Active Site Contacts in the Purine Nucleoside Phosphorylase-Hypoxanthine Complex by NMR and ab Initio Calculations †

Hypoxanthine (Hx) with specific 15 N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H 1 H and 15 N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the 1 H and 15 N chemical shifts of N-1H in the PNP‚Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (CdO to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP‚Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.