N-Benzoyl-L-serine Methyl Ester Catalysed by Bromelain and by Papain ANALYSIS OF MODIFIER MECHANISMS

1. N-Benzoyl-L-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH 7.0, plots of [SO]/vi (initial substrate concn./initial velocity) versus [SO] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [So]/vi versus [SO] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [So]/vl versus [SO] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-L-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-L-serine methyl ester at pH 7.0, I= 0.1 and 25°C is characterized by Kmj (the dissociation constant of ES)= 1.22±0.73mM, k (the rate constant for the breakdown of ES to E+products, P) =1.57x 10-2+0.32x 10-2s-1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)= 0.38 ±0.06M, and k' (the rate constant for the breakdown of SES to E+P+S) = 0.45± 0.04s-1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of a-N-benzoyl-L-arginine ethyl ester and of a-N-benzoylL-arginine amide; K,,,1 and k for the serine ester hydrolysis are somewhat similar to K,,, and kca,t. for the arginine amide hydrolysis and Ka, and k' for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kca,. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis oftheserine ester substrateare tentatively interpreted in terms ofnon-productive binding in the binary complex and a decrease of this type of binding by ternary complexformation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km = 52±4mM, kcat. = 2.80±0.1 s-' at pH7.0, I= 0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1 M have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.