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Title: Roles of Arg427 and Arg472 in the binding and allosteric effects of acetyl CoA in pyruvate carboxylase
Authors: Abdussalam Adina-Zada
Chutima Sereeruk
Sarawut Jitrapakdee
Tonya N. Zeczycki
Martin St. Maurice
W. Wallace Cleland
John C. Wallace
Paul V. Attwood
University of Western Australia
Mahidol University
The Brody School of Medicine
Marquette University
University of Wisconsin Madison, Institute for Enzyme Research
University of Adelaide
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 16-Oct-2012
Citation: Biochemistry. Vol.51, No.41 (2012), 8208-8217
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. © 2012 American Chemical Society.
ISSN: 15204995
Appears in Collections:Scopus 2011-2015

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