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Description

Malate Dehydrogenase (MDH) is the enzyme that catalyzes the ninth step of the citric acid cycle, a regenerative step in the metabolism of glucose in which malate is converted to oxaloacetate. Conformational changes in the active loop region of MDH along with the coenzyme NAD+ drive catalysis. Previous studies suggested that mutating positively charged amino acids to negatively charged amino acids at residue 130 decreased the enzymatic activity of MDH. We hypothesized that if we change arginine 130 (positively charged amino acid) to aspartate (negatively charged amino acid), then the enzymatic activity of MDH would significantly decrease. In e. Coli cells, site-directed mutagenesis was performed to create the mutation (R130D). The mutated protein was purified using nickel affinity chromatography and confirmed by Bradford assay and SDS-PAGE gel of the protein purity. The kinetics of the mutated protein were measured and compared to the wild-type to examine the effects of the mutation. We found R130D has a lower Vmax and higher Km than wild-type MDH, suggesting that the mutant MDH protein catalyzes the reaction at a much slower rate, and the substrate oxaloacetate had a reduced binding affinity to the enzyme. Learning more about which amino acid sequences of MDH are important in its metabolic function has important implications regarding the regulation of carbohydrate metabolism, specifically the citric acid cycle.

Publication Date

4-17-2024

Keywords

Malate dehydrogenase; Enzymes; Amino acids; Mutation (Biology)

Disciplines

Biochemistry

Isolation and Analysis of R130D Malate Dehydrogenase (MDH)

Included in

Biochemistry Commons

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