Quantitating direct chlorine transfer from enzyme to substrate in chloroperoxidase-catalyzed reactions

Substrate competition methods that were previously used to quantitate the involvement of free Cl2 in the chloride-dependent peroxidatic reactions catalyzed by chloroperoxidase (CPO) (Libby, R. D., Shedd, A. L., Phipps, K. A., Beachy, T. M., and Gerstberger, S. M., (1992) J. Biol. Chem. 267, 1769-1775) are extended to CPO-catalyzed halogenation reactions. Relative substrate specificities of halogen acceptor substrates (RHs) antipyrine (ap), NADH, 2-chlorodimedone (2cd), and barbituric acid (ba) are compared with previously studied peroxidatic substrates catechol (cat) and 2, 4,6-trimethylphenol (tmp) in their reactions with the CPO-H2O2-Cl system versus the hypochlorite-Cl system. Studies were carried out at pH 2.75 over a chloride concentration range of 1-100 mM and at pH 4.80 over a chloride concentration range of 100-400 mM. Competition studies involved successive pairwise comparisons of substrates of increasing enzyme specificity. The orders of specificities, ba > 2cd > ap > cat > tmp at pH 2.75 and ba > 2cd > NADH > ap > cat > tmp at pH 4.80, are the same for both the CPO-H2O2-Cl and hypochlorite-Cl systems. However, the magnitudes of the specificities are different between the two systems. In all comparisons except ap versus cat, the specificity of the CPO-H2O2-Cl system toward the preferred substrate is higher than that of the hypochlorite-Cl system. Quantitative comparisons between specificities of CPO-H2O2-Cl and hypochlorite-Cl systems indicate that at least 98% of the CPO-catalyzed halogenation reactions of ba, 2cd, NADH, and ap occur by mechanisms in which the substrate reacts directly with the enzyme. Thus, less than 2% of any of the CPO reactions could possibly involve a free oxidized halogen intermediate. All data are consistent with a mechanism in which RH binds to the CPO chlorinating intermediate (EOCl), and the chlorine atom is transferred directly from EOCl to RH. Further, the results indicate that any halogenation substrate with a higher CPO specificity than ap must also undergo direct chlorine transfer from the enzyme. These results underscore the critical need for quantitative kinetic evidence in establishing the extent of involvement of any potential reaction intermediate. Finally, this work calls into question the long held assumption of the obligatory involvement of hypochlorite as an intermediate in myeloperoxidase reactions. It supports the recent kinetic evidence presented by Marquet and Dunford for direct chlorine transfer in myeloperoxidase-catalyzed chlorination of tuarine (Marquet, L. A., and Dunford, H. B. (1994) J. Biol. Chem. 269, 7950-7956).