X-RAY CRYSTALLOGRAPHIC AND KINETIC-ANALYSIS OF HUMAN PURINE NUCLEOSIDE PHOSPHORYLASE COMPLEXES WITH -BETA-D-RIBOFURANOSYL-1,2,4-TRIAZOLE-3-CARBOXAMIDE AND ETA-D-RIBOFURANOSYL-1,2,4-TRIAZOLE-3-CARBOXAMIDINE

The three-dimensional structures of the complexes between human erythr ocytic purine nucleoside phosphorylase (PNP) and both -beta-D-ribofura nosyl-1,2,4-triazole-3-carboxamide (ribavirin) and eta-D-ribofuranosyl -1,2,4-triazole-3-carboxamidine (TCNR) have been determined using X-ra y crystallographic techniques. The structures have been refined at 2.9 Angstrom resolution using simulated annealing and conjugate-gradient minimization techniques to an R value of 21.8% for ribavirin and 20.8% for TCNR. Ribavirin and TCNR are truncated nucleosides corresponding to adenosine and inosine; respectively, and are of potential interest as PNP inhibitors. Kinetic parameters have been determined for recombi nant wild-type PNP and for a mutant PNP in which Asn 243 is converted to Asp. The K-i value for ribavirin is 4:9 mM with wild-type PNP and 4 .7 mM with the Asn243Asp mutant, while the K-i values for TCNR are 17. 6 mu M and 3.8 mu M with wild-type and mutant, respectively. X-ray cry stallographic studies showed that the binding geometry for both of the se substrate analogues was similar to that seen for natural substrates . The glycosidic torsion angles (chi) were -34 degrees for ribavirin a nd -39 degrees for TCNR which are in good agreement with values seen f or other studied nucleoside complexes with PNP, but which are unusual when compared to those seen for free nucleic acid derivatives. Based u pon the three-dimensional structure, interactions of Asn 243 and Glu 2 01 with a protonated carboxamidine of TCNR explain the stronger inhibi tion of PNP observed for TCNR over ribavirin.