Study on protease which processes gamma-glutamyltranspeptidase

加工γ-谷氨酰转肽酶的蛋白酶的研究

• 批准号: 08660106
• 负责人: SUZUKI Hideyuki
• 金额: $ 1.34万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660106/
• 关键词: gamma-glutamyltranspeptidase; gamma-glutamyltransferase; glutathione; processing; site-directed mutagenesis; post-translational processing; γ-グルタミルトランスペプチターゼ

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

GGT的N末端信号肽由25个氨基酸残留物，365个残留物的大亚基和190个残留物的小亚基组成，以此顺序在单个开放式阅读框架中编码，这是非常罕见的基因构建体。这表明除了信号肽I的裂解外，E.coli GGT还经过GLN-390和THR-391之间的翻译后裂解，并以哺乳动物GGT的形式成熟成异二聚体。进行了GGT和相关酶的处理位点相似性的序列比对。 GLN-390是大型亚基的C末端氨基酸保留，而THR-391和HIS-393和HIS-393，N端氨基酸保留的小亚基，在所有氨基酸序列已知的GGT中均配置。 T391a和H393G突变体的周质馏分未加工。因此，小亚基的N末端的THR-X-HIS序列对于处理至关重要。在哺乳动物中，有一个假设，即GGT是由……更多的膜结合的胰蛋白酶样系列蛋白酶处理的，但此后没有关于该蛋白酶的更远报道。最近，从三维结构分析的结果来看，一个假设是，小亚基的N端氨基酸保留的侧链（E.Coli GGT中的THR-391）是核植物原子，它攻击了GLN-390和Thr-391和Thr-391之间的Peppery链接的羰基碳。但是，尚未获得实验证明。从化学修饰研究中，建议一些哺乳动物GGT的碱性氨基酸保留在酶促反应的活性中心中定位。因此，在GGT中高度保守的碱性氨基酸保留（E.Coli中的ARG-513和ARG-571）是通过位置定向的诱变诱变的，并且观察到它们的效果。尽管这些残差远离GGT的主要结构中的加工位点，但突变体没有进行加工，也没有具有酶促活性。这表明该处理发生在GGT构象形成后。氨基酸保留在青霉素酰基酶的B链的C末端，受到类似的翻译后加工至关重要。这是一个证据表明，GGT是通过与青霉素酰基酶相似的方式处理的，这是拟议的N末端核水解酶超家族之一。这个家庭的所有成员都有两个由几个β链组成的反平行β式纸。这些酶使用B链的N末端氨基酸居住区的侧链作为底物羰基碳催化攻击中的核恐惧症，并建议该侧链负责其自动处理机制。尽管我们尚未完成对E.Coli GGT的X射线分析，但我们发现GGT还具有特征性的两个反平行β-折叠板。该结果强烈表明GGT的处理是自催化的，并且较少

项目成果

鈴木 秀之: "グルタチオン代謝の細胞生理の酵素分子生物学的解明と代謝酵素の構造と機能に関する研究" 日本農芸化学会誌. 71. 987-994 (1997)

Hideyuki Suzuki：“谷胱甘肽代谢的细胞生理学的酶分子生物学阐明以及代谢酶的结构和功能的研究”日本农业化学学会杂志 71. 987-994 (1997)。

W.Hashimoto, H.Suzuki, K.Yamamoto, and H.Kumagai.: "Analysis of low temperature inducible mechanism of gamma-glutamyltranspeptidase of Escherichia coli K-12." Biosci.Biotechnol.Biochem.61(1). 34-39 (1997)

W.Hashimoto、H.Suzuki、K.Yamamoto 和 H.Kumagai.：“大肠杆菌 K-12 γ-谷氨酰转肽酶的低温诱导机制分析”。

Wataru Hashimoto: "Low temperature inducible γ-glutamyltranspeptidase of Escherichia coli K-12 Bioscience,Biotechnology and Biochemistry" Bioscience,Biotechnology and Biochemistry. 61(1). 34-39 (1997)

Wataru Hashimoto：“大肠杆菌 K-12 的低温诱导型 γ-谷氨酰转肽酶生物科学、生物技术和生物化学”《生物科学、生物技术和生物化学》61(1)。

H.Suzuki, E.-S.Kim, N.Yamamoto, W.Hashimoto, K.Yamamoto, and H.Kumagai.: "Mapping, cloning, and DNA sequencing of pepB gene which encodes peptidase B of Escherichia coli K-12." J.Ferment.Biotechnol. 82. 392-397 (1996)

H.Suzuki、E.-S.Kim、N.Yamamoto、W.Hashimoto、K.Yamamoto 和 H.Kumagai.：“编码大肠杆菌 K-12 肽酶 B 的 pepB 基因的定位、克隆和 DNA 测序

鈴木秀之: "グルタチオン代謝の細胞生理の酵素分子生物学的解明と代謝酵素の構造と機能に関する研究" 日本農芸化学会誌. 71(10). 987-994 (1997)

Hideyuki Suzuki：“谷胱甘肽代谢的细胞生理学的酶分子生物学阐明以及代谢酶的结构和功能的研究”日本农业化学学会杂志71（10）（1997）。