Publication: Probing the catalytic roles of arg548 and gln552 in the carboxyl transferase domain of the rhizobium etli pyruvate carboxylase by site-directed mutagenesis
Issued Date
2010-04-20
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ISSN
15204995
00062960
00062960
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2-s2.0-77950958782
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochemistry. Vol.49, No.15 (2010), 3296-3304
Suggested Citation
Saowapa Duangpan, Sarawut Jitrapakdee, Abdussalam Adina-Zada, Lindsay Byrne, Tonya N. Zeczycki, Martin St. Maurice, W. Wallace Cleland, John C. Wallace, Paul V. Attwood Probing the catalytic roles of arg548 and gln552 in the carboxyl transferase domain of the rhizobium etli pyruvate carboxylase by site-directed mutagenesis. Biochemistry. Vol.49, No.15 (2010), 3296-3304. doi:10.1021/bi901894t Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/28734
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Title
Probing the catalytic roles of arg548 and gln552 in the carboxyl transferase domain of the rhizobium etli pyruvate carboxylase by site-directed mutagenesis
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. © 2010 American Chemical Society.
