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端信号肽由25个氨基酸残基组成，365个残基的大亚基和190个残基的小亚基按此顺序编码在单个开放阅读框中，这是一种非常罕见的基因构建体。除了信号肽酶 I 的切割外，大肠杆菌 GGT 还会在 Gln-390 和 Thr-391 之间进行翻译后切割，并且成熟为哺乳动物 GGT 的异二聚体。对 GGT 和相关酶之间的加工位点进行了序列比对，Gln-390（大亚基的 C 端氨基酸残基）不保守，而 Thr-391 和 His。 -393，小亚基的N端氨基酸残基，在其氨基酸序列已知的T391A和周质部分的所有GGT中是保守的。 H393G 突变体不进行加工，因此，小亚基 N 端的 Thr-X-His 序列对于加工至关重要，有一种假设认为，GGT 是由膜结合的胰蛋白酶样丝氨酸蛋白酶加工的。但此后并未再有关于该蛋白酶的进一步报道，最近从三维结构分析的结果来看，有一种假设是小亚基（Thr-391）的N端氨基酸残基的侧链。 E.coli GGT)是攻击Gln-390和Thr-391之间肽键的羰基碳的亲核原子，并发生加工，但一些碱性氨基酸的化学修饰研究尚未获得实验证明。哺乳动物 GGT 残基被认为位于酶促反应的活性中心，因此，GGT 中高度保守的碱性氨基酸残基（Arg-513 和 Arg-513）。通过定点诱变对大肠杆菌中的Arg-571进行诱变并观察其效果，尽管这些残基远离GGT一级结构中的加工位点，但这些突变体没有经过加工并且不具有酶促作用。这表明该加工发生在青霉素酰基转移酶B链C末端的氨基酸残基形成之后，其经历了类似的翻译后加工。这是一个证据，表明 GGT 的加工方式与青霉素酰化酶类似，青霉素酰化酶是拟议的 N 末端亲核水解酶超家族之一，该家族的所有成员都具有特征性的两个由多个 β- 组成的反平行 β 折叠片。这些酶使用 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和该侧链。虽然我们还没有完成对大肠杆菌GGT的强烈X射线分析，但我们发现GGT也具有两个反平行β折叠片的特征，这一结果表明GGT的加工过程。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）。