Phenothiazine-N-carbonyl chloride, a specific inactivator of chymotrypsin

Phenothiazine-N-carbonyl chloride inactivated chymotrypsin and trypsin by means of a 1:1 stoicheiometric reaction. Its reaction with chymotrypsin was 29 times as fast as that with trypsin and was inhibited by indole. The reaction of phenothiazine-N-carbonyl chloride with chymotrypsin resembled an enzyme-substrate reaction in which the deacylation step is rate-limiting. Slow deacylation occurred, resulting in complete regeneration of active enzyme in 15h. The pH-rate profile of the inactivation process had a maximum at pH7.8. These data and other evidence indicate that the reaction of phenothiazine-N-carbonyl chloride with chymotrypsin exhibits ;kinetic specificity'. Therefore any hypothesis that attempts to describe the topography of the active site of chymotrypsin should take into account the reactivity of phenothiazine-N-carbonyl chloride. The above findings, as well as recent reports of others, are examined within the context of a hypothesis given in an earlier paper (Erlanger, 1967).