CRYSTAL-STRUCTURE OF CALF SPLEEN PURINE NUCLEOSIDE PHOSPHORYLASE IN ACOMPLEX WITH HYPOXANTHINE AT 2.15 ANGSTROM RESOLUTION

Trimeric calf spleen purine nucleoside phosphorylase has been complexe d with hypoxanthine via phosphorolysis of inosine in the presence of p hosphate. The resulting ''Michaelis'' complex (three hypoxanthine mole cules per trimer), presumed to be formed under these conditions, cryst allized in the cubic space group P2(1)3, with unit cell dimension a=94 .11 Angstrom and one monomer in the asymmetric crystal unit; the biolo gically active trimer is located on the crystallographic 3-fold axis. High-resolution X-ray diffraction data were collected using synchrotro n radiation (EMBL outstation, Hamburg, c/o DESY). The crystal structur e has been determined by molecular replacement and refined at 2.15 Ang strom resolution to an X-value of 0.18. In the hypoxanthine binding si te, a cis-peptide bond between Asn243 and Lys244 is observed. Side-cha ins of Glu201 and Asn243, as well as one integral water molecule locat ed in the base binding site, form hydrogen bonds with the hypoxanthine N-1 H, N-7 H and O-6. A second water molecule links the base position s N-3 and N-9 with an adjacent pocket, which presumably is the phospha te-binding site. This pocket is filled completely by a cluster of six water molecules. Hence all possible donor/acceptor-positions of hypoxa nthine are saturated by hydrogen-bonding to protein side-chains or int egral water molecules. Purine nucleoside phosphorylase isolated form h uman tissues is a primary target for chemotherapeutic intervention, an d the more stable calf enzyme has similar physico-chemical and kinetic properties, as well as response to inhibitors. Hence the high-resolut ion structure presented here may serve for design of inhibitors with p otential pharmacological applications. (C) 1997 Academic Press Limited