Site-specific inactivation of papain by ascorbic acid in the presence of cupric ions.
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 (KI), 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 KI 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.
Kanazawa
H
H
Kyoto Pharmaceutical University, Japan.
Fujimoto
S
S
Ohara
A
A
D001205:Ascorbic Acid / Q000378:metabolism / Q000494:pharmacology*; D003300:Copper / Q000378:metabolism; D005609:Free Radicals; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D009942:Organometallic Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D010206:Papain / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D005609:Free Radicals; D009942:Organometallic Compounds; C015092:cupric acetate; D003300:Copper; D007501:Iron; D010206:Papain; D001205:Ascorbic Acid