Study on protease which processes gamma-glutamyltranspeptidase

GGT n端信号肽由25个氨基酸残基组成，其中365个残基的大亚基和190个残基的小亚基按此顺序编码在一个开放阅读框中，这是一种非常罕见的基因结构。这表明除了信号肽酶I的裂解作用外，E。大肠杆菌GGT经过Gln-390和Thr-391之间的翻译后裂解，成熟为哺乳动物GGT的异源二聚体。对ggt和相关酶在加工位点的相似性进行了序列比对。大亚基的c端氨基酸残基Gln-390不保守，而小亚基的n端氨基酸残基Thr-391和His-393在所有已知氨基酸序列的ggt中均保守。T391A和H393G突变体的质周部分没有发生变化。因此，小亚基n端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是由一种膜结合的胰蛋白酶样丝氨酸蛋白酶加工的，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推测，小亚基（大肠杆菌GGT中的Thr-391）的n端氨基酸残基侧链是攻击Gln-390和Thr-391之间肽链羰基碳的亲核原子，并进行加工。然而，还没有实验证明。通过对哺乳动物ggt的化学修饰研究，提出了一些碱性氨基酸残基可能位于酶促反应的活性中心。因此，采用定点诱变的方法对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，并观察其作用。虽然这些残基远离GGT初级结构中的加工位点，但这些突变体没有经过加工，也没有酶活性。这表明加工发生在GGT构象形成之后。青霉素酰化酶的b链c端氨基酸残基也经历了类似的翻译后加工过程。这是一个证据，证明GGT的加工方式与青霉素酰化酶相似，青霉素酰化酶是n端亲核水解酶超家族之一。该家族的所有成员都具有两个反平行的β -褶片的特征，由几个β -链组成。这些酶利用b链的n端氨基酸残基侧链作为亲核试剂催化攻击底物的羰基碳，这条侧链被认为是它们的自动加工机制的原因。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褶片的特征。这一结果强烈表明GGT的加工是自催化的，而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