Oxidation of Natural Hydroxybenzoic Acids by Grapevine Peroxidases: Kinetic Characteristics and Substrate Specificity

Abstract

The oxidation of natural hydroxybenzoic acids by a cellular peroxidase (EC 1.11.1.7) fraction obtained from Vitis vinifera cv. Monastrell cell cultures has been studied. Of all the hydroxybenzoic acids present in Monastrell grapes, only protocatechuic (3,4-dihydroxybenzoic), vanillic (3-methoxy-4-hydroxybenzoic), and gallic (3,4,5-trihydroxy-benzoic) acids were substrates of this enzyme fraction, showing optimal pH values for the oxidation rate in the 3.0 to 5.0 range, p-Hydroxybenzoic acid and syringic (3,5-dimethoxy-4-hydroxybenzoic) acid were not substrates under the assay conditions. Apparent K_M values for protocatechuic acid, vanillic acid, and gallic acid were 0.850, 0.120, and 1.080 mM, respectively. Using electrophoretic techniques, the differential reactivity of the peroxidase isoenzymes in this peroxidase fraction was shown against each hydroxybenzoic acid. This suggests a distinctive participation for each one of the isoenzymes in the several oxidative pathways of the hydroxybenzoic acids in Monastrell grapes.