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J. Biol. Chem., Vol. 277, Issue 24, 21906-21912, June 14, 2002
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From the The reaction of pentaerythritol tetranitrate
reductase with reducing and oxidizing substrates has been
studied by stopped-flow spectrophotometry, redox potentiometry, and
X-ray crystallography. We show in the reductive half-reaction of
pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form
an enzyme-NADPH charge transfer intermediate prior to hydride transfer
from the nicotinamide coenzyme to FMN. In the oxidative half-reaction,
the two-electron-reduced enzyme reacts with several substrates
including nitroester explosives (glycerol trinitrate and PETN),
nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and
Kinetic and Structural Basis of Reactivity of
Pentaerythritol Tetranitrate Reductase with NADPH, 2-Cyclohexenone,
Nitroesters, and Nitroaromatic Explosives*
§,§,§,¶,
,,, and**
Department of Biochemistry and Centre for
Chemical Biology, University of Leicester, University Road, Leicester
LE1 7RH and the Institute of Biotechnology, University of
Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom
,
-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of
the flavin by the nitroaromatic substrate TNT is kinetically
indistinguishable from formation of its hydride-Meisenheimer complex,
consistent with a mechanism involving direct nucleophilic attack by
hydride from the flavin N5 atom at the electron-deficient
aromatic nucleus of the substrate. The crystal structures of complexes
of the oxidized enzyme bound to picric acid and TNT are
consistent with direct hydride transfer from the reduced flavin to
nitroaromatic substrates. The mode of binding the inhibitor
2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric
acid and TNT. In this position, however, the aromatic nucleus is not
activated for hydride transfer from the flavin N5 atom, thus accounting
for the lack of reactivity with 2,4-DNP. Our work with PETN reductase
establishes further a close relationship to the Old Yellow Enzyme
family of proteins but at the same time highlights important
differences compared with the reactivity of Old Yellow Enzyme. Our
studies provide a structural and mechanistic rationale for the ability
of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with
2,4-DNP.
*
This work was funded by grants from the Biotechnology and
Biological Sciences Research Council, the Wellcome Trust, and the Lister Institute of Preventive Medicine.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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