title: Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP.
abstract: Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
authors.forename: Haiying
authors.initials: H
authors.lastname: Bao
authors.forename: Shane A
authors.initials: SA
authors.lastname: Kasten
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Yan
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Roche
mesh_terms: D000105:Acetyl Coenzyme A / Q000737:chemistry; D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D002384:Catalysis; D003999:Dichloroacetic Acid / Q000737:chemistry; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism; D019289:Pyruvic Acid / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; C000631430:PDK2 protein, human; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D000244:Adenosine Diphosphate; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D003999:Dichloroacetic Acid; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases