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Addition of a methyl group changes both the catalytic velocity and thermostability of the neutral protease from Bacillus stearothermophilus
Author(s) -
Takagi Masahiro,
Imanaka Tadayuki
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81006-3
Subject(s) - thermostability , protease , chemistry , enzyme , casein , biochemistry , mutant , substrate (aquarium) , peptide , stereochemistry , biology , ecology , gene
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‐NH 2 as a substrate, the kinetic parameters were determined. The K m values are nearly equal for both enzymes, whereas the k cat 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.