STRUCTURE, MECHANISM, AND INHIBITOR DESIGN FOR HGPRT

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Hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) is the major human enzyme of purine base salvage. In humans, the deficiency of HGPRTase causes Lesch-Nyhan syndrome which is characterized by severe gout and a poorly understood behavioral defect. In protozoan parasites the enzyme is responsible for purine utilization since these organisms are incapable of purine de novo synthesis and thus require purine salvage for growth. The protozoan parasite Plasmodium falciparum causes malaria which is estimated to cause over 2 million deaths per year. The long-term goal of this research program is to provide sufficient information on the structure and mechanisms of human and P. falciparum HGPRTases to permit the design of species-specific inhibitors for these enzymes. Both enzymes will be produced by overexpression in E. coli. The X-ray crystal structure of human HGPRTase has been determined to 2.5 Angstroms in preliminary studies for this project. Structures will be established for the human enzyme with bound inhibitors and the initial structure of the enzyme from P. falciparum will be attempted. Kinetic and binding studies will establish the catalytic mechanisms, substrate and substrate analogue binding and inhibition constants, and the steps which limit the overall catalytic cycle. Kinetic isotope effects will provide a geometric structures of the transition states stabilized by the enzymes. Isotope effects for IMP pyrophosphorolysis will be measured with a slow substrate, phosphonoacetate. Kinetic isotope effects for the chemical solvolysis of 5-P-ribosyl-1-pyrophosphate (PRPP) will be compared to those for the enzyme using PRPP as substrate in the 5-P-ribosyl transferase reaction. The comparison will establish the enzymatic contribution to stabilization of the enzymatic transition state. The transition state structure will be used to design transition state inhibitors. Comparison of crystal structures for the human and P. falciparum enzymes with and without inhibitors bound will provide fundamental information on the mechanisms for transition state stabilization. Differences in either transition- state structure or the enzymatic binding sites can be exploited to design species-specific inhibitors.