ELEMENTARY STEPS IN THE REACTION OF HORSERADISH-PEROXIDASE WITH SEVERAL PEROXIDES - KINETICS AND THERMODYNAMICS OF FORMATION OF COMPOUND-O AND COMPOUND-I

The reactions of horseradish peroxidase (HRP) with ethyl hydroperoxide, tert-butyl hydroperoxide, and peracetic acid to form compound I have been studied in 50% v/v methanol/10 mM phosphate over the +25 to -35-degrees-C temperature range using the low-temperature stopped-flow technique. All reactions were carried out under pseudo-first-order conditions with [peroxide] >> [HRP]. Arrhenius plots for k(obs) obtained under conditions where [peroxide] << K(M) are linear over the entire temperature range studied for each reaction, indicating that there is no change in mechanism over this temperature range. Above 0-degrees-C, the pseudo-first-order rate constant for compound I formation, k(obs), varies linearly with [peroxide] for each reaction. However, saturation kinetics are observed below -16-degrees-C for all of these reactions, indicating that they proceed via at least one reversible elementary step involving the formation of an intermediate. Rapid-scan optical studies have been carried out at -35-degrees-C at [peroxide] >> K(M) for each reaction in order to record the optical spectrum of the intermediate. In all three reactions, the Soret region of the intermediate exhibits bands near 360 and 410 nm and there is a weak band in the visible near 570 nm. These are the same spectral features that have been observed earlier for compound 0, an intermediate in the reaction of HRP with hydrogen peroxide. Thus, the compound I formation reaction is viewed as at least a two-step process. Double reciprocal plots obtained over the -20 to -30-degrees-C range at pH* 7.3 for these reactions give values of K(M)-1 and k(obs)max that reflect the formation of compound 0 and its rate of conversion to compound 1, respectively. The values of k(obs)max are not strongly dependent on the identity of the peroxide. However, the values of K(M)-1 decrease markedly for the bulkier peroxides. Possible structures for compound 0 are discussed.