Study on protease which processes gamma-glutamyltranspeptidase

GGT的N端信号肽由25个氨基酸残基组成，大亚基由365个氨基酸残基组成，小亚基由190个氨基酸残基组成，依次编码在一个开放的阅读框架内，这是一个非常罕见的基因结构。这表明，除了信号肽酶I的切割外，大肠杆菌GGT还在Gln-390和Thr-391之间进行翻译后切割，并成熟为异源二聚体作为哺乳动物GGT。对GGT和相关酶之间加工位点的相似性进行序列比对。大亚基的C-末端氨基酸残基Gln-390不保守，而小亚基的N-末端氨基酸残基Thr-391和His-393在氨基酸序列已知的所有GGT中保守。T391 A和H393 G突变体的周质部分不加工。因此，小亚基N端的Thr-X-His序列对加工至关重要。在哺乳动物中，有一种假设认为GGT是由 ...更多信息 一种膜结合的胰蛋白酶样丝氨酸蛋白酶，但此后没有关于这种蛋白酶的进一步报道。最近，从三维结构分析的结果中，有一种假设，即小亚基（大肠杆菌GGT中的Thr-391）的N-末端氨基酸残基的侧链是亲核原子，其攻击Gln-390和Thr-391之间的肽键的羰基碳，并进行加工。然而，还没有实验证明。化学修饰研究表明，哺乳动物GGT的一些碱性氨基酸残基位于酶促反应的活性中心。因此，通过定点诱变对GGT中高度保守的碱性氨基酸残基（大肠杆菌中的Arg-513和Arg-571）进行诱变，并观察其效果。虽然这些残基远离GGT一级结构中的加工位点，但这些突变体不经受加工，并且不具有酶活性。这表明加工发生在GGT构象形成之后。青霉素酰化酶B链C端的氨基酸残基是关键性的氨基酸残基，它们也经历类似的翻译后加工。这是一个证据表明，GGT是通过类似的方式加工的青霉素酰化酶，这是一个建议的N-末端亲核水解酶超家族。该家族的所有成员都具有特征性的两个反平行的β-折叠片层，由几个β-链组成。这些酶利用B链N端氨基酸残基的侧链作为亲核试剂催化攻击底物的羰基碳，该侧链被认为是其自加工机制的原因。虽然我们还没有完成大肠杆菌GGT的X射线分析，但我们发现GGT也具有两个反平行的β折叠片层的特征。这一结果有力地表明，GGT的加工是自催化的，并且Less

N-terminal signal peptide of GGT consists of 25 amino acid residues, the large subunit of 365 residues and the small subunit of 190 residues are en coded in a single open reading frame in this order and this is a very rare gene construct. This suggests that besides the cleavage by signal peptidase I,E.coli GGT is subjected to a post-translational cleavage between Gln-390 and Thr-391 and is matured into a heterodimer as mammalian GGTs. Sequence alignment for similarity at the processing site among GGTs and related enzymes was performed. Gln-390, the C-terminal amino acid residue of the large subunit, is not conserved, while Thr-391 and His-393, N-terminal amino acid residues of the small subunit, are conserved among all GGTs whose amino acid sequences have been known. Periplasmic fractions of T391A and H393G mutants did not process. Therefore, Thr-X-His sequence of N-terminal of the small subunit is critical for the processing. In mammals there was a hypothesis that GGT is processed by … More a membrane bound trypsin-like serine protease, but no farther report about this protease thereafter. Recently, from the results of three dimensional structure analysis there is a hypothesis that the side chain of N-terminal amino acid residue of the small subunit (Thr-391 in E.coli GGT) is the nucleophilic atom which attacks the carbonyl carbon of peptide linkage between Gln-390 and Thr-391, and the processing takes place. However, no experimental proof has been obtained. From the chemical modification study some basic amino acid residues of mammalian GGTs were suggested to locate in the active center for the enzymatic reaction. Therefore, basic amino acid residues which are highly conserved among GGTs (Arg-513 and Arg-571 in E.coli) were mutagenized by site-directed mutagenesis and their effects were observed. Although these residues are far from the processing site in the primary structure of GGT,these mutants were not subjected to processing and did not have the enzymatic activity. This suggests that the processing occurs after the conformation of GGT has formed. Amino acid residues in C-terminal of B-chain of penicillin acylase which is subjected to the similar post-translational processing are critical. This is one evidence that GGT is processed by the similar way as penicillin acylase which is one of the proposed N-terminal nucleophile hydrolase superfamily. All of members of this family have characteristic two antiparallel beta-pleated sheets consisting of several beta-strands. These enzymes use the side chain of the N-terminal amino acid residue of B-chain as the nucleophile in the catalytic attack at the carbonyl carbon of substrates and this side chain was suggested to be responsible for their autoprocessing mechanism. Although we have not completed X-ray analysis of E.Coli GGT,we have found that GGT also possesses the characteristic two antiparallel beta-pleated sheets. This result strongly suggests that the processing of GGT is autocatalytic and that the Less