Stimulatory effect of DIMBOA on NADH oxidation catalysed by horseradish peroxidase

2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) effectively enhanced the rate of NADH oxidation catalysed by horseradish peroxidase isoenzyme C (HRPC), a reaction that generates H2O2. The effect showed saturation kinetics with increasing DIMBOA concentrations and the extrapolated maximum rate gave an activating factor of 246 for this hydroxamic acid. After 80% of the NADH had been oxidized, DIMBOA had converted into a species that showed absorption bands centered at 430 and 345 nm. A product with the same absorption properties was formed upon oxidation of DIMBOA with H2O2. The V-max for the latter reaction was 3.3 times lower than the maximum rate of NADH oxidation in the presence of DIMBOA. Ferulic and p-coumaric acids were also stimulators of NADH oxidase activity of HRPC, giving saturating kinetics, Maximum rates obtained for these effecters give activating factors of 35 and 1170, respectively. The DIMBOA analogue 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) that lacks the methoxy substitution in the aromatic ring was only 16% efficient in stimulating NADH oxidase as compared with DIMBOA at the 0.1 mM level. On the other hand, the benzoxazolinones 6-methoxy-benzoxazolin-2-one (MBOA) and benxoxazolin-2-one (BOA) had negligible effect on the rate of NADH oxidation and did not alter the activating effect of DIMBOA over this reaction. (C) 1997 Elsevier Science Ltd. All rights reserved.