The Actinidin‐Catalysed Hydrolysis of N‐α‐Benzyloxycarbonyl‐l‐lysine p‐Nitrophenyl Ester

The hydrolysis of N ‐α‐benzyloxycarbonyl‐lysine p ‐nitrophenyl ester catalysed by the thiol protease, actinidin, from Actinidia chinensis has been studied under steady state and non‐steady state ([E] 0 > [S] 0 ) conditions. The Michaelis constant K m is dependent on ionising groups of p K a 3.75 and 8.1 and lowest at neutral pH. The catalytic centre activity, k cat , is almost pH independent below pH 7.0 but increases greatly at high pH with a p K a of 8.1. A biphasic reaction was observed under conditions of [E] 0 > [S] 0 indicating that the simple acylation‐deacylation mechanism does not apply and at least one additional step is necessary. The rapid phase of reaction involves production of p ‐nitrophenol bound to an acylated enzyme; p ‐nitrophenol is released in the slow phase. From the amplitude and rate constant of the rapid phase, and the steady state parameters, the rate and equilibrium constants of some of the steps of the reaction were determined. The biphasic behaviour under conditions of excess enzyme was found from pH 5.0 to 7.9 but the reaction was monophasic at pH 4.0.