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Author

Farren Billue

Date of Award

Spring 2012

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

First Advisor

Dr. Ellis Bell

Abstract

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.

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