Mechanism of nitrite-stimulated catalysis by lactoperoxidase

Abstract

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite (NO2-) were investigated. Reduction of compound I by NO2- was rapid (k2 = 2.3 × 107 M-1·S-1; pH = 7.2) and compound II was not an intermediate, indicating that NO2- radicals are not produced when NO2- reacts with compound I. The second-order rate constant for the reaction of compound II with NO2- at pH = 7.2 was 3.5 × 105 M-1·S-1. The reaction of compound III with NO2- exhibited saturation behaviour when the observed pseudo first-order rate constants were plotted against NO2- concentrations and could be quantitatively explained by the formation of a 1:1 ratio compound III/NO2- complex. The Km of compound III for NO2- was 1.7 × 10-4 M and the first-order decay constant of the compound III/NO2- complex was 12.5 ± 0.6 s-1. The second-order rate constant for the reaction of the complex with NO2- was 3.3 × 103 M-1·S-1. Rate enhancement by NO2- does not require NO2- as a redox intermediate. NO2- accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead-end intermediate compound III to form. Under these conditions, the 'rescue' reaction of NO2- with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.