title: Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations.
abstract: Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. hdeng@aecom.yu.edu
authors.forename: Hua
authors.initials: H
authors.lastname: Deng
authors.forename: Sean M
authors.initials: SM
authors.lastname: Cahill
authors.forename: José-Luis
authors.initials: JL
authors.lastname: Abad
authors.forename: Andrzej
authors.initials: A
authors.lastname: Lewandowicz
authors.forename: Robert H
authors.initials: RH
authors.lastname: Callender
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
authors.forename: Roger A
authors.initials: RA
authors.lastname: Jones
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002417:Cattle; D019295:Computational Biology; D006801:Humans; D006860:Hydrogen Bonding; D019271:Hypoxanthine / Q000737:chemistry* / Q000378:metabolism; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions / Q000737:chemistry
chemical_list: D009587:Nitrogen Isotopes; D012996:Solutions; D019271:Hypoxanthine; D011683:Purine-Nucleoside Phosphorylase