Reaction mechanism of mRNA guanylyltransferase from rat liver: isolation and characterization of a guanylyl-enzyme intermediate.

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Abstract
Rat liver RNA guanylyltransferase catalyzes a GTP-PPi exchange reaction in the absence of acceptor RNA suggesting that the reaction proceeds through the formation of a covalent guanylylated intermediate. More direct evidence for the existence of the enzyme-GMP intermediate is presented: the enzyme-[32P]GMP intermediate was formed on incubation of rat liver guanylyltransferase with [.alpha.-32P]GTP and migrated as a single radioactive band with MW 69,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the intermediate isolated on gel filtration can transfer its GMP moiety to ppGpCpC-poly(A2, U2, G) to form the capped RNA molecule or it can react with PPi to regenerate GTP. The formation of the intermediate was dependent on Mg2+ and was strongly inhibited by PPi. The addition of pyrophosphatase markedly increased the amount of the intermediate complex. On blue dextran-Sepharose affinity column chromatography, the activity of guanylyltransferase to form an enzyme-[32P]GMP intermediate comigrated with activities of cap formation and GTP-PPi exchange. A phosphoamide type linkage between GMP and enzyme is suggested by its acid-labile and alkali-stable nature and also by the susceptibility to acidic hydroxylamine. The reaction catalyzed by rat liver guanylyltransferase probably occurs through the following 2 partial steps: E + GTP .dblarw. E-pG + PPi; and E-pG + ppN .cntdot..cntdot..cntdot..cntdot..cntdot. .fwdarw. GpppN .cntdot..cntdot..cntdot..cntdot..cntdot. + E.