Crystal structures of γ-glutamyltranspeptidase from Escherichia coli, a key enzyme in glutathione metabolism, and its reaction intermediate
Crystal structures of γ-glutamyltranspeptidase from Escherichia coli, a key enzyme in glutathione metabolism, and its reaction intermediate
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DOI：
10.1073/pnas.0511020103
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发表时间：
2006-04-25
影响因子：
11.1
通讯作者：
Fukuyama, K
中科院分区：
文献类型：
--
作者：
Okada, T;Suzuki, H;Fukuyama, K
γ-谷氨酰转肽酶（GGT）是一种异源酶，由前体蛋白通过翻译后加工产生，并催化γ-谷氨酰化合物（如谷胱甘肽）中γ-谷氨酰键的水解和/或γ-谷氨酰基团转移至其他氨基酸和肽。我们已经确定了GGT的晶体结构，从大肠杆菌K-12在1.95埃分辨率。GIST具有堆叠的α β β α折叠，其包括大小亚基，类似于在N-末端亲核水解酶超家族成员中看到的折叠。活性位点Thr-391（小亚基的N-末端残基）位于沟中，γ-谷氨酰部分结合的口袋位于沟中。我们已经进一步确定了γ-谷氨酰-酶中间体的结构，所述γ-谷氨酰-酶中间体通过快速冷却浸泡在谷胱甘肽溶液中的GIST晶体而被捕获，以及GIST与L-谷氨酸盐复合物的结构。这些结构揭示了γ-谷氨酰部分和L-谷氨酸是如何被酶识别的。在待水解的中间体中的γ-谷氨酰基-Thr-391 O γ键的羰基碳上观察到水分子。值得注意的是，GIST活性所必需的残基（E. coliGGT）均参与γ-谷氨酰部分的结合。大肠杆菌GGT的结构，这里提出的，连同GGT的序列比对，可能适用于解释其他GGT的生化和遗传数据。
gamma-Glutamyltranspeptidase (GGT) is a heterodimic enzyme that is generated from the precursor protein through posttranslational processing and catalyzes the hydrolysis of gamma-glutamyl bonds in gamma-glutamyl compounds such as glutathione and/or the transfer of the gamma-glutamyl group to other amino acids and peptides. We have determined the crystal structure of GGT from Escherichia coli K-12 at 1.95 angstrom resolution. GIST has a stacked alpha beta beta alpha fold comprising the large and small subunits, similar to the folds seen in members of the N-terminal nucleophile hydrolase superfamily. The active site Thr-391, the N-terminal residue of the small subunit, is located in the groove, from which the pocket for gamma-glutamyl moiety binding follows. We have further determined the structure of the gamma-glutamyl-enzyme intermediate trapped by flash cooling the GIST crystal soaked in glutathione solution and the structure of GIST in complex with L-glutamate. These structures revealed how the y-glutamyl moiety and L-glutamate are recognized by the enzyme. A water molecule was seen on the carbonyl carbon of the gamma-glutamyl-Thr-391 O gamma bond in the intermediate that is to be hydrolyzed. Notably the residues essential for GIST activity (Arg-114, Asp-433, Ser-462, and Ser-463 in E. coli GGT) shown by site-directed mutagenesis of human GGT are all involved in the binding of the gamma-glutamyl moiety. The structure of E coli GGT presented here, together with sequence alignment of GGTs, may be applicable to interpret the biochemical and genetic data of other GGTs.