title: A Trojan horse transition state analogue generated by MgF3- formation in an enzyme active site.
abstract: Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. beta-Phosphoglucomutase catalyses the isomerization of beta-glucose-1-phosphate to beta-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using (19)F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF(3)(-) mimics the transferring PO(3)(-) moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of beta-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Baxter
authors.forename: Luis F
authors.initials: LF
authors.lastname: Olguin
authors.forename: Marko
authors.initials: M
authors.lastname: Golicnik
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Feng
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Bermel
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
authors.forename: Nicholas H
authors.initials: NH
authors.lastname: Williams
mesh_terms: D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000737:chemistry; D005958:Glucosephosphates / Q000737:chemistry; D013294:Lactococcus lactis / Q000201:enzymology*; D017616:Magnesium Compounds / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010733:Phosphoglucomutase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000577:Amides; D005958:Glucosephosphates; D017616:Magnesium Compounds; D019298:Glucose-6-Phosphate; C031288:magnesium fluoride; C031590:glucose-1-phosphate; D010733:Phosphoglucomutase; D005459:Fluorides