Understanding the broad substrate repertoire of nitroreductase based on its kinetic mechanism.

Autor:	Pitsawong W; From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055., Hoben JP; From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055., Miller AF; From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055 afm@uky.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2014 May 30; Vol. 289 (22), pp. 15203-14. Date of Electronic Publication: 2014 Apr 04.
DOI:	10.1074/jbc.M113.547117
Abstrakt:	The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s(-1), indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s(-1)). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s(-1) and a dissociation constant of 0.51 ± 0.04 mM. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines. (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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