ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 206, No. 2, February, pp. 362-371 1981 Effect of Hydroxynitrobenzylation of Tryptophan-177 on Reactivity of Active Site Cysteine-25 in Papain1 BETTIE L. B. EVANS2 JAMES A. KNOPP, AND H. ROBERT HORTON Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27650 Received July 25, 1980 It is known that the enzymatic activity of papain (EC 3.4.22.2) toward a-N-benzoyl-L- arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4- nitrophenyl (N-carbobenzoxy)glycinate (S.-M. T. Chang and H. R. Horton, 1979, Biochem- istry 18, 1559-1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactiva- tion by S,2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloro- acetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulthydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the in- creased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, a-N-benzoyl-L-phenylalanyl-L-valyl-L-arginine p-nitroanilide, and toward an ester sub- strate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxy- nitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward a-N-benzoyl-L-arginine p-nitroanilide, apparently by blocking nonproduc- tive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159). Selective chemical modifications of a pro- tein’s functional groups have provided con- siderable information relating structure to biological function, in many diverse systems. It is rare that chemical modification of an enzyme’s active site region enhances cata- lytic activity, however. Tryptophan has been implicated in the action of papain (EC 3.4.22.2) through photooxidation (1, 2>, reaction with N- ’ This investigation was supported in part by the National Science Foundation under Grant BMS 72- 01999 AOl. Any opinions, findings, and conclusions are those of the authors and do not necessarily reflect the views of the National Science Foundation. This is Paper No. 5956 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N. C. * Present address: Biology Department, Brook- haven National Laboratory, Upton, L. I., N. Y. 11973. bromosuccinimide (3-51, and reaction with the active site-directed reagent, 2-chloro- methyl-knitrophenyl (N-carbobenzoxy)- glycinate (Z-Gly-ONB-C1)3 (6, 7). The pres- ence of several of papain’s five tryptophyl residues within the vicinity of the enzyme’s active site had been discovered by X-ray crystallography (8- 10). The location of Trp- 177 in the crystallographic structure is of particular interest, since it appears to form ’ Abbreviations used: Z-Gly-ONB-Cl, 2-chloro- methyl-4-nitrophenyl (N-carbobenzoxy)glycinate; HNB-, 2-hydroxy-5-nitrobenzyl-; BzArgNan, cu-N- benzoyl-L-arginine p-nitroanilide; BzPheValArgNan, a-N-benzoyl-L-phenylalanyl-L-valyl-L-arginine p- nitroanilide; Z-Gly-ONp, N-carbobenzoxyglycine p- nitrophenyl ester; NEM, N-ethylmaleimide; Tos-Phe- CH&l, L-1-tosylamido-2-phenylethyl chloromethyl ketone; Tos-LysCH,Cl, L-1-tosylamido-5-aminopentyl chloromethyl ketone. 0003-9361/81/020362-10$02.00 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. 362