Inhibition of Ascorbate Oxidase by Phenolic Compounds. Enzymatic and Spectroscopic Studies

Abstract

Competitive inhibition by phenolic compounds of the ascorbic acid oxidation reaction catalyzed by ascorbate oxidase was investigated at pH 7.0 and 23.0 degrees C. Inhibition of p-nitrophenol is pH dependent over the range 5.0-8.0, with inhibitor binding favored at higher pH. Bulky substituents on the phenol nucleus reduce or prevent the inhibitory effect. The presence of phenol affects the binding characteristics of azide to the trinuclear cluster of the enzyme. In particular, binding of azide to type 2 copper is prevented, and the affinity of azide to type 3 copper is reduced. In addition, reduction of type 1 copper is observed upon prolonged incubation of ascorbate oxidase with excess phenol and azide, but not with phenol alone. It is proposed that binding of phenolic inhibitors occurs at or near the site where the substrate (ascorbate) binds. NMR relaxation measurements of the protons of phenols in the presence of ascorbate oxidase show paramagnetic effects due to the proximity of the bound inhibitor to a copper center, likely type 1 copper. Copper-proton distance estimates between this paramagnetic center and p-cresol or p-nitrophenol bound to ascorbate oxidase are between 4.4 and 5.9 A.

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

“…These features make substrate binding to AO very tight and explains the rather high substrate specificity exhibited by this enzyme (Casella et al, 1999;Wimalasena & Dharmasena, 1994). In addition, participation of the type 1 Cu His ligand in the substrate binding interaction confirms that this residue represents the site of electron entry into the enzyme during the catalytic activity (Bertrand et al, 2002;Gaspard et al, 1997).…”

“…Like the phenolic compounds studied previously (Gaspard et al, 1997), the three isomeric fluorophenols inhibit the ascorbic acid oxidation catalyzed by AO. To determine the type of inhibition, the enzymatic reaction was studied over a range of ascorbic acid concentrations in the presence of several fixed amounts of the inhibitor.…”

“…Ascorbate oxidase can be inhibited by two types of donor molecules: (i) small inorganic anions such as azide and fluoride, which bind to the trinuclear copper cluster of the enzyme, where the interaction with dioxygen occurs (Messerschmidt, 1997;Casella et al, 1999;Cole, Avigliano, Morpurgo, & Solomon, 1991;Huang, Sakurai, Maritano, Marchesini, & Suzuki, 1999;Gromov, Marchesini, Farver, Pecht, & Goldfarb, 1999), and (ii) organic donor molecules acting as competitive inhibitors toward ascorbate, which have been proposed to bind in a pocket adjacent to the type 1 Cu center (Casella et al, 1999;Gaspard et al, 1997). From the analysis of a Connolly surface for the X-ray structure model of AO, Messerschmidt et al (1992) identified this pocket as the possible substrate binding site.…”

“…In spite of the assumptions underlying the use of this equation, it proved to be useful in our previous studies on peroxidases (Casella, Poli, Gullotti, Selvaggini, Beringhelli, & Marchesini, 1994;Redaelli, Monzani, Santagostini, Casella, Sanangelantoni, Pierattelli, & Banci, 2002) and ascorbate oxidase (Gaspard et al, 1997). In the case of copper enzymes, it is generally assumed that the overall correlation time is determined by the electronic relaxation, and a τ C value of 3.5×10 −9 s has been estimated for a variety of copper enzymes (Bertini, Briganti, Luchinat, Mancini, & Spina, 1985;Williams & Falk, 1986).…”

“…This problem has received limited attention so far, and only recently the X-ray structure of a fungal laccase showed that binding of an arylamine occurs near type 1 copper (Bertrand et al, 2002). In the case of AO, the problem of substrate binding has been so far approached through kinetic and inhibition studies (Casella et al, 1999;Wimalasena & Dharmasena, 1994;Marchesini, Cappelletti, Canonica, Danieli, & Tollari, 1977;Gaspard, Monzani, Casella, Gullotti, Maritano, & Marchesini, 1997). In this paper, we gain a better definition of the ascorbate binding site in AO by reporting kinetic and 19 F NMR relaxation studies, and docking calculations on the competitive inhibitory interaction of fluorophenols with the enzyme.…”