title: α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction.
abstract: β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-D-glucose 1-phosphate (βG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-D-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-D-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom;
authors.forename: Yi
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Department of Chemistry, College of Pharmacy, Dalhousie University, Halifax, NS, Canada B3H 4R2;
authors.forename: Debabrata
authors.initials: D
authors.lastname: Bhattasali
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom; Structural Biology Group, European Synchrotron Radiation Facility, 38042 Grenoble, Cedex 9, France; European Molecular Biology Laboratory, Grenoble Outstation, 38042 Grenoble, Cedex 9, France;
authors.forename: Erika
authors.initials: E
authors.lastname: Pellegrini
authors.affiliation: Department of Chemistry, College of Pharmacy, Dalhousie University, Halifax, NS, Canada B3H 4R2;
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Forget
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom;
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Baxter
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom; Manchester Institute of Biotechnology, Manchester M1 7DN, United Kingdom; and.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cliff
authors.affiliation: Structural Biology Group, European Synchrotron Radiation Facility, 38042 Grenoble, Cedex 9, France; European Molecular Biology Laboratory, Grenoble Outstation, 38042 Grenoble, Cedex 9, France; Unit of Virus Host Cell Interactions, University of Grenoble Alpes-European Molecular Biology Laboratory-Centre National de la Recherche Scientifique, 38042 Grenoble, Cedex 9, France.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Bowler
authors.affiliation: Department of Chemistry, College of Pharmacy, Dalhousie University, Halifax, NS, Canada B3H 4R2; david.jakeman@dal.ca g.m.blackburn@sheffield.ac.uk j.waltho@sheffield.ac.uk.
authors.forename: David L
authors.initials: DL
authors.lastname: Jakeman
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom; david.jakeman@dal.ca g.m.blackburn@sheffield.ac.uk j.waltho@sheffield.ac.uk.
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom; Manchester Institute of Biotechnology, Manchester M1 7DN, United Kingdom; and david.jakeman@dal.ca g.m.blackburn@sheffield.ac.uk j.waltho@sheffield.ac.uk.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D005461:Fluorine / Q000737:chemistry; D019298:Glucose-6-Phosphate / Q000737:chemistry / Q000378:metabolism; D005958:Glucosephosphates / Q000737:chemistry / Q000378:metabolism; D006601:Hexoses / Q000737:chemistry* / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D063065:Organophosphonates / Q000737:chemistry* / Q000378:metabolism*; D010733:Phosphoglucomutase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D005958:Glucosephosphates; D006601:Hexoses; D063065:Organophosphonates; D011994:Recombinant Proteins; D005461:Fluorine; D019298:Glucose-6-Phosphate; C031590:glucose-1-phosphate; D010733:Phosphoglucomutase; D008274:Magnesium
keywords: 19F NMR; X-ray crystallography; phosphonate analogs; phosphoryl transfer mechanism; water-mediated substrate recognition
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