2-aroylbenzoyl serine proteases: Photoreversible inhibition or photoaffinity labeling?

Phenyl esters of 2-benzoylbenzoates were determined to be inhibitors of the serine protease enzymes chymotrypsin and thrombin. Thus, p-guanidinophenyl 2-benzoylbenzoate (Ib) inhibited thrombin while the corresponding p-nitrop henyl ester (la) inhibited chymotrypsin activity. Other p-nitrophenyl ester s were prepared that display activity as chymotrypsin inhibitors, three hav ing methoxy group substitution on the benzoyl ring: 2-methoxy (2a), 2,5-dim ethoxy (3a), and 2,4,5-trimethoxybenzoyl (4). After incubation with Ib, an acyl thrombin was isolated that showed no lytic activity but which slowly r egained activity in pH 7.4 buffer. Irradiation of this acyl enzyme with 366 nm light led to an enzyme that showed no gain of lytic activity over time. Incubation of chymotrypsin with 1a-3a and 4 led to acyl enzymes which show ed no activity but which regained activity slowly. Irradiation of these ina ctive acyl enzymes with 366 nm light led to a rapid increase in enzyme acti vity. The formation of acylchymotrypsins that can be photochemically deacyl ated is suggested by these data. Experiments that relate to the mechanism o f enzyme acylation and the subsequent photochemistry of the acylenzymes are reported.