Subtilisin Structure and Catalysis in Non - Aqueous Media

................................................................................................. 3 Acknowledgments ................................................................................. 5 Introduction ............................................................................. 7 I. The Crystal Structure of Subtilisin Carlsberg in Anhydrous Dioxane and Its Comparison with Those in Water and Acetonitrile ........................................... 10 A. Introduction ................................................................................... 10 B. Results and Discussion .................................................................. 11 C. Materials and Methods ................................................................... 22 D. References ................................................................................... 31 II. The Mechanistic Dissection of the Plunge in Enzymatic Activity upon Transition from Water to Anhydrous Solvents ............................................................. 34 A . Introduction ................................................................................. 34 B. Results and Discussion .................................................................... 35 C. Concluding Remarks.....................................................47 D. Materials and Methods .................................................................. 48 E. References ................................................................................... 54 III. Comparison of X-ray Crystal Structures of an Acyl-Enzyme Intermediate of Subtilisin Carlsberg Formed in Anhydrous Acetonitrile and in Water .............. .. 57 A . Introduction ................................................................................. 57 B. Results and Discussion .................................................................... 59 C. Concluding Remarks.....................................................70 D. Materials and Methods .................................................................... 71 E. References ............................................................................... 80 IV. Organic Solvent Binding to Crystalline Subtilisin in Mostly Aqueous Mixtures vs. in N eat Solvents ........................................................................................ 84 A . Introduction ................................................................................. 84 B. Results and Discussion .................................................................... 85 C. Materials and Methods .................................................................... 89 D . R eferences ................................................................................... 95 V. Biographical Note ................................................................. ..... 98