Studies on molecular mechanism of glutathione one salvage pathway of Escherichia coli

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1. We found that aminopeptidases A, B and N, and dipeptidase D with broad substrate specificity are the four cysteinylglycinases of Escherichia coli K-12 and there is no peptidase specific for the cleavage of cysteinylglycine.2. Aminopeptidase B was purified to electrophoretic homogeneity and its enzymatic characteristics were determined. The data indicates that aminopeptidase B is a metallopeptidase. Aminopeptidase is a metallopeptidase. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. Cysteinylglycine was the best substrates among those we tested.3. We identified that the catalytic nucleophile of Escherichia coli γ-glutamyltranspeptidase by a novel affinity labeling agent. After the modification of the enzyme, it was separated into the large subunit and the small subunit followed by the fragmentation by lysyl endopeptidase. The fragments were analyzed by MS-MS and it was found that is the Thr-residue at the N-terminal of the small subunit is the active center of this enzyme. This result strongly suggests that γ-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family.