Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity.

Clare F Megarity,David J Timson

doi:10.1042/bsr20191874

Clare F Megarity, David J Timson

Open Access

https://doi.org/10.1042/bsr20191874

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Abstract

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme’s ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound’s well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

Highlights

Human NAD(P)H quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC 1.6.5.2) is an oxidoreductase flavoenzyme [1–4]
The two-electron reduction catalysed by NQO1 ensures the reduction of quinones directly to hydroquinones thereby avoiding the production of semiquinones
The data presented here demonstrate that the cancer-associated polymorphisms in NQO1 do not affect the enzyme’s ability to exhibit negative cooperativity towards the inhibitor dicoumarol

Summary

Introduction

Human NAD(P)H quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC 1.6.5.2) is an oxidoreductase flavoenzyme [1–4]. It catalyses the two-electron reduction of quinone substrates through a substituted enzyme (or “ping-pong”) mechanism where the electron donor (NAD(P)H) enters the active site, reduces the FAD cofactor by donation of two hydride ions, leaves the active site in its oxidized form (NAD+) and is replaced by the quinone substrate, which is subsequently reduced [5–7]. The two-electron reduction catalysed by NQO1 ensures the reduction of quinones directly to hydroquinones thereby avoiding the production of semiquinones. NQO1 directly scavenges superoxide free radicals and plays a minor role in the redox cycling of vitamin K by detoxifying vitamin K3 to the corresponding hydroquinone [14–17]. Inhibition of the proteasome is involved in the pathogenesis of Parkinson’s disease and NQO1 helps to protect against this inhibition by reducing the endogenous proteasome inhibitor, License 4.0 (CC BY)

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