Yeast and viral RNA 5′ triphosphatases comprise a new nucleoside triphosphatase family
Yeast and viral RNA 5′ triphosphatases comprise a new nucleoside triphosphatase family
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DOI：
10.1074/jbc.273.51.34151
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发表时间：
1998-12-18
影响因子：
4.8
通讯作者：
Shuman, S
中科院分区：
文献类型：
--
作者：
Ho, CK;Pei, Y;Shuman, S
酿酒酵母 Cet1p 催化 mRNA 加帽的第一步，即三磷酸末端 RNA 的 γ 磷酸水解形成 5' 二磷酸末端。 Cet1p 的 RNA 三磷酸酶活性是镁依赖性的，周转数为 1 s(-1)。在此我们表明，纯化的重组 Cet1p 在锰存在下具有强大的 ATP 酶活性 (K-m = 2.8 mu M; V-max = 25 s(-1))。钴：也是一种有效的辅助因子，但镁、钙、铜和锌则不是。 Cet1p 在将核糖核苷三磷酸和脱氧核苷三磷酸转化为其各自的二磷酸方面表现出广泛的特异性。 Cet1p 的锰和钴依赖性核苷三磷酸酶类似于杆状病毒 LEF-4 和痘苗病毒 D1 加帽酶的核苷三磷酸酶活性。 Cet1p、LEF-4 和 D1 共享三个共线序列基序，突变分析确定保守的谷氨酸和精氨酸：这些基序内的侧链对于 Cet1p 体外的 RNA 三磷酸酶和 ATP 酶活性以及 Cet1p 体内的功能至关重要。这些发现与痘苗加帽酶类似位置处的单一丙氨酸突变的影响一致。我们认为，由酵母和 DNA 病毒编码的金属依赖性 RNA 三磷酸酶包含一个具有共同活性位点的新型磷酸水解酶家族。
Saccharomyces cerevisiae Cet1p catalyzes the first step of mRNA capping, the hydrolysis of the gamma phosphate of triphosphate-terminated RNA to form a 5' diphosphate end. The RNA triphosphatase activity of Cet1p is magnesium-dependent and has a turnover number of 1 s(-1), Here we show that purified recombinant Cet1p possesses a robust ATPase activity (K-m = 2.8 mu M; V-max = 25 s(-1)) in the presence of manganese. Cobalt is: also an effective cofactor, but magnesium, calcium, copper, and zinc are not. Cet1p displays broad specificity in converting ribonucleoside triphosphates and deoxynucleoside triphosphates to their respective diphosphates. The manganese- and cobalt-dependent nucleoside triphosphatase of Cet1p resembles the nucleoside triphosphatase activities of the baculovirus LEF-4 and vaccinia virus D1 capping enzymes. Cet1p, LEF-4, and D1 share three collinear sequence motifs, Mutational analysis establishes that conserved glutamate and arginine: side chains within these motifs are essential for the RNA triphosphatase and ATPase activities of Cet1p in vitro and for Cet1p function in vivo. These findings are in accord with the effects of single alanine mutations at analogous positions of vaccinia capping enzyme. We suggest that the metal-dependent RNA triphosphatases encoded by yeast and DNA viruses comprise a novel family of phosphohydrolase enzymes with a common active site.