Deuterium Isotope Effects on Papain Acylation

Abstract

Kinetic 2H isotope effects on acylation of papain with various substrates when conducted in H2O and 2H2O were presented. With alkyl esters of N-acylamino acids there is no or very little isotope effect, whereas with N-acylamino acid amides the ratio kH2O/k2H2O is less than 1, i.e. there is an inverse isotope effect. Similarly, alkylation of papain with methyl bromoacetate exhibits no kinetic isotope effect, whereas for the analogous alkylation with bromoacetamide an inverse isotope effect is observed. General base catalysis does not occur in the acylation of papain. Kinetic 2H isotope effects can be affected substantially by interaction between the substrate leaving group and the enzyme, which has not been considered in previous mechanistic investigations.