Ho C K, Pei Y, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
J Biol Chem. 1998 Dec 18;273(51):34151-6. doi: 10.1074/jbc.273.51.34151.
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 (Km = 2.8 microM; Vmax = 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.
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三磷酸酶活性依赖于镁，周转数为1 s-1。在此我们表明，纯化的重组Cet1p在锰存在的情况下具有强大的ATP酶活性（Km = 2.8 microM；Vmax = 25 s-1）。钴也是一种有效的辅因子，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各自的二磷酸时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。突变分析表明，这些基序内保守的谷氨酸和精氨酸侧链对于Cet1p在体外的RNA三磷酸酶和ATP酶活性以及在体内的Cet1p功能至关重要。这些发现与痘苗加帽酶类似位置的单个丙氨酸突变的影响一致。我们认为，酵母和DNA病毒编码的金属依赖性RNA三磷酸酶构成了一个具有共同活性位点的新型磷酸水解酶家族。
