title: Prioritization of charge over geometry in transition state analogues of a dual specificity protein kinase.
abstract: The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using (19)F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF(4)(-)), ADP, and p38α MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF(4)(-) moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF(4)(-) complex involving the alternative acceptor residue in p38α (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF(4)(-) species for both acceptor residues, rather than the trigonal bipyramidal AlF(3)(0) previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.
authors.affiliation: Krebs Institute and Department of Molecular Biology & Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Liu
authors.initials: L
authors.lastname: Xiaoxia
authors.forename: James P
authors.initials: JP
authors.lastname: Marston
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Baxter
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Hounslow
authors.forename: Zhao
authors.initials: Z
authors.lastname: Yufen
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cliff
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
mesh_terms: D017607:Aluminum Compounds / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017607:Aluminum Compounds; D047428:Protein Kinase Inhibitors; C050992:tetrafluoroaluminate; D011494:Protein Kinases; D005459:Fluorides