Abstract
Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation.
Highlights
Purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, EC 2.4.2.1) has a crucial role in the purine salvage pathway[1]
The crystal structure of the E. coli enzyme, which served for the formulation of the above mechanism[5], was the first to capture the coexistence of the two conformations of the active site, open and closed, in the hexameric PNPs
The crystal structure of the binary complex of Arg24Ala mutant with the phosphate showed that all six active sites remained in the open conformation, indicating that the binding of phosphate in the absence of Arg[24] is not sufficient for a conformational change to take place
Summary
Purine nucleoside phosphorylase (PNP, purine nucleoside orthophosphate ribosyl transferase, EC 2.4.2.1) has a crucial role in the purine salvage pathway[1]. The arrangement of subunits in the structure of a PNP hexamer is such that two of them donate two amino acids (His[4] and Arg[43] in E. coli) to each other, completing the active site of its neighbour, effectively forming a dimer which possesses an approximate 2-fold symmetry. The movement of the segmented part of the H8 α-helix is essential as it brings the side chain of the conserved amino acid Arg[217] within a hydrogen bonding distance to the conserved Asp[204] residue as the initial step in the catalysis[5] This triggers the proton transfer from Asp[204] side-chain, which is initially in the acidic form, to the purine base at the N7 position. Three different methods were used to validate this model: site-directed mutagenesis, kinetic studies, and X-ray crystallography[14] These experiments supported earlier predictions that the binding of phosphate and its interaction with Arg[24] are necessary for the conformational change to occur. In crystal structures of ternary complexes reported up to now[5,13,19], it has never been observed that only part of the active sites were occupied by nucleosides
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