title: Neighboring group participation in the transition state of human purine nucleoside phosphorylase.
abstract: The X-ray crystal structures of human purine nucleoside phosphorylase (PNP) with bound inosine or transition-state analogues show His257 within hydrogen bonding distance of the 5'-hydroxyl. The mutants His257Phe, His257Gly, and His257Asp exhibited greatly decreased affinity for Immucillin-H (ImmH), binding this mimic of an early transition state as much as 370-fold (Km/Ki) less tightly than native PNP. In contrast, these mutants bound DADMe-ImmH, a mimic of a late transition state, nearly as well as the native enzyme. These results indicate that His257 serves an important role in the early stages of transition-state formation. Whereas mutation of His257 resulted in little variation in the PNP x DADMe-ImmH x SO4 structures, His257Phe x ImmH x PO4 showed distortion at the 5'-hydroxyl, indicating the importance of H-bonding in positioning this group during progression to the transition state. Binding isotope effect (BIE) and kinetic isotope effect (KIE) studies of the remote 5'-(3)H for the arsenolysis of inosine with native PNP revealed a BIE of 1.5% and an unexpectedly large intrinsic KIE of 4.6%. This result is interpreted as a moderate electronic distortion toward the transition state in the Michaelis complex with continued development of a similar distortion at the transition state. The mutants His257Phe, His257Gly, and His257Asp altered the 5'-(3)H intrinsic KIE to -3, -14, and 7%, respectively, while the BIEs contributed 2, 2, and -2%, respectively. These surprising results establish that forces in the Michaelis complex, reported by the BIEs, can be reversed or enhanced at the transition state.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Murkin
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Birck
authors.forename: Agnes
authors.initials: A
authors.lastname: Rinaldo-Matthis
authors.forename: Wuxian
authors.initials: W
authors.lastname: Shi
authors.forename: Erika A
authors.initials: EA
authors.lastname: Taylor
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011683:Purine-Nucleoside Phosphorylase