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)

Five mitochondrial protein kinases, all members of a new family of protein kinases, have now been identified, cloned, expressed as recombinant proteins, and partially characterized with respect to catalytic and regulatory properties. Four members of this unique family of eukaryotic protein kinases correspond to pyruvate dehydrogenase kinase isozymes which regulate the activity of the pyruvate dehydrogenase complex, an important regulatory enzyme at the interface between glycolysis and the citric acid cycle. The fifth member of this family corresponds to the branched-chain alpha-ketoacid dehydrogenase kinase, an enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, the most important regulatory enzyme in the pathway for the disposal of branched-chain amino acids. At least three long-term control mechanisms have evolved to conserve branched chain amino acids for protein synthesis during periods of dietary protein insufficiency. Increased expression of the branched-chain alpha-ketoacid dehydrogenase kinase is perhaps the most important because this leads to phosphorylation and nearly complete inactivation of the liver branched-chain alpha-ketoacid dehydrogenase complex. Decreased amounts of the liver branched-chain alpha-ketoacid dehydrogenase complex secondary to a decrease in liver mitochondria also decrease the liver's capacity for branched-chain keto acid oxidation. Finally, the number of E1 subunits of the branched-chain alpha-ketoacid dehydrogenase complex is reduced to less than a full complement of 12 heterotetramers per complex in the liver of protein-starved rats. Since the E1 component is rate-limiting for activity and also the component of the complex inhibited by phosphorylation, this decrease in number further limits overall enzyme activity and makes the complex more sensitive to regulation by phosphorylation in this nutritional state. The branched-chain alpha-ketoacid dehydrogenase kinase phosphorylates serine 293 of the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Site-directed mutagenesis of amino acid residues surrounding serine 293 reveals that arginine 288, histidine 292 and aspartate 296 are critical to dehydrogenase activity, that histidine 292 is critical to binding the coenzyme thiamine pyrophosphate, and that serine 293 exists at or in close proximity to the active site of the dehydrogenase. Alanine scanning mutagenesis of residues in the immediate vicinity of the phosphorylation site (serine 293) indicates that only arginine 288 is required for recognition of serine 293 as a phosphorylation site by the branched-chain alpha-ketoacid dehydrogenase kinase. Phosphorylation appears to inhibit dehydrogenase activity by introducing a negative charge directly into the active site pocket of the E1 dehydrogenase component of the branched-chain alpha-ketoacid dehydrogenase complex. A model based on the X-ray crystal structure of transketolase is being used to predict residues involved in thiamine pyrophosphate binding and to help visualize how phosphorylation within the channel leading to the reactive carbon of thiamine pyrophosphate inhibits catalytic activity. The isoenzymes of pyruvate dehydrogenase kinase differ greatly in terms of their specific activities, kinetic parameters and regulatory properties. Chemically-induced diabetes in the rat induces significant changes in the pyruvate dehydrogenase kinase isoenzyme 2 in liver. Preliminary findings suggest hormonal control of the activity state of the pyruvate dehydrogenase complex may involves tissue specific induced changes in expression of the pyruvate dehydrogenase kinase isoenzymes.
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PMID:Studies on the regulation of the mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases. 938 74

