Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Biochemistry, Biophysics and Molecular Biology

First Advisor

Herbert J. Fromm


Enzymes are catalysts of biological reactions, and are characterized by their substrate specificity, specificity of reaction and ability to be regulated. Aspects of enzymes studied are the catalytic mechanisms, association states, origins of substrate specificity, and enzymatic regulation of the enzyme. One enzyme studied as a model system is adenylosuccinate synthetase (E.C., L-aspartate:IMP ligase, GDP forming; AMPSase). Interactions which appear to be important for enzyme association and substrate binding were studied by producing site-directed mutants of AMPSase as guided by the unligated and ligated crystal structures. The wild-type and mutant enzymes were compared by utilizing spectrophotometric assays, to determine their kinetic parameters, circular dichroism, sedimentation equilibrium analytical ultracentrifagation, and matrix-assisted laser desorption ionization. Associative states of the enzyme were studied by generating mutants at positions Arg143, and Asp231. Arg143 forms part of the active site of the opposite monomer, while Asp231 makes interface contacts. The native enzyme is exclusively a dimer in the presence of ligands, though reported as a monomer during purification. There is a direct relationship between dimer formation and activity. Due to intracellular concentrations of AMPSase and ligands, this is postulated to be a method of regulation for the enzyme. The mutations Asp21, Asn38, Thr42 and Arg419 were made to investigate their role in interactions with the 5'-phosphoryl group of IMP. The unexpected results yielded insights as to the role of a conformational change of Loop 42 - 53, a dynamic loop in the enzyme. The binding energy for interaction of Asn38 - 5'-phosphoryl of IMP goes directly to transition state stabilization, and not towards substrate recognition. Finally, the role of a collection of beta-branched side chains in substrate recognition is examined. Thr128, Thr129 , Thr300 and Thr301 have direct interactions with either L-aspartate or IMP, while Val273 has no bonded contacts with L-aspartate. The effect on the steady-state kinetics by the mutant enzymes reflects the stabilization of the bound complexes by direct hydrogen bonding and conformational limitations on the enzyme for IMP and L-aspartate recognition. The role of the non-bonding contacts is to prevent binding of nonproductive conformations of substrates, as seen by inhibition studies.



Digital Repository @ Iowa State University,

Copyright Owner

Andrea Gorrell



Proquest ID


File Format


File Size

96 pages

Included in

Biochemistry Commons