Transition state theory for enzymatic reactions indicates that molecules which resemble the enzyme-bound transition state will capture a fraction of the transition state binding energy and provide powerful and specific noncovalent inhibitors. This theory has gained wide acceptance but applications have been limited by the lack of methods to experimentally determine reliable transition state structures for enzymatic reactions. Recent advances in the application of kinetic isotope effects have permitted unprecedented understanding of the geometry and electronic nature of enzyme-stabilized transition states. The inosine-preferring nucleoside hydrolase from the trypanosome Crithidia fasciculata has provided a test case to establish that transition state information can be determined with sufficient accuracy to permit the design of powerful inhibitors. A family of inhibitors is now available to establish the interactions between the enzyme and the transition state. The inhibitors can also be used to explore a surprising diversity of transition state structures which exist in the nucleoside hydrolases. Nucleoside hydrolases are found in protozoa including Trypanosoma (Chagas' disease and sleeping sickness), Leishmania (leishmaniasis), Entamoeba (amebic dysentery), Plasmodium (malaria), Giardia (giardiasis) and Trichomonas (vaginitis). Protozoan parasites are incapable of de novo purine synthesis and require purine salvage for DNA and RNA synthesis. Mammalian tissues lack nucleoside hydrolases. Transition state inhibitors for inosine-preferring nucleoside hydrolase are highly specific and do not inhibit mammalian purine nucleoside phosphorylase or guanosine-preferring nucleoside hydrolase from C. fasciculata. The hypothesis for this proposal is that transition state inhibitors for nucleoside hydrolases are isozyme- and Protozoan-specific. The basis of this specificity will be established by transition state analysis of the guanosine-preferring nucleoside hydrolase from C. fasciculata. At least one additional nucleoside hydrolase will be obtained from cDNA and genomic libraries of Trypanosoma or Plasmodium. Inhibitors will be designed based on the geometric and electrostatic potential surface of the transition state. The genes for nucleoside hydrolases will be isolated from cDNA and genomic libraries and overexpressed in E. coli. The basis for specificity will be established by X-ray crystallography, pH profiles and transition state specificity studies. The results will provide novel information on transition state structure, catalysis, and transition state inhibitors which may be antimetabolites for protozoan parasites.
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