PURINE NUCLEOSIDE PHOSPHORYLASE .2. CATALYTIC MECHANISM

X-ray crystallography, molecular modeling, and site-directed mutagenes is were used to delineate the catalytic mechanism of purine nucleoside phosphorylase (PNP), PNP catalyzes the reversible phosphorolysis of p urine nucleosides to the corresponding purine base and ribose 1-phosph ate using a substrate-assisted catalytic mechanism, The proposed trans ition state (TS) features an oxocarbenium ion that is stabilized by th e cosubstrate phosphate dianion which itself functions as part of a ca talytic triad (Glu89-His86-PO4=). Participation of phosphate in the TS accounts for the poor hydrolytic activity of PNP and is likely to be the mechanistic feature that differentiates phosphorylases from glycos idases, The proposed PNP TS also entails a hydrogen bond between N7 an d a highly conserved Asn. Hydrogen bond donation to N7 in the TS stabi lizes the negative charge that accumulates on the purine ring during g lycosidic bond cleavage, Kinetic studies using N7-modified analogs pro vided additional support for the hydrogen bond. Crystallographic studi es of 13 human PNP-ligand complexes indicated that PNP uses a ligand-i nduced conformational change to position Asn243 and other key residues in the active site for catalysis. These studies also indicated that p urine nucleosides bind to PNP with a nonstandard glycosidic torsion an gle (+anticlinal) and an uncommon sugar pucker (C4'-endo). Single poin t energy calculations predicted the binding conformation to enhance ph osphorolysis through ligand strain. Structural data also suggested tha t purine binding precedes ribose 1-phosphate binding in the synthetic direction whereas the order of substrate binding was less clear for ph osphorolysis, Conservation of the catalytically important residues acr oss nucleoside phosphorylases with specificity for 6-oxopurine nucleos ides provided further support for the proposed catalytic mechanism.