title: Effects of neutral salts and alcohols on the activity of Streptomyces caespitosus neutral protease.
abstract: Streptomyces caespitosus neutral protease (ScNP) is one of the smallest metalloproteinase with a molecular mass of 14 kDa. Effects of solvent composition on ScNP activity were examined using a peptide substrate. The k(cat)/K(m) values of ScNP exhibited bell-shaped pH-dependence with the optimal pH of 6.4-7.0 and the pK(a) values of 5.0 +/- 0.1 and 8.3 +/- 0.1. ScNP activity increased in an exponential fashion with increasing [NaCl]. The relative k(cat)/K(m) value at 3.6 M NaCl to that at 0 M NaCl was 3.7, and the degree of the activation at x M NaCl was expressed as 1.2 (x) (x < 2.0) and 1.4(x) (x > 2.0). On the other hand, ScNP activity decreased with increasing concentrations of LiCl, KCl, NaBr, LiBr, KBr and NaClO(4). Alcohols inhibited ScNP activity with the IC(50) values, the concentration required for decreasing the activity at 50% of the maximum, of 0.77-6.54 M. The order of the inhibitory potency was 1-butanol, 2-methyl-1-propanol, 2-methyl-2-butanol > 2-methyl-2-propanol, 2-butanol, 1-propanol > 2-propanol >> ethanol >> methanol. The activities recovered completely by the dilution of alcohols, suggesting that the ScNP inhibition by alcohols is reversible. These characteristics of ScNP are compared with those of human matrix metalloproteinase 7 and thermolysin.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan. inouye@kais.kyoto-u.ac.jp
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
authors.forename: Takashi
authors.initials: T
authors.lastname: Shimada
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
mesh_terms: D000438:Alcohols / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D012492:Salts / Q000494:pharmacology*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D012492:Salts; D008666:Metalloendopeptidases; C106507:zinc endoprotease, Streptomyces caespitosus