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In Molecular Cell Biology (8th edition) there's a fragment in chapter 5.2 that says:

The energetics of the polymerization reaction strongly favor the addition of ribonucleotides to the growing RNA chain because the high-energy bond between the α and β phosphates of rNTP monomers is replaced by the lower-energy phosphodiester bond between nucleotides. The equilibrium for the reaction is driven further toward chain elongation by pyrophosphatase, an enzyme that catalyzes cleavage of the released PPi into two molecules of inorganic phosphate.

How does the pyrophosphatase make the reaction more favorable? Does it make "harder" for triphosphates to form back again (now you have to attach phosphates one by one, not two at once)? If so, isn't one additional phosphate just as bad as two? To remove either you have to break one bond (between the first phosphate and the second), and you have a specialized enzyme to remove two of them (the RNA polymerase). Isn't forming a diphosphate (which needs to form in order to later make triphosphate) actually worse? Or maybe it somehow "reuses" energy stored in the bond between the two phosphates left?

Sorry if I didn't use the proper vocabulary, I'm new to biology.

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  • $\begingroup$ No problem being new to biochemistry and molecular biology. You might find it helpful to understand Sethi's answer if you read this section from Berg et al. Biochemistry which explains the thermodynamic concept of Gibbs Free Energy which is often applied to biochemical reactions. There is a worked calculation on the pyrophosphatase topic in relation to a different reaction in the same book. $\endgroup$
    – David
    Commented Jul 7, 2020 at 10:22

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This question can be answered by considerations of chemical equilibrium. For any chemical reaction:

$$\ce{A + B <=> C + D}$$

It is a property of nature that removal of product ($C$ or $D$) from the reaction mixture drives the reaction in the forward direction. This is an example of le Chatelier's principle and can be explained as follows:

In a reaction mixture containing $A$, $B$, $C$ and $D$, the rate of forward reaction ($A + B \rightarrow C + D$) depends on the frequency of collision of $A$ and $B$ molecules. Similarly, the rate of backward reaction ($C + D \rightarrow A + B$) depends on the rate of collision of $C$ and $D$ molecules. The collision frequencies, in turn, increase with concentration of the relevant species. An abrupt reduction in the concentration of $C$ hinders the backward reaction while leaving the forward reaction unchanged. Thus the forward reaction begins to dominate.

The reaction catalysed by RNA polymerase is:

$$\ce{RNA + NTP <=> Longer \ RNA + PP_i}$$

By removing $\text{PP}_i$ from the reaction mixture, pyrophosphatase does exactly that and drives the reaction in favour of RNA polymerisation. Note that hydrolysis of pyrophosphate is favoured because of the negative free energy change associated with it (thanks to David for pointing that out).

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  • $\begingroup$ And why can’t the pyrophosphatase reaction just be reversed? Seems to me that is the key point that needs to be explained. $\endgroup$
    – David
    Commented Jul 6, 2020 at 19:11
  • $\begingroup$ @David The hydrolysis of pyrophosphate is favoured because of the negative free energy change associated with it. (Should I add this point to my answer?) $\endgroup$
    – Adhish
    Commented Jul 7, 2020 at 7:27
  • $\begingroup$ Yes. That is why I raised the point. The standard deltaG for the polymerization is ca –3 kcal per mol. That for the pyrophosphorylase reaction is an additional –4.6 kcal per mol. There is actually a worked example of the latter point for acetylCoA formation in Berg et al, available at NCBI Books online. $\endgroup$
    – David
    Commented Jul 7, 2020 at 7:42
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    $\begingroup$ @David Done. Here is another interesting point: if the standard deltaG for pyrophosphate hydrolysis were zero, or even positive, having the pyrophosphatase enzyme would still help. This is because we are starting with PPi alone, and to reach equilibrium, at least some of it must be converted to Pi. Thus, in any case, some amount of PPi would be removed. $\endgroup$
    – Adhish
    Commented Jul 7, 2020 at 9:00
  • $\begingroup$ I think there are aspects other than the thermodynamics of the reaction. If I am not mistaken, polymerization is almost irreversible. Also, in vitro RNA/DNA synthesis can happen without pyrophosphatase. There are some old studies on PPi being a competitive inhibitor of polymerization. Perhaps you can check them out. $\endgroup$
    – WYSIWYG
    Commented Jul 7, 2020 at 9:07

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