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

1.我们发现大肠杆菌K-12的四种半胱氨酸甘氨酸酶是氨基肽酶A、B、N和二肽酶D，它们具有广泛的底物专一性，并不存在半胱氨酸甘氨酸特异性的肽酶。对氨基肽酶B进行了纯化，并对其酶学性质进行了测定。结果表明，氨基肽酶B是一种金属多肽酶。氨基肽酶是一种金属多肽酶。用上述一种二价阳离子饱和的氨基肽酶B的活性在加入极少量的第二种二价阳离子时得到增强。半胱氨酸甘氨酸是我们所测试的底物中最好的。我们用一种新型亲和标记剂鉴定了大肠杆菌γ-谷氨酰转肽酶的催化亲核性。对酶进行修饰后，将其分离为大亚基和小亚基，然后用赖氨酰内肽酶进行裂解。经MS-MS分析发现，该酶小亚基N-末端的苏氨酸残基是该酶的活性中心。这一结果有力地表明γ-谷氨酰转肽酶是N端亲核水解酶家族的一个新成员。

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.