID:R-lipoic acid inhibits mammalian pyruvate dehydrogenase kinase. 1551 96

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.
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PMID:Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex. 1586 Nov 26


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