198 Biochimica et Biophysica Acta, 523 (1978) 198--206 © Elsevier/North-Holland Biomedical Press BBA 68372 RATE CONSTANTS OF INDIVIDUAL STEPS IN PAPAIN-CATALYSED REACTIONS YONGYUTH YUTHAVONG and WICHAI SUTTIMOOL * Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 4 (Thailand) (Received August 31st, 1977) Summary Rate constants for acylation of papain (EC 3.4.22.2) by specific substrates and its subsequent deacylation are derived from kinetic analysis of the reac- tions in the presence of aminoacetonitrile and methanol. Methyl and ethyl hip- purate and methyl N-benzyloxycarbonylglycinate have marginally higher values of rate constants for acylation than for deacylation, while the reverse is true for ethyl N-benzoyl-L-argininate. Both acylation and deacylation are rate-determin- ing for these substrates, while only deacylation is rate-determining for methyl- N-acetyl-L-phenylalanylglycinate. Deacylation is the only rate-determining step for p-nitrophenyl esters of hippuric acid, N-benzyloxycarbonylglycine and N-acetyl-L-phenylalanylglycine. These results are discussed in relation to those from inactivation of the enzyme by alkylating agent in the presence of sub- strate. Introduction It is now widely accepted (e.g., see ref. 1) that the minimal mechanistic scheme for papain (EC 3.4.22.2)-catalysed reactions is Eqn. 1: kl k2 k~3 E+S~-ES -+ES' -+ E + P: (1) ~-i +pl where k~ and k 1 are rate constants for formation and dissociation of enzyme • substrate complex, k2 the rate constant for acylation of the enzyme and k~ (or k3[H~O]) the rate constant for deacylation through hydrolysis: ES is the * Present address: Massachusetts College of Pharmacy, 179 Longwood Ave., Boston, Mass. 02115, U.S.A. Abbreviations: L-BAEE, ethyl ~-N-benzoyi-L-axgininate;D-BAEE, ethyl a-N-benzoyl-D-argininate.