Steady-State Kinetic Characterization of Kinase Activity and Requirements for Mg2+ of Interleukin-1 Receptor-Associated Kinase-4
Steady-State Kinetic Characterization of Kinase Activity and Requirements for Mg2+ of Interleukin-1 Receptor-Associated Kinase-4
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DOI：
10.1021/bi901609m
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发表时间：
2010-02-23
期刊：
影响因子：
2.9
通讯作者：
Swinney, David C.
中科院分区：
文献类型：
--
作者：
Hekmat-Nejad, Mohammad;Cai, Terrence;Swinney, David C.
白细胞介素-1受体相关激酶-4（IRAK-4）是一种丝氨酸/苏氨酸特异性蛋白激酶，在Toll样受体和白细胞介素-1（IL-1）受体介导的细胞内信号级联中起关键作用。尽管越来越多的信息油IRAK-4的生理功能，它的激酶活性仍然很少研究。本研究需要表征通过与MgATP孵育预活化的全长重组人IRAK-4的稳态动力学特性和Mg 2+需求。在20 mM Mg 2+存在下，本文的活化的IRAK-4被证明磷酸化源自IRAK-1的活化环的肽底物（IRAK-1肽），对于ATP和肽，k（cat）为30 +/-2.9s（-1），K-m值分别为668 +/-120和852 +/-273 μ M。双底物，死端和产品的抑制数据，使用类似物的ATP，是一致的顺序有序的动力学机制与ATP结合的酶之前的肽和顺序随机机制。对磷酸转移活性或IRAK-4的Mg 2+需求的研究表明，不止一种Mg 2+离子与酶相互作用，并且酶在5-10 mM游离Mg 2+存在下具有最大活性。而一种二价金属作为与ATP的螯合物的一部分，对于催化是必需的，动力学证据表明，未络合的Mg 2+通过使k（cat）增加约3倍和使K-降低约6倍而进一步增强IRAK-4的催化活性。m的ATP，并使核苷酸和肽底物结合位点之间的相互作用的拮抗性较低。
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 oil the physiological functions of IRAK-4, its kinase activity remains poorly studied. The present study entails characterization of the steady-state kinetic properties and Mg2+ requirements of full-length, recombinant human IRAK-4 preactivated by incubation with MgATP. In the presence of 20 mM Mg2+, 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 mu M 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 Mg2+ requirements for phosphoryl transfer activity or IRAK-4 revealed that more than one Mg2+ ion interacts with the enzyme and that the enzyme is maximally active in the presence of 5-10 mM free Mg2+ While one divalent metal, as part of a chelate complex with ATP, is essential for catalysis, kinetic evidence is provided to show that uncomplexed Mg2+ further enhances the catalytic activity of IRAK-4 by bringing about an similar to 3-fold increase in k(cat) and an similar to 6-fold reduction in the K-m for ATP and by rendering the interaction between the nucleotide and peptide substrate binding sites less antagonistic.