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Critical Role of the Cysteine 323 Residue in the Catalytic Activity of Human Glutamate Dehydrogenase Isozymes  

Yang, Seung-Ju (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Department of Biomedical Laboratory Science, College of Health Science, Yonsei University)
Cho, Eun Hee (Department of Science Education, College of Education, Chosun University)
Choi, Myung-Min (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine)
Lee, Hyun-Ju (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine)
Huh, Jae-Wan (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine)
Choi, Soo Young (Department of Genetic Engineering, College of Life Sciences, Hallym University)
Cho, Sung-Woo (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine)
Publication Information
Molecules and Cells / v.19, no.1, 2005 , pp. 97-103 More about this Journal
Abstract
The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the $K_m$ of the enzyme for NADH and ${\alpha}-ketoglutarate$ increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency ($k_{cat}/K_m$) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in $k_{cat}$ values. There was a linear relationship between incorporation of [$^{14}C$]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [$^{14}C$] incorporated per mol of monomer for wild type hGDHs. No incorporation of [$^{14}C$]p-chloromercuribenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.
Keywords
Cassette Mutagenesis; Chemical Modification; Enzyme Efficiency; Glutamate Dehydrogenase; Isozymes; Reactive Cysteine;
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