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

Ronald L. Koder, Anne Frances Miller

Research output: Contribution to journalArticlepeer-review

114 Scopus citations

Abstract

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. Copyright (C) 1998 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)395-405
Number of pages11
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Volume1387
Issue number1-2
DOIs
StatePublished - Sep 8 1998

Bibliographical note

Funding Information:
This work was supported by PRF Grant ACS-PRF 28379 (A.F.M.) and a National Science Foundation Graduate Research Fellowship (R.L.K.).

Keywords

  • Bioremediation
  • Flavoprotein
  • Nitroreductase
  • Ping-pong
  • Stereospecificity

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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