Inhibitory Effects of Alcohols on Thermolysin Activity as Examined Using a Fluorescent Substrate

Abstract

Alcohols inhibit the thermolysin-catalyzed hydrolysis of N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH₂ and decrease the NaCl-induced activation of thermolysin in a concentration-dependent manner [K. Inouye et al. (1997) J. Biochem. 122, 358-364]. In this study, the inhibitory effects of alcohols on thermolysin activity were examined in detail using 10 different alcohols and a fluorescent substrate, (7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N³-(2, 4-dinitrophenyl)-L-2, 3-diamino-propionyl]-L-Ala-L-Arg-NH₂. The inhibition by all alcohols examined is completely reversible, and thermolysin activity is recovered by dilution. The inhibitor constants (K_i) are in the range of 35-430 mM, and the order of the inhibitory effect is 1-pentanol, 1-propanol, 2-butanol, 2-methyl-l-pro-panol > 1-butanol > 2-propanol > ethanol, tert-amyl alcohol >> tert-butyl alcohol >> methanol. Linear and secondary alcohols whose mains chains consist of more than 3 carbons inhibit thermolysin effectively. Thermolysin activity is decreased by decreasing the dielectric constant, D, of the reaction medium containing the alcohol, and the decrease depending on the D value was almost the same manner for all alcohols except methanol, tert-butyl alcohol, and tert-amyl alcohol. Alcohols may inhibit thermolysin activity both by binding to the active site, most possibly to the S1' subsite, of thermolysin and by altering the electrostatic and hydrophobic environment around the thermolysin molecule.