pH and Temperature Dependences of Thermolysin Catalysis

Abstract

The pH dependences of k Cat /K m , k cat and K m of thermolysin‐catalyzed hydrolysis of N‐furylacryloylglycyl‐ l ‐leucinamide (Fua‐Gly‐LeuNH 2 ) and N‐furylacryloylglycyl‐ l ‐phenylalaninamide (Fua‐Gly‐PheNHz) were in‐ vestigated. Taking the buffer dependences into account, the k cat /K m profile was explained by a simple bell‐shaped curve with pK a1 = 5.0 and pK a2 = 8.25, at 25°C. Both k cat and K m increased with pH at lower pH and took larger values for Fua‐Gly‐LeuNHz than for Fua‐Gly‐PheNHz at 25°C. The pH dependence of inhibitory actions by amino acids and dipeptides, such as carbobenzyloxy‐L‐phenylalanine and L‐phenylalanyl‐I–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 k cat /K m , K i and the two p K a values in the k cat /K m profile were measured. The k cat /K m showed non‐linear dependence while K i increased linearly with temperature on a logarithmic scale. The calculated AH values of deprotonation for pKal and pK a1 were 33.4 kJ/mol and 35.1 kJ/mol, respectively. The value for pK al 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