Conventional assays of the oxidation of primary alcohols by alcohol dehydrogenases (ADHs) rely on measuring spectrophotometrically the increase in the concentration of NADH which mask or overlook the enzymes ability to oxidize aldehydes. In contrast, alcohol dehydrogenases operate physiologically under conditions where NADH is maintained at low and nearly constant concentration, conditions that do not permit direct spectrophotometric assays. The oxidation of primary alcohols by the horse liver ADH using an NADH recycling system to maintain a low concentration of NADH has been monitored by ~1H NMR spectroscopy. These conditions result in the formation of considerable amounts of the corresponding carboxylic acid. While the kinetics of alcohol oxidation and aldehyde reduction have been extensively studied, the introduction of aldehyde oxidation into the reaction scheme greatly increases the complexity of the kinetic analysis. The progress curves for the reaction of ethanol oxidation with NADH recycling and acetaldehyde dismutation with and without NADH recycling have been fitted to a minimal kinetic scheme. No set of kinetic values can be obtained that fit all the progress curves that have been analyzed and a much more complex scheme will need to be developed. Simulations demonstrate that changes even just one rate constant can alter the catalytic properties of the dehydrogenase with potential impact on the physiological functioning of these enzymes.
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