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J. Biol. Chem., Vol. 279, Issue 17, 17269-17277, April 23, 2004

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The Catalytic and Kinetic Mechanisms of NADPH-dependent Alkenal/one Oxidoreductase^*

Ryan A. Dick and Thomas W. Kensler¶||

From the Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine and the ^¶Division of Toxicological Sciences, Department of Environmental Health Sciences, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205

NADPH-dependent alkenal/one oxidoreductase (AOR) from the rat is a phase 2/antioxidative enzyme that is known to catalyze the reduction of the carbon-carbon double bond of ,-unsaturated aldehydes and ketones. It is also known for its leukotriene B₄ 12-hydroxydehydrogenase activity. In order to begin to understand these dual catalytic activities and validate its classification as a reductase of the medium-chain dehydrogenase/reductase family, an investigation of the mechanism of its NADPH-dependent activity was undertaken. Recombinant AOR and a 3-nonen-2-one substrate were used to perform steady-state initial velocity, product inhibition, and dead end inhibition experiments, which elucidated an ordered Theorell-Chance kinetic mechanism with NADPH binding first and NADP⁺ leaving last. A nearly 20-fold preference for NADPH over NADH was also observed. The dependence of kinetic parameters V and V/K on pH suggests the involvement of a general acid with a pK of 9.2. NADPH isomers stereospecifically labeled with deuterium at the 4-position were used to determine that AOR catalyzes the transfer of the pro-R hydride to the -carbon of an ,-unsaturated ketone, illudin M. Two-dimensional nuclear Overhauser effect NMR spectra demonstrate that this atom becomes the R-hydrogen at this position on the metabolite. Using [4R-²H]NADPH, small primary kinetic isotope effects of 1.16 and 1.73 for V and V/K, respectively, were observed and suggest that hydride transfer is not rate-limiting. Atomic absorption spectroscopy indicated an absence of Zn²⁺ from active preparations of AOR. Thus, AOR fits predictions made for medium-chain reductases and bears similar characteristics to well known medium-chain alcohol dehydrogenases.

Received for publication, January 14, 2004 , and in revised form, February 11, 2004.

^* This work was supported by National Institutes of Health (NIH) Grant CA39416. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by NIH Grant T32CA09243.

^|| To whom correspondence should be addressed: Dept. of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, 615 N. Wolfe St., Rm. 7032, Baltimore, MD 21205, Tel.: 410-955-4712; Fax: 410-955-0116; E-mail: tkensler{at}jhsph.edu.

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