Eukaryotic mRNA capping enzyme-guanylate covalent intermediate.

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Abstract
Incubation of [human cervical carcinoma] HeLa cell mRNA guanylyltransferase (EC 2.7.7.45) with [.alpha.-32P]GTP and a divalent cation in the absence of an RNA acceptor results in the formation of a covalent enzyme-guanylate complex. The complex, after purification by phosphocellulose chromatography, can transfer its bound GMP moiety to PPi, regenerating GTP, or to the 5''-diphosphate end of poly(A), forming a cap structure G(5'')pppA(pA)n. The GMP-polypeptide has a MW of 65,000 and is stable to heating in the presence of sodium dodecyl sulfate. On the basis of the alkali-stable and acid-labile nature of the bond and its susceptibility to nucleophilic attack by hydroxylamine at low pH, the GMP-polypeptide linkage appears to be a phosphoamide bond. After digestion with trypsin, a single GMP-peptide was resolved by 2-dimensional electrophoresis and chromatography.