Shibagaki Y, Gotoh H, Kato M, Mizumoto K
School of Pharmaceutical Sciences, Kitasato University, Tokyo.
J Biochem. 1995 Dec;118(6):1303-9. doi: 10.1093/oxfordjournals.jbchem.a125023.
The yeast mRNA capping enzyme is composed of 52 (alpha) and 80 kDa (beta) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the alpha subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the alpha subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]N epsilon-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the alpha subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.
酵母mRNA加帽酶由52 kDa（α）和80 kDa（β）多肽组成，它们分别负责mRNA鸟苷酸转移酶和RNA 5'-三磷酸酶活性。我们分离了编码α亚基的基因（CEG1），并表明CEG1对酵母细胞生长至关重要[Shibagaki等人，（1992年）《生物化学杂志》267，9521 - 9528]。在本研究中，CEG1在大肠杆菌中表达，α亚基蛋白被纯化至接近均一。源自重组酶 - [³²P]GMP共价反应中间体的[³²P]GMP结合胰蛋白酶肽通过高碘酸盐氧化随后β - 消除转化为[³²P]磷酰肽。用碱水解[³²P]磷酰肽产生[³²P]Nε - 磷酰赖氨酸作为唯一的磷酸氨基酸，表明酶 - GMP复合物中的GMP通过磷酰胺键与赖氨酸残基结合。[³²P]GMP - 肽的微量测序表明GMP结合位点位于氨基酸60至75之间的区域，该区域在位置70处含有一个内部抗胰蛋白酶的赖氨酸。对CEG1进行定点诱变，突变蛋白在大肠杆菌中表达。用组氨酸或异亮氨酸替代赖氨酸70完全消除了酶 - GMP形成活性，并且这种突变在体内对酵母是致死的，支持α亚基中的活性位点位于赖氨酸70的观点。用精氨酸替代赖氨酸70降低了形成酶 - GMP复合物的能力；然而，携带该等位基因的酵母细胞无法存活。还在活性位点（赖氨酸70 - 苏氨酸 - 天冬氨酸 - 甘氨酸基序）附近引入了一系列突变，包括8个氨基酸替换和3个插入，并检测了突变多肽的体外催化活性以及体内酵母细胞活力。除了用谷氨酸替代天冬氨酸72的情况外，突变蛋白的体外和体内功能之间存在良好的相关性，用谷氨酸替代天冬氨酸72允许形成酶 - GMP复合物但不能支持细胞生长。赖氨酸70到精氨酸和天冬氨酸72到谷氨酸替代的结果表明，这些突变蛋白中鸟苷酸转移到RNA和/或帽形成本身之外的其他作用受损。
