Probing the Catalytic Mechanism of the Insulin Receptor Kinase with a Tetrafluorotyrosine-containing Peptide Substrate

• Published in: The Journal of biological chemistry Vol. 275; no. 39; pp. 30394 - 30398
• Main Authors: Ablooglu, Ararat J., Till, Jeffrey H., Kim, Kyonghee, Parang, Keykavous, Cole, Philip A., Hubbard, Stevan R., Kohanski, Ronald A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29.09.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Catalytic Domain; Crystallography, X-Ray; Electrons; Hydrogen Bonding; Kinetics; Models, Molecular; Peptides - metabolism; Receptor, Insulin - metabolism; Tyrosine - analogs & derivatives; Tyrosine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M003524200

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 p Ka 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 Asp1132 and Arg1136, consistent with elevation of the p Ka . These findings strongly support a reaction mechanism favoring a dissociative transition state.