Jia Z, Hasnain S, Hirama T, Lee X, Mort J S, To R, Huber C P
Institute for Biological Sciences, National Research Council of Canada, Ottawa.
J Biol Chem. 1995 Mar 10;270(10):5527-33. doi: 10.1074/jbc.270.10.5527.
The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an important role in protein catabolism and has also been implicated in various disease states. The crystal structures of two forms of native recombinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liver enzyme. The structure of the native enzyme containing an underivatized active site cysteine (Cys29) showed the active enzyme conformation to be similar to that determined previously for the oxidized form. In a second structure Cys29 was derivatized with the reversible blocking reagent pyridyl disulfide. In this structure large side chain conformational changes were observed for the two key catalytic residues Cys29 and His199, demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cathepsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethylketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and inhibitor. In particular the interaction between Glu245 and the P2 Arg residue was clearly demonstrated, and it was found that the benzyl group of the P1 substrate residue occupies a large hydrophobic pocket thought to represent the S'1 subsite. This may have important implications for structure-based design of cathepsin B inhibitors.
溶酶体半胱氨酸蛋白酶组织蛋白酶B（EC 3.4.22.1）在蛋白质分解代谢中起重要作用，并且也与多种疾病状态有关。已测定了两种形式的天然重组大鼠组织蛋白酶B的晶体结构。结果表明，大鼠组织蛋白酶B的整体折叠与人类肝脏酶的非常相似。含有未衍生化活性位点半胱氨酸（Cys29）的天然酶结构显示活性酶构象与先前确定的氧化形式相似。在第二种结构中，Cys29用可逆封闭试剂吡啶二硫化物进行了衍生化。在该结构中，观察到两个关键催化残基Cys29和His199的大侧链构象变化，证明了这些侧链的潜在灵活性。此外，还测定了大鼠组织蛋白酶B与抑制剂苄氧羰基-精氨酸-丝氨酸（O-苄基）氯甲基酮之间的复合物结构。复合物结构表明，抑制剂结合后酶中发生的构象变化非常小。它还使酶与抑制剂之间的相互作用可视化。特别是清楚地证明了Glu245与P2精氨酸残基之间的相互作用，并且发现P1底物残基的苄基占据了一个大的疏水口袋，该口袋被认为代表S'1亚位点。这可能对基于结构的组织蛋白酶B抑制剂设计具有重要意义。
