HIGH RESOLUTION X-RAY STRUCTURE OF BOVINE PURINE NUCLEOSIDE PHOSPHORYLASE WITH INHIBITORS. Weiru Wang and Steven E. Ealick, Section of Biochemistry, Molecular and Cell Biology, 209 Biotechnology Building, Cornell University, Ithaca, NY 14853

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is a salvage enzyme important to the T-cell-mediated part of the immune system and as such is an important therapeutic target. This paper describes a series of high resolution x-ray structures of PNP inhibitors-9(((phosphono)-alkyl)-benzyl)-guanines, (((guaninyl)-alkyl)-phosphinico)-methyl)-phosphonic acids and 9-deazaguanine derivatives, complexed with bovine PNP. These inhibitors are multi-substrate analogues and are able to bind the purine bind site, the ribose binding site and the phosphate bind site simultaneously in the PNP active site. These competitive inhibitors have high binding affinity with Ki ~10nM and lower. X-ray diffraction data were obtained at low temperature with the Macromolecular Diffraction Facility at the Cornell High Energy Synchrotron Source (MacCHESS). Enzyme-inhibitor structures were refined to resolutions ranging from 1.7 to 2.0 Å with XPLOR. Detailed inhibitor-enzyme interactions were investigated based on these high resolution structures. Residue His64 which is close to the phosphate binding site moves in and out of phosphate binding site surface when different inhibitors are bound. This movement changes the number of positive charge in the phosphate binding site and suggests that electrostatic interactions in the phosphate binding site are different for different inhibitors. A phosphonyl group attached to a 9-deazaguanine derivative interacts with two positively charged residues on the pocket surface without His64 while a free phosphate, or di-phosphate analogue group interacts with three positively charged residues including His64. The difference in electrostatic interactions might contribute to difference in binding energy. Interactions between the inhibitor and enzyme in the purine binding site and the ribose binding site are similar to the results from previous study. Small conformational and positional deviation will be described. Conformational changes of the residues in the PNP active site were also observed. These changes are complementary to the inhibitor conformation and may also contribute to binding energy.