CALF SPLEEN PURINE NUCLEOSIDE PHOSPHORYLASE COMPLEXED WITH SUBSTRATESAND SUBSTRATE-ANALOGS

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine sa lvage pathway, which provides an alternative to the de novo pathway fo r the biosynthesis of purine nucleotides. PNP catalyzes the reversible phosphorolysis of 2'-deoxypurine ribonucleosides to the free bases an d 2-deoxyribose 1-phosphate. Absence of PNP activity in humans is asso ciated with specific T-cell immune suppression. Its key role in these two processes has made PNP an important drug design target. We have in vestigated the structural details of the PNP-catalyzed reaction by det ermining the structures of bovine PNP complexes with various substrate s and substrate analogues. The preparation of phosphate-free crystals of PNP has allowed us to analyze several novel complexes, including th e ternary complex of PNP, purine base, and ribose 1-phosphate and of t he completely unbound PNP. These results provide an atomic view for th e catalytic mechanism for PNP proposed by M. D. Erion et al. [(1997) B iochemistry 36, 11735-11748], in which an oxocarbenium intermediate is stabilized by phosphate and the negative charge on the purine base is stabilized by active site residues. The bovine PNP structure reveals several new details of substrate and inhibitor binding, including two phosphate-induced conformational changes involving residues 33-36 and 56-69 and a previously undetected role for His64 in phosphate binding. In addition, a well-ordered water molecule is found in the PNP active site when purine base or nucleoside is also present. In contrast to h uman PNP, only one phosphate binding site was observed. Although binar y complexes were observed for nucleoside, purine base, or phosphate, r ibose l-phosphate binding occurs only in the presence of purine base.