A mechanistic model of peroxidase-catalyzed oxidation of indole-3-acetic acid (IAA) at neutral pH has been developed, characterized, and compared with experiments. The model is based on experimental facts showing that IAA is oxidized in the presence of HRP by two pathways: (i) the standard peroxidase cycle, which is accompanied by (ii) a nonenzymatic free radical chain reaction. The peroxidase cycle normally requires the addition of a hydroperoxide, whereas IAA oxidation does not. Therefore, the model includes the enzymatic peroxidase cycle which is initiated by organic hydroperoxide (ROOH) derived from autoxidation of IAA. It also includes a nonenzymatic free radical chain which utilizes oxygen, oxidizes IAA, and recycles ROOH required for the enzymatic cycle. Available experimental values of rate constants were used. Unavailable rate constants were initially estimated analytically using the steady state assumption and then optimized by computer simulation. There is a unique set of rate constants which satisfies the model. The average deviation of simulated kinetic traces from experimental ones was less than 5%. Critical values of the rate constants were determined; for values smaller than the critical values IAA oxidation stops. In the pre-steady state the concentration of ROOH rises exponentially. Thus, a decrease of the initial concentration of ROOH by 3 orders of magnitude gives rise to only a 1 min delay of reaction. For all intermediates except ROOH the steady state was reached in 1 min; for ROOH, 10 min after reaction initiation. More than half of the oxidation of IAA occurs by the chain reaction, a nonenzymatic pathway. The model satisfactorily describes all available experimental kinetic data and predicts some previously unobserved behavior which should stimulate further experiments.
The acceleration by the phenol umbelliferone (7-hydroxycoumarin) of the horseradish peroxidase (HRP) catalyzed oxidation of indole-3-acetic acid (IAA) was studied at pH 7.4 using spectral and kinetic approaches. For the system 0.1 mM IAA/1 µM HRP/variable umbelliferone concentration, an increase in rate by a factor of 8 was reached in the presence of 1 µM umbelliferone; further increase of the umbelliferone concentration had no further effect. The rate constants for the peroxidase compounds I and II (HRP-I and HRP-II) reductions by umbelliferone in the absence of IAA were measured in the transient state as functions of the umbelliferone concentration. The plot of the pseudo-first-order rate constant k obs vs [umbelliferone] for HRP-I reduction was curved upward. This result implies that umbelliferone catalyzes its own oxidation by HRP-I. The bimolecular rate constant of the reduction of 1 µM HRP-I by 1 µM umbelliferone was estimated to be 1.1 × 10 5 M -1 s -1 . The plot of k obs vs [umbelliferone] was linear for HRP-II reduction, yielding a bimolecular rate constant of 1.7 × 10 5 M -1 s -1 . The influence of umbelliferone on the rates of HRP-I and HRP-II reduction by IAA was also studied. It was found that 1 µM umbelliferone accelerates the reduction of 1 µM HRP-I by a factor of 10 but did not influence the reduction of HRP-II by IAA. The influence of umbelliferone on the HRP-I reduction by umbelliferone and/or IAA suggests that HRP-I can bind umbelliferone at a site different from the active site, where it provides a beneficial conformational change. However, the binding of umbelliferone to native HRP was not observed. A detailed mechanism for the HRP-catalyzed oxidation of IAA, both in the absence and in the presence of umbelliferone, is presented. There are three umbelliferoneinduced accelerating effects: (i) the reduction of the rate-limiting HRP species, HRP-II, by umbelliferone, which increases the rate of enzyme turnover and hence the rate of IAA oxidation, (ii) the nonenzymatic oxidation of IAA by free radicals of umbelliferone formed in the HRP-catalyzed oxidation of umbelliferone, and (iii) umbelliferone-induced acceleration of the HRP-I reduction by IAA. The magnitudes of these three effects are similar.
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