Kinetic and Structural Characterization of Human Histidine Triad Nucleotide Binding Enzymes

Abstract

Human histidine triad nucleotide binding protein 1 (hHINT1) has been studied for its catalytic role in activating antiviral prodrugs like Remdesivir as well as the enzyme’s function as a modulator of opioid tolerance when its active site is competitively inhibited. Compared to hHINT1, relatively little is known about the isozyme hHINT2 which is similar in sequence and structure. A kinetic characterization of hHINT2 catalysis would be an important first step in building a broader understanding of the differences between these closely related enzymes, especially as further endeavors to selectively inhibit hHINT1 for pain modulation is pursued. In this thesis we first present a thermodynamic and crystallographic analysis of hHINT1 active site inhibition to better understand how hHINT1 cross-regulates the interaction between the mu-opioid and N-methyl-D-aspartate receptors in mice. Second, in the interest of exploring the differences between the hHINT1 and hHINT2 active sites, the substrate specificity of eleven substrates were kinetically assessed. Third, CPMG-NMR was used to probe the backbone dynamics of hHINT1, illuminating two residues distant from the active site which when mutated dramatically impact the enzyme’s kinetics and inhibitor binding capabilities. Analogous mutations in hHINT2 caused similar kinetic and inhibitor binding effects, demonstrating this distant and dynamic site is conserved and important to HINT catalysis. Finally, to build upon the tools used to study HINTs, a new kinetic assay was created using a switch-on fluorescent inhibitor as a reporter, removing the need for a fluorophore within HINT substrates. In summary, this work provides a structural view of hHINT1 inhibition, provides the first kinetic characterization of hHINT2, demonstrates the importance of distant dynamics on HINT active site function, and adds to the kinetic toolkit for further HINT analyses.