The functional involvement of Lys-38 in the heavy subunit of rat kidney gamma-glutamylcysteine synthetase: chemical modification and mutagenesis studies.
Rat kidney gamma-glutamylcysteine synthetase (gamma GCS) was inactivated by reaction with trinitrobenzene sulfonate (TNBS), and the reaction followed pseudo-first-order kinetics. Inactivation kinetics revealed that only one of the amino acid residues modified by TNBS was essential for gamma GCS activity. The addition of 10 mM Mg2+ to the TNBS inactivation reaction resulted in a 16-fold increase in the rate of inactivation. Chromatographic analysis on the tryptic hydrolyzates of trinitrophenylated (TNP) derivatives showed that Lys-38 in the gamma GCS heavy subunit was significantly modified in the presence of Mg2+. In contrast to small changes in the catalytic properties observed by mutation of Lys-38 to Arg, the mutants K38N and K38E had a marked decrease in enzymatic activity and about twofold increase in Km for glutamate. These results suggest that the positively charged Lys-38 may be involved in the binding of glutamate to gamma GCS.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent; D004274:DNA, Recombinant; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007202:Indicators and Reagents / Q000737:chemistry; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014302:Trinitrobenzenesulfonic Acid / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D002413:Cations, Divalent; D004274:DNA, Recombinant; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D014302:Trinitrobenzenesulfonic Acid; D014357:Trypsin; D005721:Glutamate-Cysteine Ligase; D008239:Lysine