Site-Specific Inactivation of Papain by Ascorbic Acid in the Presence of Cupric Ions

Abstract

The mechanism of inactivation of papain (EC 3.4.22.2) by ascorbic acid (AsA) in the presence of cupric ions (Cu²⁺) was investigated. The aerobic combination of Cu²⁺ and AsA resulted in an irreversible loss of enzyme activity. The inactivation was found to be an apparent first order reaction. The prior mixing of Cu²⁺ and AsA caused the complete disappearance of the inactivation. The addition of iron ions led to significant suppression against the inactivation. Cu²⁺ was bound to the enzyme in a molar ratio of 1 : 1. At lower concentrations of Cu²⁺ (molar ratio of enzyme to Cu²⁺ of 1 : <1), the extent of inactivation showed the same dependence against the extent of oxidation of AsA. The rate of inactivation increased as the concentration of AsA was increased. Saturation kinetics were observed with respect to the concentration of AsA. Changes in the concentration of Cu²⁺ had no effect on the dissociation constant of the enzyme-AsA complex (K_I), though the rate constant of inactivation (k₂) showed a linear relationship with the concentration of Cu²⁺. At various pH values tested, no change of k₂ was found, whereas the value of K_I increased when the pH bacame lower. At higher concentrations of Cu²⁺, the rate of inactivation fell beyond a certain concentration of AsA.The present results suggest that both Cu²⁺ and AsA bind to the enzyme to form a ternary complex and that free radicals are site-specifically formed and react preferentially with the enzyme, at the site of their formation, impairing its activity.