Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate.
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.
Ablooglu
A J
AJ
Mount Sinai School of Medicine, Department of Biochemistry and Molecular Biology, New York, New York 10029, USA.
Till
J H
JH
Kim
K
K
Parang
K
K
Cole
P A
PA
Hubbard
S R
SR
Kohanski
R A
RA
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
D010455:Peptides; C090828:2,3,5,6-tetrafluorotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin