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 K35E had a marked decrease in enzymatic activity and about twofold increase in K-m for glutamate. These results suggest that the positively charged Lys-38 may be involved in the binding of glutamate to gamma GCS.