Gillmor S A, Craik C S, Fletterick R J
Graduate Group in Biophysics, University of California, San Francisco 94143-0448, USA.
Protein Sci. 1997 Aug;6(8):1603-11. doi: 10.1002/pro.5560060801.
The structure of cruzain, an essential protease from the parasite Trypanosoma cruzi, was determined by X-ray crystallography bound to two different covalent inhibitors. The cruzain S2 specificity pocket is able to productively bind both arginine and phenylalanine residues. The structures of cruzain bound to benzoyl-Arg-Ala-fluoromethyl ketone and benzoyl-Tyr-Ala-fluoromethyl ketone at 2.2 and 2.1 A, respectively, show a pH-dependent specificity switch. Glu 205 adjusts to restructure the S2 specificity pocket, conferring right binding to both hydrophobic and basic residues. Kinetic analysis of activated peptide substrates shows that substrates placing hydrophobic residues in the specificity pocket are cleaved at a broader pH range than hydrophilic substrates. These results demonstrate how cruzain binds both basic and hydrophobic residues and could be important for in vivo regulation of cruzain activity.
克氏锥虫（Trypanosoma cruzi）的一种必需蛋白酶——克氏锥虫蛋白酶（cruzain）的结构，通过与两种不同的共价抑制剂结合的X射线晶体学方法得以确定。克氏锥虫蛋白酶的S2特异性口袋能够有效地结合精氨酸和苯丙氨酸残基。分别在2.2 Å和2.1 Å分辨率下，克氏锥虫蛋白酶与苯甲酰-精氨酸-丙氨酸氟甲基酮和苯甲酰-酪氨酸-丙氨酸氟甲基酮结合的结构显示出pH依赖性的特异性转换。谷氨酸205进行调整以重构S2特异性口袋，赋予对疏水和碱性残基的正确结合。对活化肽底物的动力学分析表明，在特异性口袋中放置疏水残基的底物比亲水底物在更宽的pH范围内被切割。这些结果证明了克氏锥虫蛋白酶如何结合碱性和疏水残基，并且对于克氏锥虫蛋白酶活性的体内调节可能很重要。
