Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2.
Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.
Hiromasa
Yasuaki
Y
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
Hu
Liangyan
L
Roche
Thomas E
TE
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D019281:Dimerization; D015536:Down-Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D019289:Pyruvic Acid / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D008024:Ligands; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D000244:Adenosine Diphosphate; D019289:Pyruvic Acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases