Accelerating Effect of Umbelliferone on Peroxidase-Catalyzed Oxidation of Indole-3-acetic Acid at Neutral pH

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 umbelliferone-induced 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.