Crystal Structure of Calf Spleen Purine Nucleoside Phosphorylase with Two Full Trimers in the Asymmetric Unit: Important Implications for the Mechanism of Catalysis

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3 Å resolution in space group P212121. Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P213, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations.