| |
| author |
Narisa Diana Lee
| | title |
Investigating Substrate Inhibition and the Effects of pH in an Aldo-keto Reductase from a Yeast Strain using Electron Withdrawing Substrates
| | abstract |
Aldo-keto reductases (ARKs) are NADPH-dependent oxidoreductases, catalysing the
conversion of aldehydes and ketones into alcohols. AKR's have extensive physiological roles
due to their high chemo-, enantio- and regio-selectivity, and broad substrate activity. Many
synthetic chemical processes require countless steps to achieve a compound of the right
enantioselectivity. Through the use of chemoenzymatic synthesis, these processes can
proceed much easier, allowing products to be synthesized stereo-selectively under high yield
using fewer steps. With numerous biocatalytic applications, people are on a constant look out
for AKR's across a wide range of organisms. Previous research investigating the
ketoreductase activity using Saccharomyces cerevisiae (SC108) extracted from a 45-million-
year-old amber successfully characterized AKR163 with the highest expression. In this study,
AKR163 was purified to investigate the role of substrate inhibition and the effects of pH on
enzyme activity. By analysing the underlying mechanisms in the ordered binding kinetics, the
goal of this project was to determine what is causing substrate inhibition. Additionally,
defining the pKa value was of interest to determine specific amino acid interactions for
potential mutation studies in the future. Enzyme kinetics were accomplished using non-
halogenated substrates like Ethyl-4-nitrobenzoylacetate (E4NBA) and Ethyl acetoacetate
(EAA). Results from the kinetic runs were compared to halogenated substrates such as Ethyl-
4-chloroacetoacetate (E4ClAA) and Ethyl-2-floroacetoacetate (E2FlAA) to decipher the
effects of electron withdrawing properties on substrate inhibition. The kinetic parameters
including Vmax, KM and KI were closely examined from each run. Results from the pH
dependence study from pH 5.99-6.81 were overall inconclusive due to variability seen in the
data and high error values associated with each kinetic parameter. Nevertheless, it seems like
more potent inhibition was seen at increased pH levels. Despite this, further investigation is
required to conclusively determine the relationship between pH changes and substrate
inhibition. On the other hand, the kinetic data investigating substrate inhibition in electron
withdrawing compounds demonstrated a clear relationship between electron withdrawing
substrates and its effect on substrate inhibition. This was specifically seen with E4NBA, a
non-halogenated electron withdrawing compound which exhibited a characteristic substrate
inhibition curve. When comparing this data to EAA, a non-halogenated non-electron-
withdrawing substrate, it can be seen that substrate inhibition was not observed. Altogether,
the kinetic data from this analysis provided a structural basis in understanding the underlying
mechanism leading up to substrate inhibition. Moreover, the results gathered throughout this
investigation confirmed that substrate inhibition is a known kinetic phenomenon which can
occur in halogenated and non-halogenated electron withdrawing compounds.
| | school |
The College of Liberal Arts, Drew University
| | degree |
B.S. (2023)
|
| advisor |
Adam Cassano
|
| full text | NLee.pdf |
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