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Date of Award
Restricted Thesis: Campus only access
Bachelor of Science
Biochemistry & Molecular Biol.
Dr. Ellis Bell
Malate Dehydrogenase is a dimeric enzyme that catalyzes the oxidation of malate to oxaloacetate in a reaction involving both a hydride transfer [to NAD+] and a proton abstraction, and involves subunit interactions in activity and regulation [by citrate]. Little is known concerning the mechanism of communication between active sites in the dimer. The subunit interface contains three conserved methionine residues [M62, M64 & M91] in close proximity to residues from the opposing subunit [P59, L63, Y273 & I88 as well as between M66 on one subunit and M62 & M91 on the opposing subunit]. These residues together with a methionine close to the active site [M128] have been mutated using QuikChange mutagenesis to a variety of different amino acids to test their role in subunit communication and catalytic activity. Proteins
are expressed, purified using Ni-NTA affinity chromatography and characterized by MALDI-tof. Circular Dichroism in conjunction with thermal melts has been used to examine both stability and folding cooperativity using a 4 parameter Hill equation to analyze the ellipticity at 222 nm as a function of temperature. Initial rate kinetic studies demonstrate changes in both Vmax and Km of various mutants as well as indicating changes in citrate regulation of the protein.
Billue, Farren, "Determining the role of Methionines in the activity and subunit interactions of Glyoxysomal Malate Dehydrogenase" (2012). Honors Theses. 84.