Centennial Honors College
Western Illinois University
Undergraduate Research Day 2012
Poster Presentation
Kinetics Study of Recombinant Secondary Alcohol Dehydrogenases from
Micrococcus luteus WIUJH20
Sam Wrobel
Faculty Mentors: Jenq-Kuen Huang and Lisa Wen
Chemistry
Secondary alcohol dehydrogenases (2o-ADH) are a class of enzymes catalyzes
oxidation of secondary alcohols and reduction of ketones. Because the enzymes are
active on a broad range of substrates/reactants, they were considered excellent targets
to engineer into industrial catalysts. Previously, Drs. Huang and Wen purified a novel
2o-ADH from Micrococcus luteus WIUJH20 and determined its N-terminal amino acid
sequence. This 2o-ADH belongs to the L-3-hydroxyacyl-CoA Dehydrogenase (L-3-HAD)
due to high sequence homology. L-3-HAD isolated from several organisms were well
characterized and known to display tight substrate specificity active on a few unique
substrates, while 2o-ADH has broad substrate specificity. We are interested in research
to understand how enzymes’ specificity determined.
Drs. Huang and Wen’s lab has begun to map the amino acids in 2o-ADH involved in
substrate specificity. The objective of my research was to perform the enzyme assay of
three mutant proteins and the wild-type protein. A wide range of substrates were used in
assays to see if the mutation altered substrate specificity. Detailed enzyme kinetic
studies were performed using the same amount of each enzyme but varying substrate
concentration. Enzyme activity was monitored using a spectrophotometer. The data
collected was used to calculate maximum velocity (Vmax), Michaelis constant (KM), and
catalytic constant (Kcat). The kinetic constants are critical as they help explain how
enzymes work. The knowledge obtained from the kinetics study will further our
understanding the basis of enzyme specificity and selectivity.