MECHANISM OF ACTION OF PAPAIN WITH A SPECIFIC ANILIDE SUBSTRATE

The reaction between papain and Nα-carbobenzoxy-l-lysine p-nitroanilide has been studied at subzero temperatures in fluid aqueous dimethyl sulfoxide solvents. When the reaction was initiated by mixing the enzyme and the substrate at temperatures in the 0 to -65 °C region, a series of four reactions (reactions 1-4) prior to turnover were detected by changes in the substrate spectrum. Three of the intermediates had spectra similar to that of the substrate, and one had a spectrum that closely resembled that of the product p-nitroaniline. The slowest of these preturnover reactions, reaction 4, was also detected in aqueous solution at ambient temperatures by using rapid reaction techniques. Reaction 2 has pAT*s (pk* = apparent pK in the cryosolvent) of 4.4 and ≥8.0 at -25 °C. Reaction 3 has a pK* of 4.0 at -25 °C and reaction 4 has pk*s of 4.3 and ≥8.3 at -3 °C. Both reactions 2 and 4 were absent with the Cys-25 S-methylthio derivative of papain. The concentrations of the products of reactions 2 and 4 were pH* dependent. In the former, the yield of theintermediate decreased with increasing pH*, whereas in the latter the amount of intermediate accumulated increased with increasing pH*. The experimental observations have led to the following interpretation of these reactions. Reaction 1 corresponds to the binding of the substrate yielding the initial Michaelis complex. Reactions 2 and 3 are both associated with conformational changes of the active-site groups and, in particular, with rotation of the imidazole of His-159 so as to break and re-form a salt bridge with the carboxylate of Asp-158. Kinetic evidence is presented for reaction 2 to show that the conformation change precedes substrate binding; i.e., it reflects an isomerization of the free enzyme, whereas reaction 3 represents an isomerization of the enzyme-substrate complex. From the value of the equilibrium constant for reaction 2, it is found that papain is present predominantly in the ion-pair state in the absence of ligand at neutral pH. Reaction 4 involves the thiol of Cys-25 and is ascribed to the formation of a tetrahedral intermediate. © 1979, American Chemical Society. All rights reserved.