title: Addition of a methyl group changes both the catalytic velocity and thermostability of the neutral protease from Bacillus stearothermophilus.
abstract: Specific activity was compared between wild-type (WT) neutral protease from Bacillus stearothermophilus and mutant protease (M1; Gly144 replaced by Ala144) with enhanced thermostability. When casein was used as a substrate, M1 showed 1.5-times higher specific activity than that of WT. In contrast, the specific activities of M1 for soluble reduced lysozyme and insulin B chain were lower than those of WT by 17.2 and 13.2%, respectively. After digestion of the insulin A chain by these enzymes, the peptide products were purified and the N-terminal amino acid sequences were determined. WT enzyme cleaved insulin A chain at three sites, whereas no digestion was observed with M1. Using Z-Gly-Leu-NH2 as a substrate, the kinetic parameters were determined. The Km values are nearly equal for both enzymes, whereas the kcat of M1 (240 min-1) was much smaller compared to the WT (830 min-1). The data indicate that the mutation (addition of a methyl group) exerts an effect by changing both the catalytic velocity and thermostability.
authors.affiliation: Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Takagi
authors.forename: T
authors.initials: T
authors.lastname: Imanaka
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006358:Hot Temperature; D007328:Insulin / Q000032:analysis; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification*; D013820:Thermolysin / Q000032:analysis
chemical_list: D007328:Insulin; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D013820:Thermolysin