Subject Area
Chemistry
Description
Human cytosolic malate dehydrogenase (MDH1) is a key enzyme involved in the malate-aspartate shuttle of eukaryotic cells, and which catalyzes the reaction of oxaloacetate to malate. As MDH1 is responsible for maintaining redox homeostasis by regeneration of NAD+ for glycolysis, its regulation is of particular interest in pharmaceutical applications, specifically in treating cancer cells that proliferate quickly and hence rely greatly on glycolysis. While the regulation of MDH1 by the phosphorylation of key residues has been previously documented, work still remains in identifying the specific residues involved in regulation of the enzyme by phosphorylation. In this study, we used the phosphomimetic substitution of Ser260 to Asp260 in the third isoform of MDH1 (MDH1v3) as a model for phosphorylation at the S260 residue of the enzyme, using site-directed mutagenesis to induce the mutation in a pET28a plasmid expressing the WT-MDH1v3 enzyme, producing the MDHv3-S260D mutant. We report that the mutation of Ser260 to Asp260 in MDH1v3 significantly inhibits the enzyme’s activity by several orders of magnitude, suggesting cellular regulation of MDH1v3 activity by phosphorylation at this position. Our results highlight a more than 1000-fold decrease in efficiency of the mutant enzyme (kcat/KM of 1.38 ± 0.05 × 104 M-1 s-1) as compared to that of WT-MDH1v3, and similardecreases in the kcat (2.38 ± 0.08 s-1) and KM (172 ± 9 μM) ofMDH1v3-S260D. Our findings invite further investigationinto the phosphorylation of other residues of MDH1 todevelop a broader characterization of MDH1 regulation.
Publisher
Providence College
Publication Date
5-12-2025
Type
Article
Language
English
