Purification and characterization of glutathione synthetase from Escherichia coli B.

Abstract

Glutathione synthetase was purified about 60-fold with 8.5% of activity yield from the cell extracts of Escherichia coli C600 cells transformed with a recombinant plasmid for the glutathione synthetase gene of E. coli B. The purified enzyme had a Mr of 152,000 and was composed of four identical subunits each with a Mr of 38,000. The Km values of the enzyme for gamma-glutamylcysteine, glycine, and ATP were 2.6, 2.0, and 1.8 mM, respectively. The enzyme was most active at pH 8.5 and at 45 degrees C and required divalent cations such as Mg2+, Mn2+, and Co2+ for activity. The activity was inhibited by oxidized glutathione (Ki = 4.4 mM). Reduced glutathione showed no effect on glutathione synthetase activity.