title: Roles of Arg427 and Arg472 in the binding and allosteric effects of acetyl CoA in pyruvate carboxylase.
abstract: Mutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation constant (K(a)) of acetyl CoA, with the increase being greater for the Arg472 mutants. These results indicate that while both these residues are involved in the binding of acetyl CoA to the enzyme, Arg472 is more important than Arg427. The mutations had substantially smaller effects on the k(cat) for pyruvate carboxylation. Part of the effects of the mutations was to increase the K(m) for MgATP and the K(a) for activation by free Mg(2+) determined at saturating acetyl CoA concentrations. The inhibitory effects of the mutations on the rates of the enzyme-catalyzed bicarbonate-dependent ATP cleavage, carboxylation of biotin, and phosphorylation of ADP by carbamoyl phosphate indicate that the major locus of the effects of the mutations was in the biotin carboxylase (BC) domain active site. Even though both Arg427 and Arg472 are distant from the BC domain active site, it is proposed that their contacts with other residues in the allosteric domain, either directly or through acetyl CoA, affect the positioning and orientation of the biotin-carboxyl carrier protein (BCCP) domain and thus the binding of biotin at the BC domain active site. On the basis of the kinetic analysis proposed here, it is proposed that mutations of Arg427 and Arg472 perturb these contacts and consequently the binding of biotin at the BC domain active site. Inhibition of pyruvate carboxylation by the allosteric inhibitor l-aspartate was largely unaffected by the mutation of either Arg427 or Arg472.
authors.affiliation: School of Chemistry and Biochemistry, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.
authors.forename: Abdussalam
authors.initials: A
authors.lastname: Adina-Zada
authors.forename: Chutima
authors.initials: C
authors.lastname: Sereeruk
authors.forename: Sarawut
authors.initials: S
authors.lastname: Jitrapakdee
authors.forename: Tonya N
authors.initials: TN
authors.lastname: Zeczycki
authors.forename: Martin
authors.initials: M
authors.lastname: St Maurice
authors.forename: W Wallace
authors.initials: WW
authors.lastname: Cleland
authors.forename: John C
authors.initials: JC
authors.lastname: Wallace
authors.forename: Paul V
authors.initials: PV
authors.lastname: Attwood
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011766:Pyruvate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000244:Adenosine Diphosphate; D001710:Biotin; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D001120:Arginine; D011766:Pyruvate Carboxylase