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 journalArticle

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
StatePublished - Sep 29 2000

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Insulin Receptor
Phosphotransferases
Protein-Tyrosine Kinases
Peptides
Substrates
Enzymes
Crystallography
Phosphorylation
Hydrogen Bonding
Phenol
Hydroxyl Radical
Anions
Hydrogen bonds
X-Rays
Ions
X rays
Kinetics
tetrafluorotyrosine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ablooglu, A. J., Till, J. H., Kim, K., Parang, K., Cole, P. A., Hubbard, S. R., & Kohanski, R. A. (2000). Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate. Journal of Biological Chemistry, 275(39), 30394-30398.

Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate. / Ablooglu, Ararat J.; Till, Jeffrey H.; Kim, Kyonghee; Parang, Keykavous; Cole, Philip A.; Hubbard, Stevan R.; Kohanski, Ronald A.

In: Journal of Biological Chemistry, Vol. 275, No. 39, 29.09.2000, p. 30394-30398.

Research output: Contribution to journalArticle

Ablooglu, AJ, Till, JH, Kim, K, Parang, K, Cole, PA, Hubbard, SR & Kohanski, RA 2000, 'Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate', Journal of Biological Chemistry, vol. 275, no. 39, pp. 30394-30398.
Ablooglu AJ, Till JH, Kim K, Parang K, Cole PA, Hubbard SR et al. Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate. Journal of Biological Chemistry. 2000 Sep 29;275(39):30394-30398.
Ablooglu, Ararat J. ; Till, Jeffrey H. ; Kim, Kyonghee ; Parang, Keykavous ; Cole, Philip A. ; Hubbard, Stevan R. ; Kohanski, Ronald A. / Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 39. pp. 30394-30398.
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