title: Specific ion influences on self-association of pyruvate dehydrogenase kinase isoform 2 (PDHK2), binding of PDHK2 to the L2 lipoyl domain, and effects of the lipoyl group-binding site inhibitor, Nov3r.
abstract: Association of the PDHK2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase (E2)) dimers was enhanced by K+ with higher affinity K+ binding than occurs at the PDHK2 active site. Supporting a distinct K+ binding site, the NH4+ ion did not effectively replace K+ in aiding GST-L2 binding. With 50 mM K+, Pi enhanced interference by ADP, ATP, or pyruvate of PDHK2 binding to GST-L2. The inclusion of Pi with ADP or ATP plus pyruvate greatly hindered PDHK2 binding to GST-L2 and promoted PDHK2 forming a tetramer. Reciprocally, GST-L2 interference with ATP/ADP binding also required elevated K+ and was increased by Pi. Potent inhibition by Nov3r of E2-activated PDHK2 activity (IC50 of approximately 7.8 nM) required elevated K+ and Pi. Nov3r only modestly inhibited the low activity of PDHK2 without E2. By binding at the lipoyl group binding site, Nov3r prevented PDHK2 binding to E2 and GST-L2. Nov3r interfered with high-affinity binding of ADP and pyruvate via a Pi-dependent mechanism. Thus, GST-L2 binding to PDHK2 is supported by K+ binding at a site distinct from the active site. Pi makes major contributions to ligands interfering with PDHK2 binding to GST-L2, the conversion of PDHK2 dimer to a tetramer, and Nov3r (an acetyl-lipoate analog) interfering with binding of ADP and pyruvate. Pi is suggested to facilitate transmission within PDHK2 of the stimulatory signal of acetylation from the distal lipoyl-group binding site to the active site.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Hiromasa
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Yan
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Roche
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000813:Anilides / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D019281:Dimerization; D005453:Fluorescence; D006801:Humans; D007477:Ions / Q000494:pharmacology; D050356:Lipid Metabolism / Q000187:drug effects; D008055:Lipids / Q000737:chemistry; D054878:Lipoylation / Q000187:drug effects; D008958:Models, Molecular; D010710:Phosphates / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000187:drug effects; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
chemical_list: C523124:AZD 7545; D000813:Anilides; D007477:Ions; D008055:Lipids; D010710:Phosphates; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D000644:Quaternary Ammonium Compounds; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000123:Acetyltransferases; D017346:Protein Serine-Threonine Kinases; D011188:Potassium