Cancer cells often use an altered metabolic pathway in which glycolysis, uncoupled from the citric acid cycle, serves as the primary source of ATP. To support cancer cell proliferation and growth, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) must have a constant source of NAD. While lactate dehydrogenase (LDH) in its conversion of pyruvate to lactate is a well-known source of cytosolic NAD for GAPDH activity, cytosolic malate dehydrogenase (MDH1) also plays a role in cell proliferation through its generation of cytosolic NAD by the conversion of OAA to malate. This development has implicated MDH1 in cancer cell metabolism and characterizing the interactions of its different isoforms with alternate substrates serves as a potential route to cancer treatments. In this study, we created a G148T mutant of human cytosolic malate dehydrogenase isoform 3 (hMDH1) using site-directed mutagenesis to survey potentially altered substrate specificity with oxaloacetate (OAA) and α-ketoglutarate (αKG). We produced a sample of both the mutant enzyme and the wild type enzyme (WT hMDH1) by overexpression in Escherichia coli and purification by Ni-NTA affinity chromatography. We assessed the purity of the enzyme samples using SDS-PAGE and determined the protein concentration of each sample using a bicinchoninic acid (BCA) assay. We performed kinetics assays of the G148T hMDH1 mutant with OAA αKG to determine enzyme activity relative to the wild type using a modified Michaelis Menten model. This study demonstrates that the G148T hMDH1 mutant enzyme has decreased turnover rate and proficiency with OAA compared to the wild type enzyme and that αKG is likely not a viable substrate for hMDH1 isoform 3.
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