Can Crystal Symmetry and Packing Influence the Active Site Conformation of Homohexameric Purine Nucleoside Phosphorylases?

Abstract

Abstract:It is generaly believed that enzymes retain most of their functionality in the crystal form due to the large solvent content of protein crystals. This is facilitated by the fact that their natural environment in solution is not too far from the one found in the crystal form. Nevertheless, if the nature of the enzyme is such to require conformational changes, overcoming of the crystal packing constraints may prove to be too difficult. Such conformational change is present in one class of enzymes (purine nucleoside phosphorylases), that is the subject of our scientific interest for many years. The influence of crystal symmetry and crystal packing on the conformation of the active sites in the case of homohexameric purine nucleoside phosphorylases is presented and analysed.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features

“…This arrangement of active sites was previously noted only in the case of two other crystal structures of HpPNP‐Zg . In a general survey of all structures of PNPs deposited in the PDB, these represent the only structures with a single closed active site , which seems to be a unique feature of H. pylori PNPs.…”

“…Notably, all of the crystal structures with bound ligands crystallized in the same space group P 2 1 2 1 2 1 and have similar unit cell axes, and are therefore expected to have similar crystal packing environments. Our previous investigations on all of the crystal structures of hexameric PNPs suggest no correlation between crystal packing and the distribution of the open and closed active sites .…”

“…To compare the activity of both HpPNPs and E. coli PNP, assays were conducted as described earlier , at pH 7.0 in 50 m m HEPES‐NaOH buffer at 25 °C and in the presence of 50 m m P i . Nucleoside substrate concentration was as follows: 0.5 m m or 1 m m Ino, 0.2 or 0.4 m m Ado, 0.18 m m Guo and 0.28 or 0.45 m m m 7 Guo.…”

“…For many years we have been studying a homohexameric enzyme purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2.4.2.1.html) and the conformational changes of its six active sites upon ligand binding, with the final goal of understanding the highly complex mechanism of this enzyme action . PNP catalyses the reversible phosphorolytic cleavage of the glycosidic bond of purine ribo‐ and deoxyribonucleosides:…”

The Role of Phosphate Binding in Purine Nucleoside Phosphorylase of Helicobacter pylori

“…[1] Although much progress has been made over the years of our research on PNP from E. coli, the complete blueprint of the catalytic mechanism of PNP is yet to be drawn. [2][3][4][5][6][7][8] What makes PNP an attractive target for the fight against this severe pathogen is the fact that this bacterium relies exclusively on the purine salvage pathway for the synthesis of purine nucleosides, and in this pathway the PNP plays a key role. [9] For that reason inhibiting this enzyme would cut down the only source of nucleosides and ultimately would lead to its eradication.…”