Localization and in vitro mutagenesis of the active site in the Saccharomyces cerevisiae mRNA capping enzyme.
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.
Shibagaki
Y
Y
School of Pharmaceutical Sciences, Kitasato University, Tokyo.
Gotoh
H
H
Kato
M
M
Mizumoto
K
K
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D005800:Genes, Fungal; D006157:Guanosine Monophosphate / Q000378:metabolism; D006639:Histidine; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D000098556:mRNA Guanylyltransferases
D046911:Macromolecular Substances; D010748:Phosphopeptides; D011994:Recombinant Proteins; C025308:N-epsilon phospholysine; D006639:Histidine; D006157:Guanosine Monophosphate; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D008239:Lysine