pH-dependence and structure-activity relationships in the papain-catalysed hydrolysis of anilides.
The pH-dependence of the Michaelis-Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, K(s), is independent of pH between 3.7 and 9.3, whereas the acylation constant, k(+2), shows bell-shaped pH-dependence with apparent pK(a) values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett rho value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett rho values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.
D000813:Anilides; D005998:Glycine; D006626:Hippurates; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010206:Papain; D010455:Peptides; D010649:Phenylalanine; D013329:Structure-Activity Relationship; D013424:Sulfanilamides
D000813:Anilides; D006626:Hippurates; D010455:Peptides; D013424:Sulfanilamides; D010649:Phenylalanine; D010206:Papain; D005998:Glycine