Characterization of a Mechanism-Based Inhibitor of NAD(P)H:Quinone Oxidoreductase 1 by Biochemical, X-ray Crystallographic, and Mass Spectrometric Approaches
文摘
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 witha need for metabolic activation. That ES936 was an efficient inhibitor was demonstrated in these studiesby the low partition ratio (1.40 ± 0.03). The orientation of ES936 in the active site of NQO1 was examinedby X-ray crystallography and found to be opposite to that observed for other indolequinones acting assubstrates. ES936 was oriented in such a manner that, after enzymatic reduction and loss of a nitrophenolleaving group, a reactive iminium species was located in close proximity to nucleophilic His 162 and Tyr127 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 tetrapeptidespanning amino acids 126-129 which was adducted with the reactive iminium species derived fromES936. LC/MS/MS analysis of the peptide fragment confirmed adduction of either Tyr 127 or Tyr 129residues. This work demonstrates that ES936 is a potent mechanism-based inhibitor of NQO1 and maybe a useful tool in defining the role of NQO1 in cellular systems and in vivo.