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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/28734
Title: 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
Sarawut Jitrapakdee
Abdussalam Adina-Zada
Lindsay Byrne
Tonya N. Zeczycki
Martin St. Maurice
W. Wallace Cleland
John C. Wallace
Paul V. Attwood
Mahidol University
University of Western Australia
Marquette University
University of Wisconsin Madison
University of Adelaide
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 20-Apr-2010
Citation: Biochemistry. Vol.49, No.15 (2010), 3296-3304
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.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=77950958782&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/28734
ISSN: 15204995
00062960
Appears in Collections:Scopus 2006-2010

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