在烟酰胺腺嘌呤二核苷酸（NADH）存在的情况下，辣根过氧化物酶化合物I、II和III氧化东莨菪亭的瞬态动力学和稳态动力学。

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

作者信息

Marquez L A, Dunford H B

机构信息

Department of Chemistry, University of Alberta, Edmonton, Canada.

出版信息

Eur J Biochem. 1995 Oct 1;233(1):364-71. doi: 10.1111/j.1432-1033.1995.364_1.x.

DOI:10.1111/j.1432-1033.1995.364_1.x

PMID:7588768

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.

摘要

东莨菪素是一些植物中天然存在的荧光成分，也是一种经证实的植物生长抑制剂，它是一种已知的过氧化物酶反应物。然而，尽管东莨菪素广泛用于过氧化氢的过氧化物酶测定，但该反应基本步骤的动力学从未被研究过，干扰物质的定量影响也从未被详细探讨过。在这项工作中，我们采用瞬态和稳态方法来测定辣根过氧化物酶（HRP）中间体化合物I和II氧化东莨菪素的二级速率常数：在20℃、pH 6.0和离子强度0.1 M条件下，分别为(3.7±0.1)×10⁶ M⁻¹ s⁻¹和(8.5±0.5)×10⁵ M⁻¹ s⁻¹。我们研究了NADH对东莨菪素与HRP反应的可能抑制作用，以及东莨菪素对NADH反应的影响。在NADH存在下，HRP-I与东莨菪素反应的速率常数略有下降，降至(2.8±0.1)×10⁶ M⁻¹ s⁻¹。因此，尽管NADH也是一种过氧化物酶底物，但它不能有效地竞争酶的氧化形式。另一方面，东莨菪素能刺激HRP/H₂O₂系统对NADH的氧化，显然是通过形成苯氧基自由基，然后将NADH氧化为NAD⁺自由基。我们提供的光谱证据表明，在pH 7.0（无外源添加H₂O₂）的HRP与NADH的需氧反应中，HRP-II是主要的酶中间体，HRP-III也可检测到。向东莨菪素/HRP/NADH系统中添加东莨菪素会导致双相反应，其中HRP-II和HRP-III消失。两个阶段的速率常数都与东莨菪素浓度呈线性相关。我们将较快的阶段归因于HRP-II与东莨菪素的反应，速率常数为(6.2±0.1)×10⁵ M⁻¹ s⁻¹，较慢的阶段归因于HRP-III与东莨菪素的反应，速率常数为(5.0±0.4)×10⁴ M⁻¹ s⁻¹。我们目前的工作不仅提供了HRP-I、II和III氧化东莨菪素的速率常数，还阐明了在东莨菪素存在下NADH氧化过程中可能在生理上发生的相互作用。