Active site studies of two target enzymes: reverse transcriptase and aspartate transcarbamoylase

Abstract

<p>Two target enzymes for chemotherapy were investigate by probing their active sites with various methods. The reaction catalyzed by Aspartate Transcarbamoylase from Escherichia coli is thought to involve a tetrahedral transition state. Several bisubstrate analogs with a charged tetrahedral moiety were synthesized and tested for inhibition. The results suggest that the enzyme exhibits no preference for a tetrahedral over a trigonal moiety which casts some doubt as to the actual reaction mechanism. The carbamoyl region of the active site of the enzyme was further probed with various anionic inhibitors to investigate the structural basis for the strong inhibition observed with phosphonoformate and carbonyldiphosphonate. A second target enzyme, reverse transcriptase taht is also effectively inhibited by these two anionic compounds was studied. The active site of this enzyme was probed with a variety of anionic ligands to determine the structural features necessary for inhibition. Phosphonoformate prevailed as the most effective inhibitor of three different reverse transcriptases. Combined inhibitor studies with the enzyme from Human Immunodeficiency Virus Type-1 were performed to further investigate the mode of binding of PFA with the active site. Finally, the interaction of the metal chelator ortho-phenanthroline with the enzyme was investigated. A kinetic analysis and protection/inactivation studies were performed to probe the site of metal chelation and its proximity to the binding sites for various active site ligands.</p>