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

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Abstract
The maximum rate of production of p-nitrophenol (Vmax.) for both papain-and ficin-catalysed hydrolyses of p-nitrophenyl hippurate is independent of methanol concentration up to 2[image] for papain and 1.5[image] for ficin. The observed catalytic constant (ko)Tor the production of "hippuric acid for both papain-and ficin-catalysed hydrolyses of methyl hippurate decreases with increasing methanol concentration, 1/k0 being linearly dependent on the methanol concentration. The kH2O ratio is determined. 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. 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. 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 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.