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 bond. Crystallographic studies of 13 human PNP-ligand complexes indicated that PNP uses a ligand-induced conformational change to position Asn243 and other key residues in the active site for catalysis. These studies also indicated that purine nucleosides bind to PNP with a nonstandard glycosidic torsion angle (+anticlinal) and an uncommon sugar pucker (C4'-endo). Single point energy calculations predicted the binding conformation to enhance phosphorolysis through ligand strain. Structural data also suggested that purine binding precedes ribose 1-phosphate binding in the synthetic direction whereas the order of substrate binding was less clear for phosphorolysis. Conservation of the catalytically important residues across nucleoside phosphorylases with specificity for 6-oxopurine nucleosides provided further support for the proposed catalytic mechanism.
Erion
M D
MD
Central Research Laboratory, Ciba-Geigy Ltd., Basel, Switzerland. mark.erion.gensia.com
Stoeckler
J D
JD
Guida
W C
WC
Walter
R L
RL
Ealick
S E
SE
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006801:Humans; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics / Q000378:metabolism*; D011688:Purinones / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D008024:Ligands; D011688:Purinones; D011683:Purine-Nucleoside Phosphorylase