Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase

• Published in: Biochimica et biophysica acta 1998-09, Vol.1387 (1), p.395-405
• Main Authors: Koder, Ronald L., Miller, Anne-Frances
• Format: Article
• Language: English
• Subjects: Acetates - pharmacology; Binding Sites - physiology; Bioremediation; Dicumarol - pharmacology; Dinitrobenzenes - metabolism; Electron Transport - physiology; Enterobacter cloacae - enzymology; Enzyme Inhibitors - pharmacology; Flavoprotein; Kinetics; Magnetic Resonance Spectroscopy; Molecular Conformation; NAD - chemistry; Nitrobenzenes - metabolism; Nitroreductase; Nitroreductases - chemistry; Ping-pong; Spectrophotometry; Stereospecificity; Substrate Specificity
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(98)00151-4

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a k cat/ K M an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a K i of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD + does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD + could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.