Proteolytic enzymes. Models for hydrolyses catalysed by papin

Abstract

The effect of pH on the reaction of mercaptoacetic acid with 3-nitrophenyl acetate and with 4-chlorophenyl N-methylsulphonylglycinate gave equivocal evidence for the involvement of the unionised thiol as a nucleophile. The reaction of ethanethiol with 4-nitrophenyl 5-nitrosalicylate exhibited a sigmoid pH-dependence (pK_a= 6·00) but it was not possible to distinguish unequivocally between mechanisms involving the reaction of uncharged ester with thiolate anion or the ionised ester with thiol. The pH-dependence for the reaction of ethanethiol with 4-nitrophenyl quinoline-8-carboxylate exhibited two inflections (pK_a= 3·48 and 11·33). The alkaline limb corresponds to attack of thiolate anion on uncharged ester and the other limb involves participation of tertiary nitrogen. The sensitivity (ρ= 0·99) of the rate constant to the substituent on the phenol leaving group in the latter reaction is utilised as evidence for the reaction of thiolate anion with protonated ester. The kinetics of hydrolysis of a series of substituted phenyl formates and evidence from the literature indicates a higher selectivity to leaving group for the reaction of water as opposed to the reaction of hydroxide ion at a carboxy-centre. Arguments based on diffusion rate constants are used to eliminate the zwitterion type mechanism in papain-catalysed reactions.