Catalytic oxidation of p-cresol by ascorbate peroxidase

Transient and steady state kinetics, together with a range of chromatograph ic and spectroscopic techniques, have been used to establish the mechanism and the products of the H2O2-dependent oxidation of p-cresol by ascorbate p eroxidase (APX). HPLC, GC-MS, and NMR analyses are consistent with the form ation of 2,2'-dihydroxy-5,5'-dimethylbiphenyl (II) and 4 alpha,9 beta-dihyd ro-8,9 beta-dimethyl-3(4K)-dibenzofuranone (Pummerer's ketone, III) as the major products of the reaction. In the presence of cumene hydroperoxide, tw o additional products were observed which, from GC and MS analyses, were sh own to be 1,1-dimethylbenzylalcohol (TV) and bis-(1-methyl-1-phenyl-ethyl)- peroxide (V), The product ratio II:III was dependent on enzyme concentratio n: at low concentrations Pummerer's ketone (III) predominates and at high c oncentrations formation of the biphenyl compound (II) is favored. Steady-st ate data showed a sigmoidal dependence on [p-cresol] that was consistent wi th the presence of 2.01 +/- 0.15 binding sites for the substrate (25.0 degr ees C, sodium phosphate, pH 7.0, mu = 2.2 mM) and independent of ionic stre ngth in the range 2.2-500 mM. Single turnover kinetic experiments (pH 7.0, 5.0 degrees C, mu = 0.10 M) yielded second-order rate constants for Compoun d I reduction by p-cresol, k(2), of 5.42 +/- 0.10 x 10(5) M-1 s(-1), respec tively. Rate-limiting reduction of Compound II by p-cresol, k(3), showed sa turation kinetics, giving values for K-d = 1.54 +/- 0.12 x 10(-3) M and k(3 ) = 18.5 +/- 0.7 s(-1). The results are discussed in the more general conte xt of APX-catalyzed aromatic oxidations. (C) 2000 Academic Press.