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The kinetics of papain- and ficin-catalysed hydrolyses in the presence of alcohols.

ABSTRACT: 1. The maximum rate of production of p-nitrophenol (V(max.)) for both papain- and ficin-catalysed hydrolyses of p-nitrophenyl hippurate is independent of methanol concentration up to 2m for papain and 1.5m for ficin. 2. The observed catalytic constant (k(0)) for the production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate decreases with increasing methanol concentration, 1/k(0) being linearly dependent on the methanol concentration. The k(MeOH)/k(H2O) ratio is determined. 3. These results provide strong evidence against general base catalysis for the rate-determining step in the deacylation of hippuryl-papain and hippuryl-ficin and probably for other specific acyl-papains and acyl-ficins. 4. The rate-determining step for the deacylation of the non-specific trans-cinnamoyl-papain appears to be different from that for the specific hippuryl-papain, and is probably subject to general base catalysis. It is possible, however, to accommodate all these observations in a single four-step reaction pathway. 5. Propan-2-ol did not influence the rate of production of hippuric acid for the papain-catalysed hydrolysis of methyl hippurate. A similar result has previously been reported for the ficin-catalysed hydrolysis of methyl hippurate. Ethanol and of course methanol (see 2) decrease the rate of production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate. It is suggested that the secondary alcohol is incapable for structural reasons of approaching the bond to be hydrolysed.

SUBMITTER: Lake AW

PROVIDER: S-EPMC1270121 | biostudies-other | 1966 Nov

REPOSITORIES: biostudies-other

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Project description: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 pH7.0, plots of [S(0)]/v(i) (initial substrate concn./initial velocity) versus [S(0)] 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 [S(0)]/v(i) versus [S(0)] 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 [S(0)]/v(i) versus [S(0)] 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 pH7.0, I=0.1 and 25 degrees C is characterized by K(m) (1) (the dissociation constant of ES)=1.22+/-0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57x10(-2)+/-0.32x10(-2)s(-1), K(a) (2) (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 alpha-N-benzoyl-l-arginine ethyl ester and of alpha-N-benzoyl-l-arginine amide; K(m) (1) and k for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine amide hydrolysis and K(as) and k' for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of k(cat.) and K(m) 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 of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (K(m)=52+/-4mm, k(cat.)=2.80+/-0.1s(-1) at pH7.0, I=0.1, 25.0 degrees C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.

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The kinetics of papain- and ficin-catalysed hydrolyses in the presence of alcohols.

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