In the first project, structural and functional studies of TDH are described. TDH is an unusual NAD-dependent enzyme that exhibits a multiplicity of catalytic activities at a single active site. These activities arise from the capacity of this enzyme to catalyze a reaction pathway in which different substrates undergo the same initial catalytic steps, but the subsequent intermediates can dissociate from the enzyme at different stages in the catalytic cycle thereby leading to different final products.
The first crystal structure of an NAD-dependent TDH has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a ternary complex with the intermediate analogue oxalate, Mg2+ and NADH, and also as a binary complex with Mg2+ and NADH. The active site in these complexes is highly ordered, allowing identification of the substrate and cofactor binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and the divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating site reaction mechanism. Each substrate undergoes the initial hydride transfer, but differences in orientation of the substrate are proposed to account for the different reactions catalyzed by TDH.
For the second project two different approaches to treat Canavan disease are explored. Canavan disease is a fatal neurological disorder caused by mutations in the aspA gene leading to altered aspartoacylase with diminished catalytic activity. These clinical mutations are found at sites located throughout the enzyme suggesting that destabilization of the protein structure may be responsible for the loss of activity in many of these patients. In the first approach, mutations have been introduced at several of these sites and the purified enzymes were found to have low catalytic activity and, in some cases, diminished stability. Docking studies have suggested some small probe molecules that could bind at these sites. Optimization of these compounds, guided by kinetic and structural studies, will lead to pharmacological chaperones that have the potential to stabilize these defective enzyme forms and reverse the effect of these mutations in Canavan patients. As a second approach, enzyme replacement therapy for the treatment of Canavan disease is described. The structure of human aspartoacylase has allowed us to locate putative binding sites for polyethylene glycol (PEG) molecules. PEGylation has produced altered forms of the enzyme that could have decreased immunogenicity and increased in vivo stability. In the study we have tested different PEGylation reactions and evaluated their effect on the catalytic activity of aspartoacylase. PEGylated forms of this enzyme have been injected into a mouse model of Canavan disease and the effects of enzyme replacement on the activity of aspartoacylase has been measured in the tissues. Enzyme activity levels in the mice brain have increased and dose dependent studies are currently performed. In conclusion, these studies will hopefully lead to a viable approach for the treatment of Canavan disease.