Mechanism of the mRNA guanylyltransferase reaction: isolation of N epsilon-phospholysine and GMP (5' leads to N epsilon) lysine from the guanylyl-enzyme intermediate.
The mRNA capping reaction catalyzed by rat liver mRNA guanylyltransferase proceeds through an enzyme-GMP intermediate in which GMP is linked to the enzyme by a phosphoamide linkage. The studies described here show that GMP is bound to the epsilon-amino group of lysine of rat liver guanylyltransferase. The enzyme-[32P]GMP intermediate was digested with pronase to a [32P]GMP-peptide which was then converted to [32P]phosphoryl-peptide through periodate oxidation followed by beta-elimination. After alkaline hydrolysis of the [32P]phosphoryl-peptide, the major radioactive product co-electrophoresed with the authentic N epsilon-phospholysine on DEAE-cellulose paper. Neither [32P]Nimid-phosphohistidine nor Nguanido-phosphoarginine was detected in the hydrolysates. Furthermore, formation of N epsilon-guanylyl-lysine linkage on the enzyme was more directly shown by isolation of [32P]GMP(5' leads to N epsilon)lysine when the steps of periodate oxidation and beta-elimination were omitted. The results indicate that the nucleophile in the guanylyltransferase to which the guanylyl residue is linked is the epsilon-amino group of a lysine residue. [32P]Phosphoryl-lysine was also isolated from the vaccinia virus capping enzyme-[32P]GMP intermediate. Guanylyltransferase from HeLa cells, wheat germ, Artemia salina and yeast also formed the enzyme-GMP complex and, from the stability of the complex, the linkage between the enzyme and GMP was suggested to be a phosphoamide.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D006150:Guanine Nucleotides; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010748:Phosphopeptides / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011485:Protein Binding; D012315:RNA Caps / Q000378:metabolism; D051381:Rats; D000098556:mRNA Guanylyltransferases
C040458:GMP (5'-N(6))lysine; D006150:Guanine Nucleotides; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D012315:RNA Caps; C025308:N-epsilon phospholysine; D006157:Guanosine Monophosphate; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D008239:Lysine