BINDING SITES FOR SUBSTRATE LEAVING GROUPS AND ADDED NUCLEOPHILES IN PAPAIN-CATALYZED HYDROLYSES

The interactions of some alcohol and amine nucleophiles in the papain-catalyzed hydrolysis of the pnitrophenyl esters of N-acetyl-L-tryptophan and N-CBz-L-Lys have been investigated. The effects of the added nucleophile on the kinetic parameters and product distribution were found to vary with the structure of the nucleophile, and both activation and inhibition could be observed with respect to the rate of disappearance of substrate. The results for the addition of n-alkyl alcohols could be interpreted in terms of simple partition of the acyl-enzyme between water and alcohol only for the smallest members of the series. However, all the results could be satisfactorily explained by a scheme in which the added modifier binds to the free enzyme, enzyme substrate complex, and acyl-enzyme. Evidence is presented for the existence of two different modes of binding for the added nucleophile, one which may also overlap that of the substrate leaving group, the other which interferes with substrate binding. The dependence of kcat/Km upon the nucleophile concentration, and the noncompetitive inhibition by 2-pentanone are interpreted in terms of a ternary enzymesubstrate-nucleophile complex. The increased binding affinity of the added nucleophile with lengthening of the apolar chain in the case of the aliphatic alcohols suggests hydrophobic interactions to be the major force responsible for binding. The reactivity of the alcohol toward the acyl-enzyme apparently decreases with increasing chain length, whereas the formation of the ternary complex increases the rate of acylation with the more apolar nucleophiles. The results support the existence of an acyl-enzyme intermediate, with general base catalysis involved in deacylation. The specificity of binding for the added modifier suggests the possibility of using papain as a catalyst in asymmetric synthesis of acyl derivatives, and in peptide synthesis. © 1969, American Chemical Society. All rights reserved.