title: Transition state analogue structures of human phosphoglycerate kinase establish the importance of charge balance in catalysis.
abstract: Transition state analogue (TSA) complexes formed by phosphoglycerate kinase (PGK) have been used to test the hypothesis that balancing of charge within the transition state dominates enzyme-catalyzed phosphoryl transfer. High-resolution structures of trifluoromagnesate (MgF(3)(-)) and tetrafluoroaluminate (AlF(4)(-)) complexes of PGK have been determined using X-ray crystallography and (19)F-based NMR methods, revealing the nature of the catalytically relevant state of this archetypal metabolic kinase. Importantly, the side chain of K219, which coordinates the alpha-phosphate group in previous ground state structures, is sequestered into coordinating the metal fluoride, thereby creating a charge environment complementary to the transferring phosphoryl group. In line with the dominance of charge balance in transition state organization, the substitution K219A induces a corresponding reduction in charge in the bound aluminum fluoride species, which changes to a trifluoroaluminate (AlF(3)(0)) complex. The AlF(3)(0) moiety retains the octahedral geometry observed within AlF(4)(-) TSA complexes, which endorses the proposal that some of the widely reported trigonal AlF(3)(0) complexes of phosphoryl transfer enzymes may have been misassigned and in reality contain MgF(3)(-).
authors.affiliation: The Krebs Institute & The Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cliff
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Bowler
authors.forename: Andrea
authors.initials: A
authors.lastname: Varga
authors.forename: James P
authors.initials: JP
authors.lastname: Marston
authors.forename: Judit
authors.initials: J
authors.lastname: Szabó
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Baxter
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
authors.forename: Mária
authors.initials: M
authors.lastname: Vas
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D017607:Aluminum Compounds / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D055592:Biophysical Phenomena; D004583:Electrons; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism; D005988:Glyceric Acids / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D017434:Protein Structure, Tertiary
chemical_list: D017607:Aluminum Compounds; D005988:Glyceric Acids; D007527:Isoenzymes; C050992:tetrafluoroaluminate; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase; D008274:Magnesium; D005459:Fluorides