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

• Published in: The Journal of biological chemistry Vol. 279; no. 17; pp. 17269 - 17277
• Main Authors: Dick, Ryan A., Kensler, Thomas W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.04.2004; American Society for Biochemistry and Molecular Biology
• Subjects: Alcohol Dehydrogenase - chemistry; Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - metabolism; Animals; Antioxidants - pharmacology; Carbon - chemistry; Catalysis; Deuterium - chemistry; Deuterium - pharmacology; Hydrogen - chemistry; Hydrogen-Ion Concentration; Ketones; Kinetics; Magnetic Resonance Spectroscopy; Models, Chemical; NADH, NADPH Oxidoreductases - chemistry; NADP - chemistry; NADP - metabolism; Oxygen - metabolism; Phylogeny; Progesterone - chemistry; Protein Binding; Rats; Recombinant Proteins - chemistry; Spectrometry, Fluorescence; Spectrophotometry, Atomic; Stereoisomerism; Tryptophan - chemistry; Zinc - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M400427200

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 α,β-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 β-carbon of an α,β-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.