We report the characterization of 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) as a mechanism-based inhibitor of NQO1. Inactivation of NQO1 by ES936 was time- and concentration-dependent and required the presence of a pyridine nucleotide cofactor consistent with a need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studies by the low partition ratio (1.40 +/- 0.03). The orientation of ES936 in the active site of NQO1 was examined by X-ray crystallography and found to be opposite to that observed for other indolequinones acting as substrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenol leaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr 127 and Tyr 129 residues in the active site. To determine if ES936 was covalently modifying NQO1, ES936-treated protein was analyzed by electrospray ionization liquid chromatography/mass spectrometry (ESI-LC/MS). The control NQO1 protein had a mass of 30864 +/- 6 Da (n = 20, theoretical, 30868.6 Da) which increased by 217 Da after ES936 treatment (31081 +/- 7 Da, n = 20) in the presence of NADH. The shift in mass was consistent with adduction of NQO1 by the reactive iminium derived from ES936 (M + 218 Da). Chymotryptic digestion of the protein followed by LC/MS analysis located a tetrapeptide spanning amino acids 126-129 which was adducted with the reactive iminium species derived from ES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129 residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and may be a useful tool in defining the role of NQO1 in cellular systems and in vivo.
NAD(P)H͞quinone acceptor oxidoreductase (QR1, NQO1, formerly DT-diaphorase; EC 1.6.99.2) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. In this paper we report the apoenzyme structures of human (at 1.7-Å resolution) and mouse (2.8 Å) QR1 and the complex of the human enzyme with the substrate duroquinone (2.5 Å) (2,3,5,6-tetramethyl-p-benzoquinone). In addition to providing a description and rationale of the structural and catalytic differences among several species, these structures reveal the changes that accompany substrate or cofactor (NAD) binding and release. Tyrosine-128 and the loop spanning residues 232-236 close the binding site, partially occupying the space left vacant by the departing molecule (substrate or cofactor). These changes highlight the exquisite control of access to the catalytic site that is required by the ping-pong mechanism in which, after reducing the flavin, NAD(P) ؉ leaves the catalytic site and allows substrate to bind at the vacated position. In the human QR1-duroquinone structure one ring carbon is significantly closer to the flavin N5, suggesting a direct hydride transfer to this atom.DT diaphorase ͉ cancer ͉ chemoprotection ͉ chemotherapy N AD(P)H:Quinone acceptor oxidoreductase type 1 (QR1, NQO1; EC 1.6.99.2) is a flavoenzyme (homodimer of 273 residues, one FAD per monomer) that catalyzes the obligatory two-electron reduction of quinones to hydroquinones (1-3). This reaction prevents the one-electron reduction of quinones by cytochrome P450 reductase and other flavoproteins, resulting in oxidative cycling of deleterious radical species. The enzyme is inducible by a wide variety of Michael reaction acceptors and other electrophiles (4, 5). In addition to its possible role in the detoxification of dietary quinones, the enzyme has been shown to catalyze the reductive activation of quinolic chemotherapeutic compounds such as mitomycins (6), anthracyclines, and aziridinyl-benzoquinones. Overexpression of QR1 in many tumors, including those of lung, colon, liver, and breast, makes this enzyme an ideal target for the development of additional activatable cytotoxic compounds.We previously reported the crystal structures of rat liver quinone reductase (rQR1) in complex with NADP ϩ and the ternary complex with the inhibitor Cibacron Blue (CB) and the substrate duroquinone (2,3,5,6-tetramethyl-1,2-benzoquinone; DQ) (7). These structures have revealed insight into many biochemical and physiological properties of QRs, as well as the mechanism of quinone reduction. These structures have provided a wealth of information, including the overall fold, the nature of the dimer, the interactions of the FAD cofactor, and the possibility of hydride transfer between the NADH and FAD cofactors and from FADH 2 to the quinone substrate. Nonetheless many important aspects of QR1 function remain elusive. This is caused in part by the lack of structural information about the apoenzyme (apo) containing only the FAD prosthetic group (apo QR1) or of QR1...
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