Degree Name

Master of Science (MS)

Semester of Degree Completion

1999

Thesis Director

Norbert C. Furumo

Abstract

Regulatory mechanisms of malate dehydrogenase from E.coli (eMDH) involving NADH as an allosteric effector were investigated. The reaction was studied in both directions: malate oxidation and oxaloacetate reduction. When malate was the variable substrate, a plot of rate against substrate concentration was sigmoidal in the presence of 0.065 mM NADH, which indicates the presence of an allosteric site for NADH on the enzyme. Binding of NADH at the allosteric site causes conformational changes in the active site and, thereby, changes the catalytic activity of the enzyme. An increase in Km value, from 1.3 to 3.9 mM malate was observed, which indicates a decrease in the enzyme affinity for the substrate.

When eMDH was chemically modified with 5'-p-flurosulfonyl-benzoyladenosine (FSBA) in the presence of 0.15 mM NADH as a protecting agent, the allosteric behavior was abolished, which suggests that FSBA is modifying specific amino acid residues in the allosteric site and, therefore, preventing NADH from binding. eMDH was inactivated by FSBA in the absence of NADH. The inactivation appears to result from covalent modification of His 177 in the active site, which is believed to be crucial for the catalytic mechanism of eMDH.

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