Photofragmentation and photoaffinity labeling of phenacyl and naphthacyl α-chymotropsins

The photochemistry of phenacyl, 1-, and 2-napthacyl α-chymotrypsins has been studied using light of wavelengths greater than 310 nm. Irradiation of these modified enzymes, having aracyl chromophores covalently bound at Met-192, led to partial regeneration of their esterase activity. The initial rates of photoreactivation have been found to be dependent on the hydrogen ion concentration and on the presence of potential competitive inhibitors having triplet energies lower than the chromophores initially receiving the light energy. Irradiation of carbonyl-14C-labeled phenacyl α-chymotrypsin resulted in partial loss of this radiolabeled from the protein at a rate concurrent with the increase in esterase activity. These results have been interpreted and discussed in terms of a dual photochemical behavior of these modified enzymes involving (1) cleavage of the Met-192 sulfur--phenacyl α-carbon bond leading to liberation of α-chymotrypsin and substituted phenone(s) and (2) photoaffinity labeling of one or more of the functional groups in the enzyme active-site region