title: Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate.
abstract: The interaction of a synthetic tetrafluorotyrosyl peptide substrate with the activated tyrosine kinase domain of the insulin receptor was studied by steady-state kinetics and x-ray crystallography. The pH-rate profiles indicate that the neutral phenol, rather than the chemically more reactive phenoxide ion, is required for enzyme-catalyzed phosphorylation. The pK(a) of the tetrafluorotyrosyl hydroxyl is elevated 2 pH units on the enzyme compared with solution, whereas the phenoxide anion species behaves as a weak competitive inhibitor of the tyrosine kinase. A structure of the binary enzyme-substrate complex shows the tetrafluorotyrosyl OH group at hydrogen bonding distances from the side chains of Asp(1132) and Arg(1136), consistent with elevation of the pK(a). These findings strongly support a reaction mechanism favoring a dissociative transition state.
authors.affiliation: Mount Sinai School of Medicine, Department of Biochemistry and Molecular Biology, New York, New York 10029, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Ablooglu
authors.forename: J H
authors.initials: JH
authors.lastname: Till
authors.forename: K
authors.initials: K
authors.lastname: Kim
authors.forename: K
authors.initials: K
authors.lastname: Parang
authors.forename: P A
authors.initials: PA
authors.lastname: Cole
authors.forename: S R
authors.initials: SR
authors.lastname: Hubbard
authors.forename: R A
authors.initials: RA
authors.lastname: Kohanski
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004583:Electrons; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D010455:Peptides; C090828:2,3,5,6-tetrafluorotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin