| Abstract
| - Purine nucleoside phosphorylase (PNP) is a key enzyme in thepurine salvage pathway, whichprovides an alternative to the de novo pathway for the biosynthesis ofpurine nucleotides. PNP catalyzesthe reversible phosphorolysis of 2‘-deoxypurine ribonucleosides to thefree bases and 2-deoxyribose1-phosphate. Absence of PNP activity in humans is associated withspecific T-cell immune suppression.Its key role in these two processes has made PNP an important drugdesign target. We have investigatedthe structural details of the PNP-catalyzed reaction by determining thestructures of bovine PNP complexeswith various substrates and substrate analogues. The preparationof phosphate-free crystals of PNP hasallowed us to analyze several novel complexes, including the ternarycomplex of PNP, purine base, andribose 1-phosphate and of the completely unbound PNP. Theseresults provide an atomic view for thecatalytic mechanism for PNP proposed by M. D. Erion et al. [(1997)Biochemistry 36, 11735−11748], inwhich an oxocarbenium intermediate is stabilized by phosphate and thenegative charge on the purinebase is stabilized by active site residues. The bovine PNPstructure reveals several new details of substrateand inhibitor binding, including two phosphate-induced conformationalchanges involving residues 33−36 and 56−69 and a previously undetected role for His64 in phosphatebinding. In addition, a well-ordered water molecule is found in the PNP active site when purine baseor nucleoside is also present. Incontrast to human PNP, only one phosphate binding site was observed.Although binary complexes wereobserved for nucleoside, purine base, or phosphate, ribose 1-phosphatebinding occurs only in the presenceof purine base.
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