Kinetics of Binding between Thermolysin and Streptomyces

The mechanism of binding between thermolysin with its specific inhibitor, talopeptin (MKI), was studied kinetically with the stopped-flow method by monitoring the enhancement of tryptophan fluorescence caused by the complex formation. Only one relaxation obeying first-order kinetics was observed. The dependence of the apparent first-order constant, Arapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular binding step followed by a slow unimolecular step. It was found that the increase in tryptophan fluores-cence occurs solely in the slow unimolecular step. The apparent second-order rate constant, (ArOn)app, in the low inhibitor concentration range, was determined over the pH range between 5 and 8.5 and decreases with increasing pH. The activation parameters for the overall binding process were obtained from the temperature dependence of (£on)app • A protease inhibitor, talopeptin (MKI), which was purified from Streptomyces mozunensis MK-23 by Murao et al. (7), specifically inhibits metallopro-teases such as thermolysin, a thermophilic Zn(II)-containing neutral protease isolated from Bacillus thermoproteolyticus (2). The structure of the in-hibitor, Af-(6-deoxy-a-L-talopyranosyloxyphospho)-L-leucyl-L-tryptophan, resembles another specific inhibitor for thermolysin named phosphoramidon