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Lisa Gentile


Malate dehydrogenase (MDH) is an enzyme that has a key role in biological processes, like the Krebs cycle. Specifically, it reversibly catalyzes the interconversion of (S)-malate with NAD+ to oxaloacetate and NADH. Once oxaloacetate is synthesized, MDH dispatches it to citrate synthase, but it is not clear how this happens. One theory is that MDH channels it to citrate synthase by forming a metabolon, a mechanism for direct channeling, preventing diffusion of reaction intermediates into a bulk matrix. There is a lack of research in this area due to the absence of a spectroscopic probe necessary to visualize MDH’s conformational changes. Therefore, a method was tested to incorporate a fluorescent landmark into MDH’s structure and thus be used in future research to reveal the interactions between MDH and citrate synthase. Specific amino acids of MDH were mutated to tryptophan, an amino acid known to fluoresce (V189, I319, A120, I136, P119, G218). The coding sequence for the wildtype MDH and mutant MDHs were incorporated into plasmids and bacterially transformed into Escherichia coli. Both wildtype and mutant proteins were over-expressed, then purified by nickel affinity chromatography using a hexahistidine tag on the N-terminus of MDH. Data will demonstrate that I139W, V189W, and A120W had significantly lower activity than wildtype MDH, and the same is predicted for I136W. I139W and V189W emitted fluorescence at 290 nm, but I136W did not. The mutations P119W and G218W could not be overexpressed or purified. Next steps in design of a fluorescent, active MDH will be discussed.

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