REFINED CRYSTAL STRUCTURE OF BOVINE SPLEEN PURINE NUCLEOSIDE PHOSPHORYLASE AT 1.6 Å RESOLUTION. Matthew J. Pugmire, Chen Mao, Steven E. Ealick, Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

The low temperature X-ray crystal structure of bovine spleen purine nucleoside phosphorylase has been solved by molecular replacement and refined to high resolution in the native form and in complex with 9-deazainosine. Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of ribonucleosides and 2'-deoxyribonucleosides of guanine and hypoxanthine. This phosphorolysis is an important step in both the synthetic and metabolic pathways of nucleosides. PNP has become an important target for drug design due to its' relationship with various immunological diseases. Refinement and rebuilding cycles were carried out using the programs XPLOR, CHAIN, and O. The native structure has been refined to an R-factor of 18.8 and a free-R-factor of 23.4 while the complex form has been refined to an R-

factor of 20.0 and a free-R-factor of 24.8. The stereochemistry of the substrate is well defined at this high resolution as are other important structural features of the enzyme. The nucleoside shows an anti conformation of the glycosidic bond and C4' endo - O4' exo puckering of the sugar ring. In the native crystal structure a Mg-6H₂O complex has been modeled between packing units while both the native and complex forms show what appears to be a metal (possibly a first row transition metal) complex with 3 His residues and 2 water molecules that is on a 3-fold symmetry axis between packing units. Differences between the native and complex structures show that binding of substrate induces structural changes as a disordered loop becomes more ordered in an [[alpha]]-helix. These high resolution structures provide a detailed structural model of the PNP enzyme and aid in describing a possible reaction mechanism. Detailed structural information is also invaluable as inhibitors of PNP are designed as potential therapeutic agents.