Insight into the Carboxyl Transferase Domain Mechanism of Pyruvate Carboxylase from Rhizobium etli

Authors

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

Document Type

Article

Language

eng

Format of Original

9 p.

Publication Date

5-26-2009

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

DOI: 10.1021/bi9003759

Abstract

The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO₂ to form oxaloacetate and complete the reaction.

Comments

Accepted version. Biochemistry, Vol. 48, No. 20 (May 26, 2009): 4305-4313. DOI. © 2009 American Chemical Society. Used with permission.

Recommended Citation

Zeczycki, Tonya N.; Maurice, Martin St.; Jitrapakdee, Sarawut; Wallace, John C.; Attwood, Paul V.; and Cleland, W Wallace, "Insight into the Carboxyl Transferase Domain Mechanism of Pyruvate Carboxylase from Rhizobium etli" (2009). Biological Sciences Faculty Research and Publications. 290.
https://epublications.marquette.edu/bio_fac/290