Steady-state kinetic characterization of kinase activity and requirements for Mg2+ of interleukin-1 receptor-associated kinase-4.
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 presence of 5-10 mM free Mg(2+). While one divalent metal, as part of a chelate complex with ATP, is essential for catalysis, kinetic evidence is provided to show that uncomplexed Mg(2+) further enhances the catalytic activity of IRAK-4 by bringing about an approximately 3-fold increase in k(cat) and an approximately 6-fold reduction in the K(m) for ATP and by rendering the interaction between the nucleotide and peptide substrate binding sites less antagonistic.
Hekmat-Nejad
Mohammad
M
Department of Virology, Roche Palo Alto, LLC, 3431 Hillview Avenue, Palo Alto, California 94304, USA. mohammad.hekmat-nejad@roche.com
Cai
Terrence
T
Swinney
David C
DC
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D018411:Spodoptera / Q000235:genetics / Q000821:virology; D013379:Substrate Specificity / Q000235:genetics
D010455:Peptides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D000251:Adenosine Triphosphatases; D008274:Magnesium