Schmitke J L, Stern L J, Klibanov A M
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):12918-23. doi: 10.1073/pnas.95.22.12918.
The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-A resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.
已分别在无水乙腈和水中，将丝氨酸蛋白酶枯草芽孢杆菌卡尔伯格亚种的酰基酶共价中间体反式肉桂酰枯草芽孢杆菌蛋白酶的X射线晶体结构解析到2.2埃分辨率。肉桂酰枯草芽孢杆菌蛋白酶的结构在这两种溶剂中几乎相同。此外，它们的酶部分与先前测定的该游离酶在乙腈和水中的结构几乎无法区分；因此，在水性或非水性溶剂中的酰化反应不会引起明显的构象变化。然而，在乙腈和水中，结合在酰基酶和游离酶形式活性位点的溶剂分子位置是不同的。活性位点溶剂化的这种差异可能导致所观察到的酶活性变化。长时间暴露于有机溶剂或从晶格中去除间隙溶剂后，酶晶体中的通道会塌陷，导致底物可及的活性位点数量下降。我们讨论了这些发现对有机溶剂中酶催化的机理和制备方面的影响。
