Steady-state kinetic characterization of kinase activity and requirements for Mg2+ of interleukin-1 receptor-associated kinase-4

Biochemistry
Mohammad Hekmat-NejadDavid C Swinney

Abstract

Interleukin-1 receptor-associated kinase-4 (IRAK-4) is a Ser/Thr-specific protein kinase that plays a critical role in intracellular signaling cascades mediated by Toll-like and interleukin-1 (IL-1) receptors. Despite a growing body of information on the physiological functions of IRAK-4, its kinase activity remains poorly studied. The present study entails characterization of the steady-state kinetic properties and Mg(2+) requirements of full-length, recombinant human IRAK-4 preactivated by incubation with MgATP. In the presence of 20 mM Mg(2+), activated IRAK-4 herein is demonstrated to phosphorylate a peptide substrate (IRAK-1 peptide), derived from the activation loop of IRAK-1, with a k(cat) of 30 +/- 2.9 s(-1) and K(m) values of 668 +/- 120 and 852 +/- 273 microM for ATP and the peptide, respectively. Two-substrate, dead-end and product inhibition data, using analogues of ATP, are consistent with both a sequential ordered kinetic mechanism with ATP binding to the enzyme prior to the peptide and a sequential random mechanism. Investigation of the Mg(2+) requirements for phosphoryl transfer activity of IRAK-4 revealed that more than one Mg(2+) ion interacts with the enzyme and that the enzyme is maximally active in the pres...Continue Reading

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Citations

Jun 4, 2011·Journal of Enzyme Inhibition and Medicinal Chemistry·Marielle BrockhoffPatrick Chène
Jul 19, 2014·Scientific Reports·Vijayakumar Gosu, Sangdun Choi
Jan 28, 2014·The Plant Journal : for Cell and Molecular Biology·Daniel BojarMichael Hothorn
Feb 11, 2015·Pharmacological Research : the Official Journal of the Italian Pharmacological Society·Robert Roskoski

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