Horseradish Peroxidase. XVII. Reactions of Compounds I and II with p-Aminobenzoic Acid in Deuterium Oxide

Abstract

The kinetics of reactions of horseradish peroxidase compounds I and II (HRP-I and HRP-II, respectively) with p-aminobenzoic acid have been studied in ordinary water and in deuterium oxide solution over a pH (pD) range 3–10, at 25° and at an ionic strength of 0.11. Under the conditions of the experiments the rate of reaction is first order both in substrate concentration and in enzyme concentration in both solvents. An analysis of the pH dependence of the second order rate constant in H2O confirms the presence of two acid dissociation groups on the enzyme with pKa' of 8.6 and ∼0 for HRP-II, whereas for HRP-I the data suggest a pKa of 5.1 on the enzyme and reveal, as previously shown, the influence of the ionization of the substituted ammonium group of the substrate. In deuterium oxide the pD profiles are similar to those in water but significant shifts for both kinetic and acid dissociation constants are observed for both compounds.The numerical values of the isotope effects taken together with previous results in general confirm that with labile substrates the group of pKa 8.6 in H2O on HRP-II is involved in general acid catalysis. p-Aminobenzoic acid is intermediate between labile and somewhat unreactive substrates and behaves similarly to the ferrocyanide ion in that both acid dissociation groups (pKa's 8.6 and ∼0) are influential in the catalysis of substrate oxidation by HRP-II. The kinetic isotope effect for the HRP-I reaction with p-aminobenzoic acid at high pH (pD) is consistent with a rate determining proton transfer but the group of pKa 5.1 in H2O remains unidentified.