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Guanosine transfer is the second step of RNA capping. The enzyme (GTase) first displaces a pyrophosphate of a GTP molecule, forming an unstable covalent enzyme-GMP intermediate (E + Gppp ⇌ E-pG + PPi). Then the GMP moiety is transferred onto the diphosphate end of RNA (E-pG +ppRNA ⇌ GpppRNA + E) forming a triphosphate bridge. It’s obvious that this reaction is highly reversible, because the GMP moiety is transferred from a diphosphate to another diphosphate. As a result, the two intermediate reactions should be reversible as well. This is indeed the case according to this paper. enter image description here Recently a paper revealed that coronaviruses use a distinct reaction to make 5’ caps. The viral enzyme nsp9 reacts with the triphosphorylated RNA, forming an enzyme-pRNA intermediate (nsp9 + pppRNA ⇌ nsp9-pRNA + PPi). Then the pRNA is transferred onto a GDP molecule (nsp9-pRNA + Gpp ⇌ nsp9 + GpppRNA). This reaction is shared with nonsegmented negative sensed RNA viruses, and is likewise reversible (the L–pRNA intermediate is able to transfer pRNA to PPi to produce pppRNA, although less efficiently than to GDP to form GpppRNA). However, the paper claimed that “Interestingly, although inorganic pyrophosphate (PPi) was able to deAMPylate nsp9-AMP, it was unable to deRNAylate nsp9-pRNA”, “RNAylated nsp9 was not reversible in the presence of PPi”, which indicates that the first reaction is not reversible. This creates a problem. If the first reaction is irreversible, the covalent intermediate should be very stable and the reaction should be strongly thermodynamically favorable (large ΔG gain), which will make the second reaction strongly unfavorable. Although coronavirus nsp9 is different from the L proteins of negative sensed RNA viruses, the reaction thermodynamics should be irrespective of catalysts. enter image description here

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