Changing the Substrate Preference of the Hydrophobic S4 Binding Pocket of Subtilisin 309 from Bacillus Lentus

Abstract

The serine endopeptidase subtilisin 309 (Savinase® ) has a preference for substrates containing hydrophobic amino acids in the P4 position. Substitution of Leul35, situated in the hydrophobic S4 binding pocket, for the charged amino acid Glu alters the substrate specificity approximately 500-fold when comparing the ratios of kcat/Km for substrates with Phe and Arg as the P4 residue. This factor can be further increased to 1700-fold by additional replacement of Ilel07 with Val.