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

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NADP
Oxidoreductases
Kinetics
Carbon
Ketones
Hydrides
Atomic spectroscopy
Alcohol Dehydrogenase
Deuterium
Metabolites
Absorption spectroscopy
Kinetic parameters
Aldehydes
Isotopes
Isomers
NAD
Hydrogen
Catalyst activity
Spectrum Analysis
Nuclear magnetic resonance

ASJC Scopus subject areas

  • Biochemistry

Cite this

The Catalytic and Kinetic Mechanisms of NADPH-dependent Alkenal/one Oxidoreductase. / Dick, Ryan A.; Kensler, Thomas W.

In: Journal of Biological Chemistry, Vol. 279, No. 17, 23.04.2004, p. 17269-17277.

Research output: Contribution to journalArticle

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