Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli

Authors

Tonya N. Zeczycki, University of Wisconsin - MadisonFollow
Ann L. Menefee, Marquette University
Sarawut Jitrapakdee, Mahidol University
John C. Wallace, University of Adelaide
Paul V. Attwood, University of Western Australia
Martin St. Maurice, Marquette UniversityFollow
W Wallace Cleland, University of Wisconsin - MadisonFollow

Document Type

Article

Language

eng

Format of Original

14 p.

Publication Date

2011

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Abstract

While crystallographic structures of the R. etli pyruvate carboxylase (PC) holoenzyme revealed the location and probable positioning of the essential activator, Mg²⁺, and nonessential activator, acetyl-CoA, an understanding of how they affect catalysis remains unclear. The current steady-state kinetic investigation indicates that both acetyl-CoA and Mg²⁺ assist in coupling the MgATP-dependent carboxylation of biotin in the biotin carboxylase (BC) domain with pyruvate carboxylation in the carboxyl transferase (CT) domain. Initial velocity plots of free Mg²⁺ vs pyruvate were nonlinear at low concentrations of Mg²⁺ and a nearly complete loss of coupling between the BC and CT domain reactions was observed in the absence of acetyl-CoA. Increasing concentrations of free Mg²⁺ also resulted in a decrease in the K_a for acetyl-CoA. Acetyl phosphate was determined to be a suitable phosphoryl donor for the catalytic phosphorylation of MgADP, while phosphonoacetate inhibited both the phosphorylation of MgADP by carbamoyl phosphate (K_i = 0.026 mM) and pyruvate carboxylation (K_i = 2.5 mM). In conjunction with crystal structures of T882A R. etli PC mutant cocrystallized with phosphonoacetate and MgADP, computational docking studies suggest that phosphonoacetate could coordinate to one of two Mg²⁺ metal centers in the BC domain active site. Based on the pH profiles, inhibition studies, and initial velocity patterns, possible mechanisms for the activation, regulation, and coordination of catalysis between the two spatially distinct active sites in pyruvate carboxylase from R. etli by acetyl-CoA and Mg²⁺ are described.

Comments

Accepted version. Biochemistry, Vol. 50, No. 45 (2011): 9694–9707. DOI. © 2011 American Chemical Society Publications. Used with permission.

Recommended Citation

Zeczycki, Tonya N.; Menefee, Ann L.; Jitrapakdee, Sarawut; Wallace, John C.; Attwood, Paul V.; St. Maurice, Martin; and Cleland, W Wallace, "Activation and Inhibition of Pyruvate Carboxylase from Rhizobium etli" (2011). Biological Sciences Faculty Research and Publications. 124.
https://epublications.marquette.edu/bio_fac/124