Critical role of specific ions for ligand-induced changes regulating pyruvate dehydrogenase kinase isoform 2.
In the complete absence of K+ and phosphate (Pi), pyruvate dehydrogenase kinase isoform 2 (PDHK2) was catalytically very active but with an elevated Km for ATP, and this activity is insensitive to effector regulation. We find that K+ or 5-fold lower levels of NH4+ markedly enhanced quenching of Trp383 fluorescence of PDHK2 by ADP and ATP. K+ binding caused an approximately 40-fold decrease in the equilibrium dissociation constants (Kd) for ATP from approximately 120 to 3.0 microM and an approximately 25-fold decrease in Kd for ADP from approximately 950 to 38 microM. Linked reductions in Kd of PDHK2 for K+ were from approximately 30 to approximately 0.75 mM with ATP bound and from approximately 40 to approximately 1.7 mM with ADP bound. Without K+, there was little effect of ADP on pyruvate binding, but with 100 mM K+ and 100 microM ADP, the L0.5 of PDHK2 for pyruvate was reduced by approximately 14 fold. In the absence of K+, Pi had small effects on ligand binding. With 100 mM K+, 20 mM Pi modestly enhanced binding of ADP and hindered pyruvate binding but markedly enhanced the binding of pyruvate with ADP; the L0.5 for pyruvate was specifically decreased approximately 125-fold with 100 microM ADP. Pi effects were minimal when NH4+ replaced K+. We have quantified coupled binding of K+ with ATP and ADP and elucidated how linked K+ and Pi binding are required for the potent inhibition of PDHK2 by ADP and pyruvate.
Hiromasa
Yasuaki
Y
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Roche
Thomas E
TE
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D005453:Fluorescence; D006801:Humans; D007477:Ions / Q000378:metabolism / Q000494:pharmacology*; D008024:Ligands; D015636:Magnesium Chloride / Q000494:pharmacology; D008958:Models, Molecular; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D019289:Pyruvic Acid / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
D007477:Ions; D008024:Ligands; D010710:Phosphates; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D000644:Quaternary Ammonium Compounds; D015636:Magnesium Chloride; D000244:Adenosine Diphosphate; D019289:Pyruvic Acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D011188:Potassium