Degree Name

Master of Science (MS)

Semester of Degree Completion

1996

Thesis Director

Norbert C. Furumo

Abstract

Kinetic studies and chemical modification studies were performed on malate dehydrogenase from Escherichia coli (eMDH). Chemical modification studies using diethylpyrocarbonate and iodoacetic acid, along with log V/K profiles indicate that one ionizing group with a pKa between 7.9 and 8.5 acts as a general acid/base in the catalytic mechanism. Log V profiles suggest that malate binds to the correctly protonated form of the enzyme. These results imply that a histidine residue is required for catalysis.

A primary deuterium kinetic isotope effect of 1.43 (±0.14) was observed on V/K using malate-2-d, while no isotope effect was measured on Vmax. This implies that proton abstraction is partially rate determining under nonsaturating conditions. Small isotope effects were observed on Vmax and V/K (1.22 ±0.19 and 1.08 ±0.06) when using NADD, indicating that the rate determining step is the release of the cofactor.

Other kinetic studies show that eMDH is inhibited by the substrates oxaloacetate and NADH at high concentrations. Through substrate analog inhibition experiments, using citrate, malonate, ketomalonate and ATP, it was found that eMDH is very specific for it substrates.

Allosteric interactions between ligand and substrate provide a means of regulation, since they allow the activity of an enzyme to be controlled by changes in the concentrations of other species (including the products). It was determined that eMDH is allosterically inhibited in the presence of 0.065 mM NADH

Other chemical modification reagents specific for arginine residues such as, 1,2-cyclohexanedione, indicate that arginine residues are important for catalysis and most likely play a role in binding the substrate.

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