The role of Pro-239 in the catalysis and heat stability of subtilisin E.
Site-directed mutagenesis was employed to analyze the role of an alpha-helix containing catalytic Ser-221 of subtilisin E. Pro-239 located at the carboxy-terminal end of the alpha-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 alpha-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 alpha-helix containing catalytic Ser-221 in the catalysis as well as in the heat stability of subtilisin.
Takagi
H
H
Central Research Laboratories, Ajinomoto Co., Inc., Kanagawa.
Morinaga
Y
Y
Ikemura
H
H
Inouye
M
M
D002384:Catalysis; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011392:Proline / Q000032:analysis*; D011487:Protein Conformation; D013381:Subtilisins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature
D011392:Proline; D013381:Subtilisins