The administration of a low-carbohydrate/high-saturated-fat (LC/HF) diet for 28 days or starvation for 48 h both increased pyruvate dehydrogenase kinase (PDHK) activity in extracts of rat hepatic mitochondria, by approx. 2.1-fold and 3.5-fold respectively. ELISAs of extracts of hepatic mitochondria, conducted over a range of pyruvate dehydrogenase (PDH) activities, revealed that mitochondrial immunoreactive PDHKII (the major PDHK isoform in rat liver) was significantly increased by approx. 1.4-fold after 28 days of LC/HF feeding and by approx. 2-fold after 48 h of starvation. The effect of LC/HF feeding to increase hepatic PDHK activity was retained through hepatocyte preparation, but was decreased on 21 h culture with insulin (100 micro-i.u./ml). A sustained (24 h) 2-4-fold elevation in plasma insulin concentration in vivo (achieved by insulin infusion via an osmotic pump) suppressed the effect of LC/HF feeding so that hepatic PDHK activities did not differ significantly from those of (insulin-infused) control rats. The increase in hepatic PDHK activity evoked by 28 days of LC/HF feeding was prevented and reversed (within 24 h) by the replacement of 7% of the dietary lipid with long-chain omega-3 fatty acids. Analysis of hepatic membrane lipid revealed a 1.9-fold increase in the ratio of total polyunsaturated omega-3 fatty acids to total mono-unsaturated fatty acids. The results indicate that the increased hepatic PDHK activities observed in livers of LC/HF-fed or 48 h-starved rats are associated with long-term actions to increase hepatic PDHKII concentrations. The long-term regulation of hepatic PDHK by LC/HF feeding might be achieved through an impaired action of insulin to suppress PDHK activity. In addition, the fatty acid composition of the diet, rather than the fat content, is a key influence.
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PMID:Studies of the long-term regulation of hepatic pyruvate dehydrogenase kinase. 940 79

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50nmol/min per mg for PDK2 to 1250nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.
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PMID:Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex. 940 93

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.
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PMID:Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart. 940 94

It is generally believed that mammalian pyruvate dehydrogenase kinase is a heterodimer consisting of catalytic and regulatory subunits. However, the contribution of the two subunits to the kinase-mediated signal transduction has remained undefined. In the present study recombinant components of mammalian pyruvate dehydrogenase complex were employed in order to characterize the role of the kinase catalytic subunit in the regulation of pyruvate dehydrogenase reaction. The results provide the first evidence strongly suggesting that the catalytic subunit of pyruvate dehydrogenase kinase is competent to respond to known effectors of kinase activity as well as to interact with the E2-core without assistance of a regulatory subunit.
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PMID:Regulation of mammalian pyruvate dehydrogenase kinase. 942 33

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum. A cDNA coding for an A. suum pyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)+ RNA isolated from adult A. suum muscle.2 APDK exhibited significant sequence identity to mammalian PDKs. Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide spliced leader, characteristic of many nematode mRNAs, a 5'-UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3'-UTR of 101 nucleotides that included a putative polyadenylation signal. The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide. Recombinant APDK (rAPDK) was functionally expressed in Escherichia coli with a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA-agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adult A. suum muscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex. After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios within the same ranges as observed for the native APDK. Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI 6.1-6.8) on two-dimensional gels of the purified adult A. suum muscle PDC.
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PMID:Molecular cloning, functional expression, and characterization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum. 957 13

The dihydrolipoyl acetyltransferase (E2 component) is a 60-mer assembled via its COOH-terminal domain with exterior E1-binding domain and two lipoyl domains (L2 then L1) sequentially connected by mobile linker regions. E2 facilitates markedly enhanced function of the pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). Human E2 structures were prepared with only one lipoyl domain (L1 or L2) or with alanines substituted at the sites of lipoylation (Lys-46 in L1 or Lys-173 in L2). The L2 domain and its lipoyl group were shown to be essential for markedly enhanced PDP function and were required for greatly up-regulated PDK function. The complete absence of the L1 domain reduced the enhancements of both of these activities but not the maximal effector-stimulated PDK activity through acetylation of L2. With nonlipoylated L2 present, lipoylated L1 supported a lesser enhancement in PDK function with significant stimulation upon acetylation of L1. Prevention of L1 lipoylation in K46AE2 removed this competitive L1 role and enhanced L2-facilitated PDK activity beyond that of native E2 when PDK activity was measured in the absence or in the presence of stimulatory effectors. Thus, the E2-L2 domain has a paramount role in facilitating enhanced PDK and PDP function but inclusion of E2-L1 domain, even in a noninteracting (nonlipoylated) form, contributes to the marked elevation of these activities.
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PMID:Requirements for the adaptor protein role of dihydrolipoyl acetyltransferase in the up-regulated function of the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. 960 12

