Messenger RNA guanlyltransferase from Saccharomyces cerevisiae. II. Catalytic properties.
Highly purified mRNA-capping enzyme from Saccharomyces cerevisiae catalyzes (a) removal of the gamma-phosphoryl group from the 5'-end of the newly formed mRNA and (b) guanylylation of the resulting diphosphoryl end. Characteristics of the two reactions catalyzed by this enzyme are studied. Guanylyltransferase is most active at pH 7.0 in the presence of 3 mM Mg2+, and utilizes GTP as a guanylyl donor with an apparent Km of 5 microM, and ppGCC (A2, U2, G)n as a guanylyl acceptor with two Km values of 0.5 and 4 microM. It catalyzes GTP-PPi exchange in the absence of the acceptor RNA, and forms a covalent enzyme-GMP intermediate having Mr = 45,000 in sodium dodecyl sulfate gel electrophoresis. RNAs with 5'-diphosphoryl as well as 5'-triphosphoryl ends are capped, while mononucleotides such as GDP and ppGp are inert. Since guanylyltransferase can utilize ppGpC and ppGpCpC as acceptors, the presence of at least one phosphodiester bond seems to be sufficient for the acceptor activity. However, oligonucleotides of longer chain length are preferred. RNA 5'-triphosphatase associated with the purified enzyme requires Mg2+ and exhibits a broad pH optimum from 6.5 to 8.5, and an apparent Km value for pppA-terminated poly(A) is 1.4 microM. The enzyme is specific for the gamma-phosphoryl group at the 5'-terminus of RNA and does not hydrolyze ATP. It can hydrolyze the gamma-phosphoryl group of pppGp, but the RNA substrates with longer chain length are preferred.
D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009843:Oligoribonucleotides / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D009843:Oligoribonucleotides; D008345:Manganese; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; C035410:GTP-RNA guanylyltransferase; D012316:RNA Nucleotidyltransferases; D008274:Magnesium