Roles of Arg427 and Arg472 in the Binding and Allosteric Effects of Acetyl CoA in Pyruvate Carboxylase

Authors

Abdussalam Adina-Zada, University of Western Australia
Chutima Sereerukb, Mahidol University
Sarawut Jitrapakdee, Mahidol University
Tonya N. Zeczycki, University of Wisconsin - MadisonFollow
Martin St. Maurice, Marquette UniversityFollow
W. Wallace Cleland, University of Wisconsin - MadisonFollow
John C. Wallace, University of Adelaide
Paul V. Attwood, University of Western Australia

Document Type

Article

Language

eng

Format of Original

10 p.

Publication Date

2012

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

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 Mg²⁺ 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.

Comments

Accepted version. Biochemistry, Vol. 51, No. 41 (2012): 8208-8217. DOI. © 2012 ACS Publications. Used with permission.

Recommended Citation

Adina-Zada, Abdussalam; Sereerukb, Chutima; Jitrapakdee, Sarawut; Zeczycki, Tonya N.; Maurice, Martin St.; Cleland, W. Wallace; Wallace, John C.; and Attwood, Paul V., "Roles of Arg427 and Arg472 in the Binding and Allosteric Effects of Acetyl CoA in Pyruvate Carboxylase" (2012). Biological Sciences Faculty Research and Publications. 118.
https://epublications.marquette.edu/bio_fac/118