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Effect of D91 and E294 Mutations on the Kinetics of Yeast Mitochondrial Malate Dehydrogenase

  • Nguyen, Kylie
  • Zhang, Jing
  • Kim, Heejeong
  • Behrens, Mark
Publication Date
Apr 01, 2024
University of Nebraska - Lincoln
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Malate dehydrogenase (MDH) is one of the eight enzymes in the citric acid cycle (TCA), involved in the conversion of malate to oxaloacetate (OAA), and the reverse reaction of OAA to malate, using NAD+/NADH as the cofactor, respectively. MDH and citrate synthase (CS) form a metabolon, a machinery potentially driving the TCA cycle. This study explored the impact of the point mutations D91 and E294, two amino acid residues presumably involved in the interaction between MDH and CS, on the enzymatic kinetics of Yeast mitochondrial Malate Dehydrogenase (YmMDH). The wild type (WT) and mutant YmMDH expressed in the bacterial system were purified by Ni-affinity chromatography. The effectiveness of the purification was evaluated by protein gel electrophoresis. The enzyme kinetics were measured at varied OAA and NADH concentrations using spectrophotometry and the Vmax and Km of the mutant YmMDH were compared to those of the WT. The Vmax of the mutants in general decreased compared to that of the WT. The Km of the mutants for OAA and NADH had differential changes compared to that of the WT depending on the specific mutation, and it did not seem to correlate with the changes in Vmax. These results suggested that D91 and E294 are involved in the mechanisms of MDH-catalyzed reaction rates. Further research is required to verify the data including the adjustment of OAA concentrations for kinetic measurement.

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