Transient and steady-state kinetics of the oxidation of scopoletin by horseradish peroxidase compounds I, II and III in the presence of NADH

Abstract

Scopoletin, a naturally occurring fluorescent component of some plants and a proven plant growth inhibitor, is a known reactant with peroxidase. However, the kinetics of the elementary steps of the reaction have never been investigated, nor has the quantitative effect of interfering substances ever been explored in detail, despite the fact that scopoletin is widely used in a peroxidase assay for H2O2. In this work, we employed both transient-state and steady-state methods to determine the second-order rate constants for the oxidation of scopoletin by the horseradish peroxidase (HRP) intermediate compounds I and II: (3.7 +/- 0.1) x 10(6) M-1 s-1 and (8.5 +/- 0.5) x 10(5) M-1 s-1 at 20 degrees C, pH 6.0 and ionic strength of 0.1 M. We investigated the possible inhibitory effect of NADH on the reaction of scopoletin with HRP and also the effect of scopoletin on the NADH reaction. In the presence of NADH the rate constant for the reaction between HRP-I and scopoletin decreased slightly to (2.8 +/- 0.1) x 10(6) M-1 s-1. Thus, although NADH is also a peroxidase substrate, it cannot compete effectively for the oxidized forms of the enzyme. On the other hand, scopoletin stimulates the oxidation of NADH by the HRP/H2O2 system, apparently by forming a phenoxyl radical which then oxidizes NADH to NAD. radicals. We present spectral evidence showing that in the aerobic reaction between HRP and NADH at pH 7.0 (without exogenously added H2O2) HRP-II is the dominant enzyme intermediate with HRP-III also detectable. Addition of scopoletin to the HRP/NADH system leads to a biphasic reaction in which HRP-II and HRP-III disappear. The rate constants for both phases are linearly dependent on scopoletin concentration. We attribute the faster phase to the HRP-II reaction with scopoletin with a rate constant of (6.2 +/- 0.1) x 10(5) M-1 s-1 and the slower phase to the HRP-III reaction with scopoletin with rate constant (5.0 +/- 0.4) x 10(4) M-1 s-1. Our present work not only provides rate constants for the oxidation of scopoletin by HRP-I, II and III but also elucidates the interactions that possibly occur physiologically during NADH oxidation in the presence of scopoletin.