Seminarium Zakładu Biofizyki

Protein nucleoside phosphorylase (PNP) is an enzyme that catalyzes a reversible conversion process (ribosylation and phosphorolysis) between nucleobases (purines) and their nucleosides. Experimental studies showed that calf PNP ribosylates purine analogues in specific positions: 2,6-diamino-8-azapurine in position 7 or 8 and 8-azaguanine in position 9 of the triazole ring. The reason for this phenomenon can be a result of different expositions of purine substrates to the channel leading to the binding site. This hypothesis was verified by the application of molecular modeling techniques to two complexes of purine analogues 2,6-diamino-azapurine, calf PNP (pdb-code: 1LVU), and 8-azaguanine, calf PNP (pdb-code: 2AI1). The results obtained with a combination of quantum chemistry, docking, and molecular dynamics methods showed qualitative validity of our hypothesis. Binding free energies of protein–ligand systems showed that most probable binding poses expose N8 nitrogen for 2,6-diamino-8-azapurine and N9 nitrogen for 8-azaguanine into the binding channel and ruled out the exposition of N9 for 2,6-diamino-8-azapurine and N7 for 8-azaguanine, partially in agreement with the experimental data. The other important result obtained in this study is a significantly higher population of the protonated form of crucial residue Glu-201 present in the binding pocket, compared to the standard protonation of free glutamic acid in solution. This result combined with populations of tautomeric forms of both investigated systems strongly suggests that 2,6-diamino-8-azapurine and 8-azaguanine are recognized by proteins with deprotonated and protonated Glu-201 residues, respectively. A comparison of computed binding poses of the investigated ligands to the inhibitors present in crystal structures suggests that the modification of the (S)-PMPDAP inhibitor, in which a 2-(phosphonomethoxy)propyl chain is attached at position 8 instead of position 9, might increase its binding affinity.