Transient kinetic investigation of GTP hydrolysis catalyzed by interferon-γ-induced hGBP1 (human guanylate binding protein 1)
Transient kinetic investigation of GTP hydrolysis catalyzed by interferon-γ-induced hGBP1 (human guanylate binding protein 1)
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DOI：
10.1074/jbc.m604911200
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发表时间：
2006-09-29
影响因子：
4.8
通讯作者：
Herrmann, Christian
中科院分区：
文献类型：
--
作者：
Kunzelmann, Simone;Praefcke, Gerrit J. K.;Herrmann, Christian
在大GTP结合蛋白家族中，人鸟苷酸结合蛋白1（hGBP1）属于干扰素诱导蛋白的亚组。这些蛋白质的GTP水解活性比其他GTP酶家族的成员高得多，其机制尚不清楚。大的GTP结合蛋白形成自组装，导致催化活性的刺激。由hGBP1催化的GTP水解的独特结果是GDP和GMP。我们使用放射性标记的GTP以及荧光探针通过瞬态动力学方法研究了该反应机制。底物结合和hGBP1同二聚体的形成是快速的，因为在GTP水解的时间过程中没有观察到滞后期。相反，多个营业额实验表明，Pi形成的快速爆发之前的稳态阶段，表明GTP裂解后的限速步骤。这两种分子都具有催化活性，并在第一步中裂解出磷酸根离子。然后分叉成催化失活，可能是由二聚体的不可逆解离，并观察到GDP水解。第二个裂解步骤甚至比第一个步骤更快，这意味着hGBP1催化中心内的核苷酸快速重排。我们还可以证明，包括磷酸根离子在内的产物的释放是快速的，并且不限制稳态活性。我们认为GMP结合的同二聚体的缓慢解离引起爆发行为并控制稳态。hGBP1的GDP和GMP结合状态的组装形式只能通过GTP结合和水解来获得，并实现几秒钟的寿命。
Within the family of large GTP-binding proteins, human guanylate binding protein 1 (hGBP1) belongs to a subgroup of interferon-inducible proteins. GTP hydrolysis activity of these proteins is much higher compared with members of other GTPase families and underlies mechanisms that are not understood. The large GTP-binding proteins form self-assemblies that lead to stimulation of the catalytic activity. The unique result of GTP hydrolysis catalyzed by hGBP1 is GDP and GMP. We investigated this reaction mechanism by transient kinetic methods using radioactively labeled GTP as well as fluorescent probes. Substrate binding and formation of the hGBP1 homodimer are fast as no lag phase is observed in the time courses of GTP hydrolysis. Instead, multiple turnover experiments show a rapid burst of Pi formation prior to the steady state phase, indicating a rate-limiting step after GTP cleavage. Both molecules are catalytically active and cleave off a phosphate ion in the first step. Then bifurcation into catalytic inactivation, probably by irreversible dissociation of the dimer, and into GDP hydrolysis is observed. The second cleavage step is even faster than the first step, implying a rapid rearrangement of the nucleotide within the catalytic center of hGBP1. We could also show that the release of the products, including the phosphate ions, is fast and not limiting the steady state activity. We suggest that slow dissociation of the GMP-bound homodimer gives rise to the burst behavior and controls the steady state. The assembled forms of the GDP- and GMP-bound states of hGBP1 are accessible only through GTP binding and hydrolysis and achieve a lifetime of a few seconds.