Subject Area
Chemistry
Description
The cytosolic human enzyme, malate dehydrogenase (MDH1), is believed to have a significant effect on the proliferation of cancerous cells. This enzyme oxidizes NADH to NAD+ as it converts oxaloacetate (OAA) to malate. This oxidation of NADH provides the needed NAD+ to cancer cells, which require NAD+ as they upregulate glycolysis in aerobic conditions. Targeting MDH1 can reveal new therapeutic treatments for cancer by preventing the conversion of OAA to malate and depleting NAD+ stores in cancer cells, which can be done by mutating residues in the enzyme and assessing the change in activity. Here we show the replacement of a glutamate with an alanine at position 115 (E115A) of MDH1 through site-directed mutagenesis and Ni-NTA protein purification to analyze the change in the enzymatic activity on native and non-native substrates of the enzyme. A modified Michaelis-Menten equation was utilized to determine that the wild-type enzyme has a ksp of 6 ± 1 × 106 M-1s-1 on OAA and 1.12 ± 0.05 × 103 M-1s-1 on phenylpyruvate while the E115A mutant enzyme has ksp values of 6.3 ± 0.5 × 106 M-1s-1 and 400 ± 40 M-1s-1 on OAA and phenylpyruvate respectively. The data suggest that the E115A mutation has a higher affinity for OAA, lower affinity for phenylpyruvate, and decreased specificity for phenylpyruvate compared to the wild- type. More research can be conducted to address and manipulate the affinity of MDH1 for a non-native substrate to inhibit the enzyme in cancerous cells.
Publisher
Providence College
Date
Spring 4-17-2023
Type
Article
Format
Text
Language
English
