Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes.
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 Km 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 (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic 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.
Yang
Seung-Ju
SJ
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea.
Cho
Eun Hee
EH
Choi
Myung-Min
MM
Lee
Hyun-Ju
HJ
Huh
Jae-Wan
JW
Choi
Soo Young
SY
Cho
Sung-Woo
SW
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D019943:Amino Acid Substitution; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology
D007527:Isoenzymes; D007656:Ketoglutaric Acids; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D020245:p-Chloromercuribenzoic Acid; D005969:Glutamate Dehydrogenase; C061421:glutamate dehydrogenase (NAD(P)+); D003545:Cysteine