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

游离SSI （Streptomyces Subtilisin Inhibitor）的晶体结构和与目标酶Subtilisin BPN'配合的SSI晶体结构分别细化为24% （1.85 A）和21% （1.8 A）的r因子。此外，还有两个相关的结构被提炼出来，其中枯草杆菌素BPN'与突变体SSI-1或突变体SSI-2络合。在两个突变体SSI中，P1残基的抑制剂。Met 73，通过基因操作转化为赖氨酸。此外，在突变体SSI-2中，P4残基Met 70转化为Gly。对这些结构的结构和“热”参数的比较检查表明：(A) SSI的一些（但不是全部）部分在与目标酶（枯草杆菌素BPN’）形成复合体时发生了明显的硬化。(B)固化不仅发生在与靶酶直接接触的“反应位点”段，也发生在通过共价键或范德华接触与反应位点简单连接的多肽段。(C)上述硬化的SSI多肽段大致对应于C-13核磁共振光谱（M.Kainosho和他的同事使用各种富含C-13的SSI观察到的复合物形成时明显移位的片段）。(D) subtilisin BPN'的SI口袋的基本形状似乎几乎是固定的，而不考虑其“客体”（抑制剂的P1残基）的性质。(E)相比之下，S4口袋似乎具有诱导拟合的内在能力：当它的客人P4残基具有较小的侧链时，它显着变窄。

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.