Identification of the vaccinia virus mRNA guanyltransferase active site lysine.
The vaccinia virus mRNA capping enzyme is a heterodimeric protein containing subunits of 97 and 33 kDa, the products of genes D1R and D12L, respectively. The enzyme catalyzes the first three reactions in the mRNA cap formation pathway: mRNA triphosphatase, guanyltransferase and (guanine-7-)methyltransferase. The guanyltransferase reaction proceeds by way of a covalent enzyme GMP (E-GMP) intermediate (Shuman, S. and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 187-191) in which the GMP is linked to the large subunit through a lysine residue (Toyama, R., Mizumoto, K., Nakahara, Y., Tatsuno, T., and Kaziro, Y. (1983) Eur. J. Biochem. 2, 2195-2201; Roth, M. J., and Hurwitz, J. (1984) J. Biol Chem. 259, 13488-13494). In order to identify the map position of the guanyltransferase active site lysine residue, high specific activity [32P]E-GMP was prepared. Digestion of the E-GMP with hydroxylamine at pH 9.5 yielded a 31-kDa radioactive fragment derived from amino acids 1-273. Cleavage of E-GMP with cyanogen bromide produced a radioactive peptide of 14 kDa corresponding to amino acids 242-365. Lysine residues are found at positions 244 and 260. Staphylococcus aureus V8 protease digestion of cyanogen bromide-cleaved E-GMP yields a radioactive product of about 5 kDa in molecular mass corresponding to the peptide generated by cleavage at glutamic acid residues 253 and 297, demonstrating that lysine 260 is the site of linkage of GMP.
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Department of Biochemistry, State University of New York, School of Medicine and Biomedical Sciences, Buffalo 14214.
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D000595:Amino Acid Sequence; D001665:Binding Sites; D006157:Guanosine Monophosphate / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000032:analysis*; D008780:Methyltransferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D009713:Nucleotidyltransferases / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology*; D014764:Viral Proteins
D009097:Multienzyme Complexes; D014764:Viral Proteins; D006157:Guanosine Monophosphate; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008239:Lysine