Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase.
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 kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki 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.
Koder
R L
RL
Department of Biophysics, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Miller
A F
AF
D000085:Acetates / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D001728:Dicumarol / Q000494:pharmacology; D004136:Dinitrobenzenes / Q000378:metabolism; D004579:Electron Transport / Q000502:physiology; D016972:Enterobacter cloacae / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009243:NAD / Q000737:chemistry; D009578:Nitrobenzenes / Q000378:metabolism; D009601:Nitroreductases / Q000737:chemistry*; D013053:Spectrophotometry; D013379:Substrate Specificity
D000085:Acetates; D004136:Dinitrobenzenes; D004791:Enzyme Inhibitors; D009578:Nitrobenzenes; D009243:NAD; C016403:2,4-dinitrotoluene; D001728:Dicumarol; C036077:nitrobenzene; D009601:Nitroreductases