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Purine nucleoside phosphorylase. 2. Catalytic mechanism
M D Erion1, J D Stoeckler, W C Guida
1Central Research Laboratory, Ciba-Geigy Ltd., Basel, Switzerland. mark.erion.gensia.com
Biochemistry
|October 8, 1997
Summary
Purine nucleoside phosphorylase (PNP) uses a substrate-assisted mechanism involving a phosphate ion and a conserved asparagine residue to catalyze phosphorolysis. This mechanism explains PNP
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Purine nucleoside phosphorylase (PNP) is a key enzyme in purine metabolism.
- Understanding its catalytic mechanism is crucial for drug development and metabolic studies.
Purpose of the Study:
- To elucidate the detailed catalytic mechanism of purine nucleoside phosphorylase (PNP).
- To investigate the role of substrate-assisted catalysis and active site residues in PNP function.
Main Methods:
- X-ray crystallography of 13 human PNP-ligand complexes.
- Molecular modeling and computational studies.
- Site-directed mutagenesis and kinetic analyses.
Main Results:
- A substrate-assisted catalytic mechanism involving a phosphate ion and a conserved asparagine (Asn) residue was proposed.
- The transition state is stabilized by a catalytic triad (Glu89-His86-PO4=) and a hydrogen bond to N7 of the purine.
- Ligand-induced conformational changes position key residues, and nonstandard binding angles contribute to catalytic efficiency.
Conclusions:
- PNP employs a unique mechanism differentiating it from glycosidases.
- The findings provide a structural basis for PNP inhibition and drug design.
- Conserved residues across related enzymes support the proposed catalytic mechanism.