title: Effects of mutations of thermolysin, as N116 to asp and asp150 to glu, on salt-induced activation and stabilization.
abstract: Neutral salts activate and stabilize thermolysin. We previously found that two single mutations, Asn116→Asp and Asp150→Glu, increase the activity of thermolysin. In the present study, we examined their effects on NaCl-induced activation and stabilization. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, the relative activities (the ratios of the specificity constant, kcat/Km, at x M NaCl to that at 0 M NaCl) at 0.5-4.0 M NaCl of D150E and N116D/D150E were lower than those of wild-type thermolysin (WT) and N116D, respectively. In thermal inactivation at 70 °C, the relative stabilities (the ratios of the first-order rate constant, kobs, at 0 M NaCl to that at x M NaCl) at 0.5-4.0 M NaCl of D150E and N116D/D150E were lower than those of WT and N116D, respectively. These results indicate that unlike Asn116→Asp, Asp150→Glu reduced NaCl-induced activation and stabilization, suggesting that the binding of ions with certain residues of thermolysin is involved in the activation and stabilization.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Evans
authors.initials: E
authors.lastname: Menach
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D000179:Acrylates / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D004151:Dipeptides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D012965:Sodium Chloride / Q000494:pharmacology*; D013820:Thermolysin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000179:Acrylates; D004151:Dipeptides; C108649:N-(3-(2-furyl)acryloyl)glycyl-leucinamide; C051210:N-benzyloxycarbonyl-aspartylphenylalanine methyl ester; D012965:Sodium Chloride; D013820:Thermolysin