Purine Nucleoside Phosphorylase. 2. Catalytic Mechanism

X-ray crystallography, molecular modeling, and site-directed mutagenesis were used to delineate the catalytic mechanism of purine nucleoside phosphorylase (PNP). PNP catalyzes the reversible phosphorolysis of purine nucleosides to the corresponding purine base and ribose 1-phosphate using a substrate-assisted catalytic mechanism. The proposed transition state (TS) features an oxocarbenium ion that is stabilized by the cosubstrate phosphate dianion which itself functions as part of a catalytic 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 glycosidases. The proposed PNP TS also entails a hydrogen bond between N7 and a highly conserved Asn. Hydrogen bond donation to N7 in the TS stabilizes the negative charge that accumulates on the purine ring during glycosidic bond cleavage. Kinetic studies using N7-modified analogs provided additional support for the hydrogen...