Hibernation is a physiological adaptation characterized by dramatic decreases in heart rate, body temperature, and metabolism, resulting in long-term dormancy. Hibernating mammals survive for periods up to 6 mo in the absence of food by minimizing carbohydrate catabolism and using triglyceride stores as their primary source of fuel. The cellular and molecular mechanisms underlying the changes from a state of activity to the hibernating state are poorly understood; however, the selective expression of genes offers one level of control. To address this problem, we used a differential gene expression screen to identify genes that are responsible for the physiological characteristics of hibernation in the heart of the thirteen-lined ground squirrel (Spermophilus tridecemlineatus). Here, we report that genes for pancreatic lipase and pyruvate dehydrogenase kinase isozyme 4 are up-regulated in the heart during hibernation. Pancreatic lipase is normally expressed exclusively in the pancreas, but when expressed in the hibernating heart it liberates fatty acids from triglycerides at temperatures as low as 0 degreesC. Pyruvate dehydrogenase kinase isozyme 4 inhibits carbohydrate oxidation and depresses metabolism by preventing the conversion of pyruvate to Ac-CoA. The resulting anaerobic glycolysis and low-temperature lipid catabolism provide evidence that adaptive changes in cardiac physiology are controlled by the differential expression of genes during hibernation.
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PMID:Low-temperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal. 965 97

Two maize cDNAs were isolated and sequenced that had open reading frames with approximately 37% amino acid identity to mammalian pyruvate dehydrogenase kinases. Both maize kinase sequences contain the five domains with conserved signature residues typical of procaryotic two-component histidine kinases. Sequence comparisons identified six other highly conserved motifs that are proposed to be specific to pyruvate dehydrogenase kinases. In addition, specific Trp and Cys residues are also invariant in these sequences. The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated molecular masses of 38,867 and 41,327 Da that share 77% amino acid identity. Reverse transcriptase-polymerase chain reaction analysis with oligonucleotide-specific primers revealed a differential expression pattern for the two isoforms. PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His6 tags to facilitate purification. The recombinant proteins migrated at 44 and 48 kDa, respectively, during SDS-polyacrylamide gel electrophoresis. Anti-PDK1 antibodies immunoprecipitated 75% of pyruvate dehydrogenase kinase activity from a maize mitochondrial matrix fraction, and recognized a matrix protein of 43 kDa. Recombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of 32P from [gamma-32P]ATP into the alpha subunit of pyruvate dehydrogenase.
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PMID:Molecular analysis of two pyruvate dehydrogenase kinases from maize. 975 1

Recent experimental findings on the structural--functional features of pyruvate dehydrogenase phosphatase (PDP) isolated from various sources are compared. Two alternative mechanisms (a and b) of dephosphorylation of the E1 component in the pyruvate dehydrogenase complex (PDC) are discussed: a) the reaction occurs as a result of stochastic collisions of PDP and PDC, and the generation of an enzyme--substrate complex (PDP--E1--PDC) and dephosphorylation of the E1 component occur independently at different PDP binding sites on the PDC core; b) the dephosphorylation is performed simultaneously by a certain number of PDP molecules symmetrically bound on the PDC core. The second mechanism is suggested by the self-assembly theory of multicomponent enzyme systems and can be proved by kinetic experiments. Based on self-assembly principles and data on feasible binding sites of peripheral components of the PDC, the stoichiometry and mutual location of PDP, pyruvate dehydrogenase kinase, and the E1 component on the core of mammalian PDC are postulated to provide optimal functioning of the PDC. Structural mechanisms of stimulation of PDP activity by Ca2+ and polyamines are also discussed.
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PMID:A model for the spatial location of pyruvate dehydrogenase phosphatase in mammalian pyruvate dehydrogenase complex. 1020 2


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