Molecular Basis of Lysine Specificity of Lysylendopeptidase

赖氨酰内肽酶赖氨酸特异性的分子基础

• 批准号: 06680585
• 负责人: NORIOKA Shigemi
• 金额: $ 1.41万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680585/
• 关键词: Lysylendopeptidase; Substrate Specificity; Site-directed Mutagenesis; Protein Engineering; Hydrogen Bond; リジルエンドペプチダーゼ

Achromobacter protease I (API) is a lysine-specific serine protease. Crystal structural analyzes of API and API-TLCK complex revealed that the carboxyl oxygen of Asp225 formed several hydrogen bonds with its surrounding amino acid residues and a water molecule (W420) in S1-pocket. Odelta2 of Asp225 formed hydrogen bonds with Ogamma1 of Thr189, Ogamma of Ser214, and Nepsilon1 of Trp182. W420 also formed hydrogen bonds with Odelta1 of Asp225, CO of Thr189, CO of Ser214 and Ogamma of Ser214. From these observations, it was thought that besides Asp225, Thr189, Ser214 and Trp182 might also contribute to the strict specificity for lysine and the high proteolytic activity of API.In order to investigate role of the hydrogen bonds, Trp182, Thr189 and Ser214 were substituted for other amino acids. The kcat/Km of T189V,S214A,and T189VS214A were decreased to 1/4,1/3 and 1/46 of that of native API,respectively. The decreased activities were mainly due to the increase of Km value, suggesting that the partial loss of the hydrogen bond network and a local minute structural change in S1-pocket decreased substrate binding ability of these mutants. On the other hand, the similarity of enzymatic properties between W182F and native API suggested that the hydrogen bond between Odelta2 of Asp225 and Nepsilon1 of Trp182 was not directly related to the substrate binding.The strict specificity and high proteolytic activity of API were primarily based on the electrostatic interaction between Asp225 and lysine substrate. But this interaction was not sufficient for strong binding of the lysine substrate to API and the hydrogen bonding system in the S1-pocket assisted the binding of a substrate to API.

无色杆菌蛋白酶I（API）是赖氨酸特异性丝氨酸蛋白酶。对API和API-TLCK复合物的晶体结构分析表明，Asp 225的羧基氧与其周围的氨基酸残基和S1-口袋中的水分子（W 420）形成多个氢键。Asp 225的Odelta 2与Thr 189的Ogamma 1、Ser 214的Ogamma和Trp 182的Nepsilon 1形成氢键。W 420还与Asp 225的Odelta 1、Thr 189的CO、Ser 214的CO和Ser 214的Ogamma形成氢键。除Asp 225外，Thr 189、Ser 214和Trp 182也可能对API的高蛋白水解活性和对赖氨酸的严格特异性有贡献。T189 V、S214 A和T189 VS 214 A的kcat/Km分别降低至天然API的1/4、1/3和1/46。活性的下降主要是由于Km值的增加，表明氢键网络的部分损失和S1-口袋的局部微小结构变化降低了这些突变体的底物结合能力。另一方面，W182 F与天然API的酶学性质相似，表明Asp 225的Odelta 2与Trp 182的Nepsilon 1之间的氢键与底物结合没有直接关系，API的严格特异性和高蛋白水解活性主要是基于Asp 225与赖氨酸底物之间的静电相互作用。但这种相互作用不足以使赖氨酸底物与API强结合，S1-口袋中的氢键系统有助于底物与API的结合。

项目成果

Yuko Nagamine-Natsuka, Shigemi Norioka, and Fumio Sakiyama: "Mocecular cloning, nucleotide sequence, and expression of the gene encoding a trypsin-like protease from Streptomyces erythraeus." J.Biochem.118. 338-346 (1995)

Yuko Nagamine-Natsuka、Shigemi Norioka 和 Fumio Sakiyama：“红链霉菌中编码胰蛋白酶样蛋白酶的基因的分子克隆、核苷酸序列和表达。”

Yuko Nagamine-Natsuka: "Molecular cloning,nucleotide sequence,and expression of the gene encoding a trypsin-like protease from Streptomyces erythraeus." J. Biochem.118. 1007-1013 (1995)

Yuko Nagamine-Natsuka：“红链霉菌胰蛋白酶样蛋白酶编码基因的分子克隆、核苷酸序列和表达。”

Yuko Nagamine-Natsuka: "Molecular cloning, nucleotidesegucncs, and expression of the gene encoding a trypshlike protease from Stroptomyces erythraous" J.Biochemistry. 118. 338-346 (1995)

Yuko Nagamine-Natsuka：“红链霉菌中编码胰蛋白酶样蛋白酶的基因的分子克隆、核苷酸序列和表达”J.Biochemistry。

Shigemi Norioka: "Identification of three catalytic triad constituents and Asp-225 essential for function of lysine-specific serine protease,Achromobacter protease I." J. Biol. Chem.269. 17025-17029 (1994)

Shigemi Norioka：“鉴定对赖氨酸特异性丝氨酸蛋白酶、无色杆菌蛋白酶 I 的功能至关重要的三种催化三联体成分和 Asp-225。”

Shigemi Norioka: "Site-directed mutagenic study of the substrate specificity of Streptomyces erythraeus trypsin-like protease." Protein Engineering. 7. 1153-1154 (1994)

Shigemi Norioka：“红链霉菌胰蛋白酶样蛋白酶底物特异性的定点诱变研究。”