Molecular Basis of Lysine Specificity of Lysylendopeptidase

Achromobacter Protease I(API)是一种赖氨酸专一性的丝氨酸蛋白酶。API和API-TLCK络合物的晶体结构分析表明，Asp225的羧基氧与其周围的氨基酸残基和S1-口袋中的水分子(W420)形成了几个氢键。Asp225的Odelta2与Thr189的OGamma1、Ser214的OGamma和Trp182的尼泊尔1形成氢键。W420还与Asp225的Odelta1、Thr189的CO、Ser214的CO和Ser214的OGamma形成氢键。这些结果表明，除了Asp225外，Trp182、Thr189、Ser214和Trp182也可能与API对赖氨酸的高度专一性和较高的蛋白分解活性有关。为了研究氢键的作用，用Trp182、Thr189和Ser214取代了其他氨基酸。T189V、S214A和T189VS214A的kcat/kM分别为天然API的1/4、1/3和1/46。活性下降的主要原因是Km值的增加，这表明氢键网络的部分丧失和S1-Pocket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，W182F与天然API的酶学性质的相似性表明，Asp225的Odelta2与Trp182的尼泊尔1之间的氢键与底物结合没有直接关系，API的严格专一性和高蛋白水解性主要是基于Asp225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，而S1-口袋中的氢键系统有助于底物与API的结合。

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.