Purification and Characterization ofγ-Glutamylcysteine Synthetase ofEscherichia coliB

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Abstract
γ-Glutamy Icy steine synthetase was purified from E. coli B. The enzyme had a molecular weight of 5.5 × 104 and required only magnesium ion for activity. The optimal pH and temperature for reaction were 8.5 and 45°C, respectively. The Km values for l-glutamate, l-cysteine, and ATP were 0.50, 0.09, and 0.01 mm, respectively. GTP and UTP were also used as energy sources. The enzyme activity was inhibited by phosphate anions and by various sulfhydryl reagents. Unlike the enzyme from mammalian tissues, the E. coli B enzyme was not inhibited by α-alkyl analogues of methionine. The enzyme was feedback inhibited by reduced glutathione, although oxidized glutathione had no inhibitory effect.