The stability and steady-state kinetics of vanadium chloroperoxidase from the fungus Curvularia inaequalis.

Abstract

In this article we report on the steady-state kinetics of the chlorination and the stability of the vanadium chloroperoxidase from the fungus Curvularia inaequalis. The data show that the kinetics of this enzyme resemble that of the vanadium bromoperoxidase from the seaweed Ascophyllum nodosum. At low pH, chloride inhibited the enzyme, but the inhibition was of a dual nature. At pH 4.1 a mixed type of inhibition by chloride with respect to hydrogen peroxide was observed whereas at pH 3.1 the nature of the inhibition became competitive. The log Km for hydrogen peroxide decreased linearly with pH with a slope of -1 in the pH range 3-5. A reaction mechanism is presented to explain the observed data. We also showed that this class of enzymes is inhibited by nitrate. At pH 5.5, nitrate inhibits the chlorination reaction competitively with respect to chloride (Ki = 2 mM) and uncompetitively with respect to hydrogen peroxide. Furthermore, we showed that the enzyme produces HOCl as a reaction product. The enzyme exhibited a high thermostability (tm of 90 degrees C) and displayed high stability in organic solvents (solutions of 40% methanol, ethanol or 2-propanol) and moderate stability in the presence of the chaotropic agent guanidine/HCl (G1/2, the concentration of guanidine/HCl at which the enzyme activity was half the original activity was 3.7 M).