Crystallographic Studies on Protease ー Substrate Interaction Using Genetically - Engineered Inhibitors.

游离SSI（链霉菌枯草杆菌蛋白酶抑制剂）和SSI与其靶酶枯草杆菌蛋白酶BPN'复合物的晶体结构分别精确到24%（1.85 A）和21%（1.8 A）。此外，还改进了两种更相关的结构，其中枯草杆菌蛋白酶BPN'与突变体SSI-1或突变体SSI-2复合。在两种突变SSI中，抑制剂的P1残基。通过基因操作将Met 73转化为Lys。此外，在突变体SSI-2中，P4残基Met 70转化为Gly。(A)在与靶酶（枯草杆菌蛋白酶BPN '）形成复合物时，SSI的一些（但不是全部）部分发生SSI结构的显著硬化。(B)硬化不仅发生在与靶酶直接接触的“反应位点”区段，而且发生在通过共价键或货车德瓦尔斯接触简单地连接到反应位点的那些多肽区段。(C)上述SSI的硬化多肽片段大致对应于在C-13 NMR谱中观察到复合物形成时的显著位移的片段（由M.Kainosho和他的同事使用各种富含C-13的SSI观察到）。(D)枯草杆菌蛋白酶BPN'的SI口袋的基本形状似乎几乎是固定的，而不管其“客体”的性质（抑制剂的P1残基）。(E)相比之下，S4口袋似乎具有诱导适应的内在能力：当它的客人P4残基具有较小的侧链时，它会显着变窄。

Crystal structure of free SSI (Streptomyces Subtilisin Inhibitor) and that of SSI complexed with its target enzyme subtilisin BPN' were refined to R-factors of 24% (1.85 A) and 21% (1.8 A) respectively. Furthermore two more related structures were refined in which subtilisin BPN' was complexed with either mutant SSI-1 or mutant SSI-2. In both mutant SSI's, the P1 residue of the inhibitor. Met 73, was converted to Lys through gene manipulation. Additionally, in mutant SSI-2 the P4 residue, Met 70, was converted to Gly.Comparative examinations of the structural and "thermal" parameters of these structures indicate the following.(A) Marked rigidification of the SSI structure occurs to some (but not all) parts of SSI upon complex formation with the target enzyme (subtilisin BPN').(B) The rigidification occurs not only to the "reactive site" segment which is in direct contact with the target enzyme but also to those polypeptide segments which are simply connected to the reactive site through either covalent linkage or van der Waals contacts.(C) The rigidified polypeptide segments of SSI mentioned above roughly correspond to the segments where marked shifts upon complex formation were observed in C-13 NMR spectra (observed by M.Kainosho and his colleague using various C-13 enriched SSI's).(D) Basic shape of the SI pocket of subtilisin BPN' seems to be almost fixed irrespective of the nature of its "guest" (P1 residue of the inhibitor).(E) In contrast, the S4 pocket seems to have intrinsic capacity for induced fit : it is considerably narrowed when its guests, P4 residue has smaller sidechain.