Kinetic analysis of the interaction of guanine nucleotides with eukaryotic translation initiation factor eIF5B.
Kinetic analysis of the interaction of guanine nucleotides with eukaryotic translation initiation factor eIF5B.
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鸟嘌呤核苷酸与真核翻译起始因子 eIF5B 相互作用的动力学分析。
DOI：
10.1021/bi062134g
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Pestova,TatyanaV
中科院分区：
文献类型：
--
作者：
Pisareva,VeraP;Hellen,ChristopherUT;Pestova,TatyanaV
真核细胞翻译起始因子eIF 5 B是一种核糖体依赖性的GT3，它负责起始的最后一步，这涉及起始因子从起始复合物中的40 S核糖体亚基的置换及其与60 S亚基的连接。eIF 5 B结合GTP的水解对于其在亚基连接中的功能不是必需的，但对于随后从组装的80 S核糖体释放eIF 5 B是必需的。在这里，我们研究了鸟嘌呤核苷酸结合eIF 5 B的动力学，通过荧光停流技术，使用GTP和GDP的荧光衍生物和GTP类似物GTPγS和GMPPNP。eIF 5 B对mant-GTP的亲和力（Kd = 14−18 μM）比对mant-GDP的亲和力（Kd = 2.3 μM）低约7倍，两种鸟嘌呤核苷酸都能迅速从eIF 5 B上解离（k-1 mant-GTP = 22−28 s-1，k-1 mant-GDP = 10−14 s-1）。eIF 5 B的这些性质表明eIF 5 B上的快速自发GTP/GDP交换，因此与其不需要特殊的鸟嘌呤核苷酸交换因子一致。eIF 5 B对mant-GTPγS的亲和力比mant-GTP低约2倍（Kd = 6.9 μM），对mant-GMPPNP的亲和力比mant-GTP高1.5倍（Kd = 25.7 μM），表明eIF 5 B很好地耐受三磷酸部分的修饰。
Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that is responsible for the final step in initiation, which involves the displacement of initiation factors from the 40S ribosomal subunit in initiation complexes and its joining with the 60S subunit. Hydrolysis of eIF5B-bound GTP is not required for its function in subunit joining but is necessary for the subsequent release of eIF5B from assembled 80S ribosomes. Here we investigated the kinetics of guanine nucleotide binding to eIF5B by a fluorescent stopped-flow technique using fluorescent mant derivatives of GTP and GDP and of the GTP analogues GTPγS and GMPPNP. The affinity of eIF5B for mant-GTP (Kd∼ 14−18 μM) was approximately 7-fold less than for mant-GDP (Kd∼ 2.3 μM), and both guanine nucleotides dissociated rapidly from eIF5B (k-1mant-GTP∼ 22−28 s-1,k-1mant-GDP∼ 10−14 s-1). These properties of eIF5B suggest a rapid spontaneous GTP/GDP exchange on eIF5B and are therefore consistent with it having no requirement for a special guanine nucleotide exchange factor. The affinity of eIF5B for mant-GTPγS was about 2 times lower (Kd∼ 6.9 μM) and for mant-GMPPNP 1.5 times higher (Kd∼ 25.7 μM) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety well.