Reactive cysteine residue of bovine brain glutamate dehydrogenase isoproteins.
Protein chemical studies of glutamate dehydrogenase isoproteins (GDH I and GDH II) from bovine brain reveal that one cystein residue is accessible for reaction with thiol-modifying reagent. Reaction of the two types of GDH isoproteins with p-chloromercuribenzoic acid resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order kinetics with the second-order rate constant of 83 M(-1) s(-1) and 75 M(-1) s(-1) for GDH I and GDH II, respectively. The inactivation was partially prevented by preincubation of the glutamate dehydrogenase isoproteins with NADH. A combination of 10 mM 2-oxoglutarate with 2 mM NADH gave complete protection against the inactivation. There were no significant differences between the two glutamate dehydrogenase isoproteins in their sensitivities to inactivation by p-chloromercuribenzoic indicating that the microenvironmental structures of the GDH isoproteins are very similar to each other. Allosteric effectors such as ADP and GTP had no effects on the inactivation of glutamate dehydrogenase isoproteins by thiol-modifying reagents. By a combination of peptide mapping analysis and labeling with [14C] p-chloromercuribenzoic acid, a reactive cystein residue was identified as Cys323 in the overall sequence. The cysteine residue was clearly identical to sequences of other GDH species known.
Cho
S W
SW
Department of Biochemistry, University of Ulsan College of Medicine, Seoul, Korea. swcho@www.amc.seoul.kr
Cho
E H
EH
Hwang
S H
SH
Choi
S Y
SY
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002250:Carbon Radioisotopes / Q000378:metabolism; D002417:Cattle; D003545:Cysteine / Q000502:physiology*; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D051381:Rats; D016415:Sequence Alignment; D020245:p-Chloromercuribenzoic Acid / Q000378:metabolism
D002250:Carbon Radioisotopes; D007527:Isoenzymes; D020245:p-Chloromercuribenzoic Acid; D005969:Glutamate Dehydrogenase; D003545:Cysteine