1. Kinetics of Human β3 Alcohol Dehydrogenase
Joshua Benton, Jennifer Chase, Ph.D. (Department of Biology)
Alcohol-related diseases have been observed to predominate in specific people groups while
appearing suppressed in others. Multiple genetic polymorphisms exist for the alcohol
dehydrogenase (ADH) enzyme and are responsible for the unique tolerance level of each people
group to developing alcohol-related diseases. ADH is largely responsible for ethanol
metabolism in the body. High alcohol consumption in humans results in elevated ethanol levels
and can cause disease (i.e. fetal alcohol syndrome) if not effectively metabolized. It is
hypothesized that ethanol competitively inhibits the oxidation of retinol, slowing the production
of retinoic acid and ultimately deregulating cell growth. The β₃β₃ isoform of the ADH enzyme
has been said to be protective against alcohol-related diseases by oxidizing ethanol at a faster
rate than other ADH isoforms. African-Americans have been found to predominantly possess
the ADH1B3 gene that codes for the β₃β₃ ADH enzyme. Almost no literature data has
determine the kinetic properties of β₃β₃ ADH with appropriate consideration for physiological
conditions. A complete set of methodologically sound kinetic studies of β₃β₃ ADH are needed
for correctly understanding its suppression of disease. The goal was to not only verify the
hypothesized ethanol inhibition of retinol oxidation but to also investigate temperature's effect on
the determination of the kinetic constants needed to assess ethanol inhibition. Recombinant
enzyme was isolated and two kinetic studies were performed, first with varied ethanol and NAD+
substrate concentrations and second with varied acetaldehyde and NADH. Both studies were
performed at 25ºC and 37ºC. Data suggests that temperature only has a significant effect when
β₃β₃ ADH is catalyzing the oxidation of acetaldehyde. Results also indicate that β₃β₃ ADH
binds in the forward direction via an ordered bi-bi mechanism. In order to resolutely determine
ethanol inhibition of retinol oxidation, further kinetic studies with other substrates of varied
concentration and temperature (25ºC and 37ºC) are needed.