Active site localization in a viral mRNA capping enzyme.
Capping of reovirus mRNAs is catalyzed by a guanylyltransferase that corresponds to virion structural polypeptide lambda 2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. The nucleotide attachment site on lambda 2 was localized to a region between amino acids 213 and 269 by incubating virus particles with [alpha-32P]GTP followed by proteolytic cleavage and analysis of the resulting fragments using sequence-directed antibodies as probes. The 213-269 region contains as potential GMP acceptors a single lysine, 1 arginine, and 4 histidine residues, as deduced from the nucleotide sequence of the L2 gene encoding lambda 2. Digestion of 32P-labeled capping intermediate with alkali after oxidation and beta-elimination yielded phospholysine as the only phosphoamino acid, localizing the active site to a region in lambda 2 that includes the lysine at position 226.
Fausnaugh
J
J
Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854-5638.
Shatkin
A J
AJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D012089:Mammalian orthoreovirus 3 / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D010449:Peptide Mapping; D012087:Reoviridae / Q000201:enzymology*; D014771:Virion / Q000201:enzymology*
D010446:Peptide Fragments; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D010447:Peptide Hydrolases