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 th e 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 mu M HRP/variable umbelliferone concentr ation, an increase in rate by a factor of 8 was reached in the presenc e of 1 mu M umbelliferone; further increase of the umbelliferone conce ntration had no further effect. The rate constants for the peroxidase compounds I and II (HRP-I and HRP-II) reductions by umbelliferone in t he absence of IAA were measured in the transient state as functions of the umbelliferone concentration. The plot of the pseudo-first-order r ate constant k(obs) vs [umbelliferone] for HRP-I reduction was curved upward. This result implies that umbelliferone catalyzes its own oxida tion by HRP-I. The bimolecular rate constant of the reduction of 1 mu M HRP-I by 1 mu M umbelliferone was estimated to be 1.1 x 10(5) M(-1) s(-1). The plot of k(obs) vs [umbelliferone] was linear for HRP-II red uction, yielding a bimolecular rate constant of 1.7 x 10(5) M(-1) s(-1 ). The influence of umbelliferone on the rates of HRP-I and HRP-II red uction by IAA was also studied. It was found that 1 mu M umbelliferone accelerates the reduction of 1 mu M HRP-I by a factor of 10 but did n ot influence the reduction of HRP-II by IAA. The influence of umbellif erone 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 b inding of umbelliferone to native HRP was not observed. A detailed mec hanism for the HRP-catalyzed oxidation of IAA, both in the absence and in the presence of umbelliferone, is presented. There are three umbel liferone-induced accelerating effects: (i) the reduction of the rate-l imiting HRP species, HRP-II, by umbelliferone, which increases the rat e of enzyme turnover and hence the rate of IAA oxidation, (ii) the non enzymatic 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.