pH and temperature dependences of thermolysin catalysis. Catalytic role of zinc-coordinated water.

The pH dependences of kcat/Km, kcat and Km of thermolysin-catalyzed hydrolysis of N-furylacryloylglycyl-L-leucinamide (Fua-Gly-LeuNH2) and N-furylacryloylglycyl-L-phenylalaninamide (Fua-Gly-PheNH2) were investigated. Taking the buffer dependences into account, the kcat/Km profile was explained by a simple bell-shaped curve with pKa1 = 5.0 and pKa2 = 8.25, at 25 degrees C. Both kcat and Km increased with pH at lower pH and took larger values for Fua-Gly-LeuNH2 than for Fua-Gly-PheNH2 at 25 degrees C. The pH dependence of inhibitory actions by amino acids and dipeptides, such as carbobenzyloxy-L-phenylalanine and L-phenylalanyl-L-leucinamide, showed characteristic features depending on their charge states: anionic or neutral ones inhibited the enzyme more strongly at lower pH while cationic ones did so at neutral pH. Temperature dependences of kcat/Km, Ki and the two pKa values in the kcat/Km profile were measured. The kcat/Km showed non-linear dependence while Ki increased linearly with temperature on a logarithmic scale. The calculated delta H values of deprotonation for pKa1 and pKa2 were 33.4 kJ/mol and 35.1 kJ/mol, respectively. The value for pKa1 is too large to be assigned to the carboxylic group of Glu-143, in contrast to the generally accepted view. A mechanism for thermolysin catalysis is presented with particular emphasis on the binding specificity and the catalytic role of zinc-coordinated water.