title: Critical role of a lipoyl cofactor of the dihydrolipoyl acetyltransferase in the binding and enhanced function of the pyruvate dehydrogenase kinase.
abstract: Mammalian pyruvate dehydrogenase kinase binds to the lipoyl domain region of the core structure forming dihydrolipoyl acetyltransferase (E2) subunits. The bound kinase has a greatly enhanced rate in phosphorylating E2-bound pyruvate dehydrogenase (E1) tetramers versus the rate at which resolved kinase phosphorylates dissociated E1. This E2-activated kinase function was completely prevented by selective alkylation of reduced lipoyl groups while kinase and E1 binding to the E2 core were retained. Selective removal of lipoyl cofactors from intact E2 by treatment with Enterococcus faecalis lipoamidase decreased kinase activity by 4-fold and caused selective release of a major portion of the kinase from E2 in a sucrose-step gradient procedure. Selective and reversible modification of the lipoyl groups of E2 subunits also allowed the kinase to be dissociated under mildly chaotropic conditions. Thus, the lipoyl prosthetic group on one of the two lipoyl domains of E2 subunits is critically important for maintaining E2-activated kinase function and contributes to binding of the kinase to E2. Since removal of the lipoyl group weakened kinase binding to E2 more than modifying lipoyl thiols, it is suggested that the hydrophobic inner portion of the lipoyl conjugate (i.e., lysine carbons and C1 to C5 of the lipoic acid) is important in the binding of the kinase.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan 66506.
authors.forename: G A
authors.initials: GA
authors.lastname: Radke
authors.forename: K
authors.initials: K
authors.lastname: Ono
authors.forename: S
authors.initials: S
authors.lastname: Ravindran
authors.forename: T E
authors.initials: TE
authors.lastname: Roche
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000478:Alkylation; D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D066298:In Vitro Techniques; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D000581:Amidohydrolases; C023940:lipoamidase