Kim D H
Center for Biofunctional Molecules, Pohang University of Science and Technology, Korea.
Biopolymers. 1999;51(1):3-8. doi: 10.1002/(SICI)1097-0282(1999)51:1<3::AID-BIP2>3.0.CO;2-Q.
The substrate stereospecificity in enzymic reactions, which is one of characteristics of enzymes along with the substrate and regiospecificity can provide a basis for the rational design of inhibitors. This has been demonstrated using alpha-chymotrypsin, a prototypic serine protease as a model enzyme. On the basis of the structure-activity relationships for substrates as well as inhibitors and mechanism of the enzymic reaction, a schematic three-dimensional model of the S1 subsite of alpha-chymotrypsin is constructed. It was envisioned from the three-dimensional active site model that 2-benzyl-3,4-epoxybutanoic acid methyl ester (1) having a (2S)-configuration would bind the enzyme with its oxirane ring being rested at the catalytic site, in which the oxirane ring is subject to a nucleophilic attack by the Ser-195 hydroxyl to form a ether linkage. Kinetic analysis of the enzymic reaction in the presence of the potential inhibitors showed that (2S,3R)-1 inactivates alpha-chymotrypsin, while (2S,3S)-1 inhibits the enzyme competitively. The lack of inactivating activity in the case of (2S,3S)-1 may be due to the unfavorable alignment of the C3-O bond with respect to the hydroxyl of Ser-195 for the SN2-type ring cleave reaction of the oxirane moiety. When the design protocol was applied to papain, a representative cysteine protease, (2S,3S)-1 inhibited the enzyme irreversibly, while (2S,3R)-1 inhibited reversibly. On the basis of the stereospecificity shown in the inactivation of the enzymes, it was inferred that in the case of alpha-chymotrypsin, the nucleophilic attack of the Ser-195 hydroxyl at the scissile carbonyl carbon of substrates occurs in a si fashion, while the thiolate of Cys-25 in papain attacks the substrate amide bond in a re fashion. The inhibitor design protocol may be applied to other proteases.
酶促反应中的底物立体特异性是酶的特性之一，与底物特异性和区域特异性一样，可为抑制剂的合理设计提供依据。这已通过使用α-胰凝乳蛋白酶（一种典型的丝氨酸蛋白酶作为模型酶）得到证明。基于底物以及抑制剂的构效关系和酶促反应机制，构建了α-胰凝乳蛋白酶S1亚位点的示意性三维模型。从三维活性位点模型可以设想，具有(2S)-构型的2-苄基-3,4-环氧丁酸甲酯(1)将以其环氧乙烷环位于催化位点的方式与酶结合，其中环氧乙烷环会受到Ser-195羟基的亲核攻击以形成醚键。在潜在抑制剂存在下对酶促反应的动力学分析表明，(2S,3R)-1使α-胰凝乳蛋白酶失活，而(2S,3S)-1竞争性抑制该酶。(2S,3S)-1缺乏失活活性可能是由于C3-O键相对于Ser-195羟基的排列不利于环氧乙烷部分的SN2型环裂解反应。当将设计方案应用于木瓜蛋白酶（一种代表性的半胱氨酸蛋白酶）时，(2S,3S)-1不可逆地抑制该酶，而(2S,3R)-1可逆地抑制。基于酶失活中显示的立体特异性，推断在α-胰凝乳蛋白酶的情况下，Ser-195羟基对底物可裂解羰基碳的亲核攻击以si方式发生，而木瓜蛋白酶中Cys-25的硫醇盐以re方式攻击底物酰胺键。该抑制剂设计方案可应用于其他蛋白酶。
