title: The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis.
abstract: Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.
authors.affiliation: Central Research Laboratories, Ajinomoto Co., Inc., Kanagawa.
authors.forename: H
authors.initials: H
authors.lastname: Takagi
authors.forename: S
authors.initials: S
authors.lastname: Arafuka
authors.forename: M
authors.initials: M
authors.lastname: Inouye
authors.forename: M
authors.initials: M
authors.lastname: Yamasaki
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000235:genetics; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D013381:Subtilisins; D010196:Pancreatic Elastase