The Role of Pro-239 in the Catalysis and Heat Stability of Subtilisin E

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Abstract
Site-directed mutagenesis was employed to analyze the role of an α-helix containing catalytic Ser-221 of subtilisin E. Pro-239 located at the carboxy-terminal end of the α-helix was first replaced with Gly to examine the role of Pro-239 in the catalysis and stability of subtilisin E. The mutation was found to decrease both the catalytic rate (kcat) and the heat stability. This result strongly suggests that Pro-239 plays an important role in the maintenance of the α-helix, affecting the functioning of the active site. Various amino acid substitutions at position 239 were attempted to obtain the active subtilisins from Gly-239 subtilisin. Lys- and Arg-substitutions were found to result in more active and stable subtilisins than the Gly-239 subtilisin. In particular, the Arg-239 mutant showed enhanced heat stability compared with the wild type. These results demonstrate the important role of the α-helix containing catalytic Ser-221 in the catalysis as well as in the heat stability of subtilisin.