Takeuchi Y, Nonaka T, Nakamura K T, Kojima S, Miura K, Mitsui Y
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Yokohama, Japan.
Proc Natl Acad Sci U S A. 1992 May 15;89(10):4407-11. doi: 10.1073/pnas.89.10.4407.
Proteinase specificity of a proteinaceous inhibitor of subtilisin (SSI; Streptomyces subtilisin inhibitor) can be altered so as to strongly inhibit trypsin simply by replacing P1 methionine with lysine (with or without concomitant change of the P4 residue) through site-directed mutagenesis. Now the crystal structure of one such engineered SSI (P1 methionine converted to lysine and P4 methionine converted to glycine) complexed with bovine trypsin has been solved at 2.6 A resolution and refined to a crystallographic R factor of 0.173. Comparing this structure with the previously established structure of the native SSI complexed with subtilisin BPN', it was found that (i) P1 lysine of the mutant SSI is accommodated in the S1 pocket of trypsin as usual, and (ii) upon complex formation, considerable conformation change occurs to the reactive site loop of the mutant SSI. Thus, in this case, flexibility of the reactive site loop seems important for successfully changing the proteinase specificity through mere replacement of the P1 residue.
枯草杆菌蛋白酶的一种蛋白质抑制剂（SSI；链霉菌枯草杆菌蛋白酶抑制剂）的蛋白酶特异性可以通过定点诱变将P1位的甲硫氨酸替换为赖氨酸（无论P4残基是否同时改变）而发生改变，从而强烈抑制胰蛋白酶。现在，已解析出一种这样经过工程改造的SSI（P1位甲硫氨酸转变为赖氨酸且P4位甲硫氨酸转变为甘氨酸）与牛胰蛋白酶形成的复合物的晶体结构，分辨率为2.6 Å，并精修至晶体学R因子为0.173。将该结构与先前确定的天然SSI与枯草杆菌蛋白酶BPN'形成的复合物的结构进行比较，发现：（i）突变型SSI的P1位赖氨酸像往常一样容纳在胰蛋白酶的S1口袋中；（ii）在形成复合物时，突变型SSI的活性位点环发生了相当大的构象变化。因此，在这种情况下，活性位点环的灵活性对于仅通过替换P1残基成功改变蛋白酶特异性似乎很重要。
