| |
| author |
Lucas Portadin
| | title |
Product Inhibition of AKR163: an Aldoketoreductase from a Yeast from Ancient Amber
| | abstract |
Aldoketoreductases (AKRs) are an important family of biocatalysts present in all
organisms, and possess a wide substrate specificity. They are known for their ability to reduce
ketones or aldehydes to their respective alcohol products using an NADPH cofactor. Recently,
the use of AKRs in industrial biocatalysis has been being looked at more closely. They are now
being used as a more efficient way to produce vital pharmaceutical intermediates and building
blocks, rather than using reagents that are nonrenewable. AKRs, like other enzymes, can be
inhibited by other molecules either competitively, noncompetitively, or uncompetitively.
Understanding the properties of each specific enzyme is vital for ensuring the proper function of
the body. In this work, the enzyme AKR163, recovered from an ancient yeast strain, is being
studied for its inhibition properties. Specifically, the goal is to determine if different substituents
present on a substrate alter the type of inhibition that occurs. To find the answer to this question,
Michaelis-Menten and substrate inhibition fits must be applied to the velocity of the enzyme at
different substrate concentrations. This way the Km and Vmax values of the inhibited enzyme can
be compared to said values of the uninhibited enzyme to draw conclusions. Two substrates being
tested are ethyl acetoacetate (EAA) and ethyl-4-chloroacetoacetate (E4ClAA), and some
inhibitors being tested are ethyl (R)-(-)-3-hydroxybutyrate (E(R)HB), ethyl
(S)-(+)-3-hydroxybutyrate (E(S)HB), NADP+, EAA, and ethyl (S)-(-)-4-chloro-3-acetoacetate
(E(S)4Cl3H). AKR163 was not inhibited when the substrate EAA was reacted with the enzyme
and E(R)HB or NADP+, as the Km and Vmax values between the uninhibited and inhibited
reactions were not significantly different. AKR163 was noncompetitively inhibited when the
substrate E4ClAA was reacted with the enzyme and NADP+, as the Vmax of the uninhibited
reaction was around 1.5 times higher than the Vmax of the inhibited reaction, with Km values
being similar in both reactions. The results of these experiments show that product inhibition of
the enzyme AKR163 is not occurring as expected, but the enzyme can be inhibited via substrate
inhibition.
| | school |
The College of Liberal Arts, Drew University
| | degree |
B.S. (2023)
|
| advisor |
Dr. Adam Cassano
|
| full text | LPortadin.pdf |
| |
