Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate

Ararat J. Ablooglu, Jeffrey H. Till, Kyonghee Kim, Keykavous Parang, Philip A. Cole, Stevan R. Hubbard, Ronald A. Kohanski

Research output: Contribution to journalArticlepeer-review

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

Original languageEnglish (US)
Pages (from-to)30394-30398
Number of pages5
JournalJournal of Biological Chemistry
Volume275
Issue number39
DOIs
StatePublished - Sep 29 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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