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

Ryan A. Dick, Thomas W. Kensler

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)17269-17277
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number17
DOIs
StatePublished - Apr 23 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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