Subject Area
Chemistry
Description
Metabolic profiling for a variety of cancerous cells indicate significant increases in the levels of glucose consumption. To support uncontrolled cell division, cancer cells also present an uncoupling of glycolysis from the citric acid cycle to promote glucose carbons to the synthesis of biomass, therefore, requiring a constant supply of NAD+. Recent studies indicate that cancer cells exhibit upregulated cytosolic malate dehydrogenase (MDH1) activity, which catalyzes the conversion of oxaloacetate to malate with the oxidation of NADH, generating NAD+. Given its increased activity, MDH1 may serve as a valuable target for treating cancer. Here we report the effects on enzyme catalytic efficiency and specificity induced by the point-substitution, R111V, within the flexible loop of MDH1-isoform-3, constructed by site-directed mutagenesis. The mutant protein was purified by Ni-NTA chromatography and analyzed by modified Michaelis-Menten kinetics to determine changes in kinetic constants with natural substrate, OAA, and non-preferred substrate, phenylpyruvate. We found that this mutation does not significantly change enzyme specificity or catalytic efficiency for phenylpyruvate, as shown in kcat values of 1050 ± 50 and 1.1 ± 0.1 103 M-1s-1 for mutant and wild type enzyme, respectively. However, we report changes in ksp for preferred substrate, OAA, with values of 1.2 ± 0.1 107 and 5.9 ± 0.1 107 M-1s-1 for mutant and wild type enzymes, respectively. In addition, we report a Hill coefficient (n = 2) for wild type enzyme with OAA that suggests cooperativity between subunits of MDH1, which challenges preceding classification of the enzyme as non-cooperative.
Publisher
Providence College
Date
Spring 3-24-2023
Type
Article
Format
Text
Language
English
