Probing the Catalytic Roles of Arg548 and Gln552 in the Carboxyl Transferase Domain of the Rhizobium etli Pyruvate Carboxylase by Site-directed Mutagenesis

Authors

Saowapa Duangpan, Mahidol University
Sarawut Jitrapakdee, Mahidol University
Abdussalam Adina-Zada, University of Western Australia
Lindsay Byrne, University of Western Australia
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

9 p.

Publication Date

4-2010

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

10.1021/bi901894t

Abstract

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase.

Comments

Accepted version. Biochemistry, Vol. 49, No. 15 (April 2010): 3296-3304. DOI. © 2010 American Chemical Society. Used with permission.

Recommended Citation

Duangpan, Saowapa; Jitrapakdee, Sarawut; Adina-Zada, Abdussalam; Byrne, Lindsay; Zeczycki, Tonya N.; Maurice, Martin St.; Cleland, W. Wallace; Wallace, John C.; and Attwood, Paul V., "Probing the Catalytic Roles of Arg548 and Gln552 in the Carboxyl Transferase Domain of the Rhizobium etli Pyruvate Carboxylase by Site-directed Mutagenesis" (2010). Biological Sciences Faculty Research and Publications. 373.
https://epublications.marquette.edu/bio_fac/373