MECHANISM OF THIOL PROTEASE CATALYSIS - DETECTION AND STABILIZATION OF A TETRAHEDRAL INTERMEDIATE IN PAPAIN CATALYSIS

Evidence supporting the existence of a tetrahedral intermediate in the acylation reaction of papain with the specific substrate N.alpha.-carbobenzoxy-L-lysine p-nitroanilide is presented. This intermediate can be directly observed both at subzero temperatures using cryoenzymological techniques and in aqueous solution at 25.degree. C using rapid reaction techniques. The intermediate is accumulated in essentially 1:1 stoichiometry with active enzyme at high pH* [apparent pH in the cryosolvent] (.gtoreq. 9.3) and stabilized for indefinitely long periods at subzero temperatures. Both the rate of formation and concentrations of the intermediate are pH dependent (pK1 .simeq. 4.3; pK2 .simeq. 8.6). Formation of the intermediate is rate limiting at low pH, whereas breakdown is rate determining at high pH. The spectral characteristics of the tetrahedral intermediate are similar to those of p-nitroaniline except .lambda.max is blue-shifted by about 16 nm. If mercuripapain or the S-methylthio derivative of papain is used, no reaction is observed at ambient or subzero temperatures. Extrapolation of the rate of formation of the tetrahedral intermediate at subzero temperatures to 25.degree. C and 0% cosolvent yields a calculated value of Kobsd = 65 .+-. 10 s-1, in excellent agreement with the observed value of 70 .+-. 4 s-1 from stopped-flow studies. The role of stereoelectronic factors in the formation of the tetrahedral intermediate is considered in the context of the proposed catalytic mechanism.