Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Mary D. Barkley


The focus of this work is structural and functional studies of wild-type and active-site mutants of human adenosine deaminase. Two active-site amino acids, Glu217 and His238, were previously substituted with alanine by site-directed mutagenesis. The Glu217Ala and His238Ala mutants showed little or no changes in Zn content and $\rm K\sb{m}$ value, yet dramatic loss of enzyme activity. Circular dichroism studies of the solution conformation of wild-type and mutant human adenosine deaminases indicate that 30 to 50% of $\alpha$-helical structure is lost in Glu217Ala and His238Ala, respectively, whereas $\beta$-sheet structure is fully retained. Inhibitor binding is measured by a fluorescent assay. Dissociation constants for transition-state analog inhibitors are similar for wild-type and mutant enzymes, suggesting that inhibitor and substrate binding determinants are preserved in the mutants. Protein stability studies show that Glu217Ala and His238Ala are less stable to thermal, urea, and guanidinium chloride denaturation than wild-type. Because the enzyme conformational changes caused by substitution of Glu217 and His238 mainly destabilizes the protein with only minor effects on substrate and inhibitor binding, the loss of enzyme activity is probably due to the modification of functional groups that are critical for catalysis. The active-site Zn environment of wild-type, Glu217Ala, and His238Ala mutant adenosine deaminases is probed by EXAFS. There are five ligands coordinated to Zn in the free enzyme and ground- and transition-state analog inhibitor complexes of these three adenosine deaminases, suggesting the presence of a water/hydroxide or a hydroxyl group. Zn-ligand distances of the mutants are similar to those of the wild-type, indicating that the active-site of mutants are intact upon substitution. Wild-type and His238Ala form a transition state with the transition-state analog inhibitor purine riboside whereas with Glu217Ala a ground state is formed. The findings are consistent with the proposed catalytic mechanism based on the X-ray crystal structure of mouse adenosine deaminase, and provide further insight into the effects of these active site residues on the structure, stability and function of human adenosine deaminase.