title: Remote mutations alter transition-state structure of human purine nucleoside phosphorylase.
abstract: Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine (2'-deoxy)ribonucleosides to give the corresponding purine base and (2'-deoxy)ribose 1-phosphate as products. Human and bovine PNPs (HsPNP and BtPNP) form distinct transition states despite 87% identity in amino acid sequence. A PNP hybrid was produced by replacing K22 and H104 in HsPNP with the corresponding Glu and Arg residues found in BtPNP. We solved the transition-state structure of E:R-HsPNP (K22E:H104R mutant of HsPNP) using competitive kinetic isotope effects (KIE) and global density functional calculations. An array of PNP transition states was generated from optimized structure candidates with varied C1'-N9, C1'-Ophosphate distances, ribosyl pucker configurations and N7-protonation states. Isotopically labeled [1'-3H], [2'-3H], [1'-14C], [9-15N], [1'-14C, 9-15N] and [5'-3H2]inosines gave intrinsic KIE values of 1.210, 1.075, 1.035, 1.024, 1.065, 1.063 with E:R-HsPNP, respectively. The suite of E:R-HsPNP KIEs match a single structure from the array of PNP transition-state candidates. The transition state of E:R-HsPNP is fully dissociative, N7-protonated hypoxanthine (C1'-N9 distance >or= 3.0 A) with partial participation of phosphate (C1'-Ophosphate distance = 2.26 A), 2'-C-exo-ribosyl ring pucker and the O5'-C5'-C4'-O4' dihedral angle near 60 degrees . The transition state of E:R-HsPNP is altered from the fully dissociative DN*AN character for HsPNP to a late phosphate-associative character. E:R-HsPNP differs from native HsPNP by only two residues over 25 A away from the active site. New interactions caused by the mutations increase the catalytic efficiency of the enzyme for formation of a late transition state with increased participation of the phosphate nucleophile. Dynamic coupling motions from the remote mutations to the catalytic sites are proposed.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Minkui
authors.initials: M
authors.lastname: Luo
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D002384:Catalysis; D004789:Enzyme Activation; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D017433:Protein Structure, Secondary; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D044366:Transition Temperature
chemical_list: D011994:Recombinant Proteins; D011683:Purine-Nucleoside Phosphorylase