Mechanistic enzymology in non-aqueous media

Abstract

A variant of subtilisin Carlsberg thiolsubtilisin, in which the active site serine residue is replaced by a thiol-containing cysteine has been synthesized in our laboratory. From the results of analysis of the resulting enzyme it can be argued that the mechanism of subtilisin suspended in organic solvents involves Ser221, undoubtedly in the formation of an acyl-enzyme intermediate. Further, the active site of the enzyme is not interacting with bulk solvent, and the environment of Ser221 is not radically altered by changing the solvent in which the enzyme is suspended. The enzyme structure has also been analyzed with FT-IR to demonstrate that the global secondary structure is the same in organic dispersants as in water. Finally, the morphology of subtilisin particles has been studied during hydration in the gas phase using ESEM. © 1992, IUPAC