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Date of Award


Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science


Biochemistry & Molecular Biol.

First Advisor

Dr. Ellis Bell


Glutamate Dehydrogenase is a homohexamic enzyme that has complex regulatory and kinetic properties that involve subunit interactions. Previous work has demonstrated a role of ligand induced changes in conformational flexibility in modulating regulatory effects of the enzyme. Extensive analysis on the effects of ligands on the unfolding of GDH via thermal denaturation were analyzed to quantitatively monitor global stability and the cooperativity of the unfolding process using a 4- parameter sigmoidal equation. Correlation of the effects of various regulators on the unfolding process suggest that in general increased flexibility of the protein is related to the ligand producing enhanced activity. These effects have been correlated to the temperature dependence of not only the parameters of folding but also effects on both regulator cooperativity and ADP activation and GTP inhibition. Initial rate kinetics were used to study entropy and enthalpy contributions to substrate binding and relate them to conformational flexibility. Regulators were found to have opposite effects, with ADP increasing stability and decreasing flexibility and GTP decreasing stability and increasing flexibility which correlate with changes in enzyme activity. The cofactors NADH and NADPH both destabilized GDH but to different levels, with NADH having the smaller effect. Kinetics studies with NAD+ revealed an entropic basis for the reaction in the low affinity site

while GTP kinetics had almost zero entropy contribution.