I'm curious to know how just by adding a fluorine atom to uracil (to form 5-fluorouracil) permanently inhibits the enzyme thymidylate synthase when they bind together. This molecule is widely used as an antimetabolite in chemotherapy as it stops the synthesis of thymine.


What changes undergoes the enzyme-inhibitor compound so it is permanently bonded to the fluorouracil? Probably the chemistry is too complicated to be explained in this Q&A format, if that is the case, references like books or papers would be great.

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    $\begingroup$ Possibly, but I think asking it here first was warranted. Questions on biochemistry / chemical biology have been understandably complicated. The biochemistry would typically stop at the statement, "X inhibits Y", but your question is, "What is the chemistry of the X-Y interaction that results in inhibition of Y?" It's more topical for Chem.SE, though the expertise you need might be only on Bio.SE. Wait and see what answers come, I'd say. $\endgroup$ – hBy2Py Feb 8 '16 at 11:31
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    $\begingroup$ compare strucutures of the substrate, the product and the drug. It sees, that fluorine occupies position where hydrogen should be replaced with methyl in the reaction catalised by the ferment. To my knowledge, biogenic methylation is electrophilic (i.e. methyl is transfered as $\ce{Me+}$ in $S_N2$ -type reaction. For this to happen, a negative charge on the atom next to fluorine should occur, which would lead to dissociation of fluoride ion and active carbene generation, readily binding to the closest molecule. At least it is what I can come with out of the blue. $\endgroup$ – permeakra Feb 8 '16 at 11:52
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    $\begingroup$ However, for a definitive answer you should study extensively the literatute. It is pretty possible, that there is no known agreed upon mechanism of action. $\endgroup$ – permeakra Feb 8 '16 at 11:52
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    $\begingroup$ I've seen many examples where substituting F for H does havoc on biology. Not sure if there is some reaction with the enzymes, or if the just bind and try in vain to do their job. Would love to see an answer on this. $\endgroup$ – Gyro Gearloose Feb 10 '16 at 19:26
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    $\begingroup$ I think this will solve your problem: nature.com/nrc/journal/v3/n5/fig_tab/nrc1074_F2.html $\endgroup$ – ShankRam Feb 11 '16 at 1:37

The inhibition comes about because:

  1. Uracil and $5$-fluorouracil are sufficiently similar that both moieties have a strong binding affinity for the active site of thymidylate synthase.
  2. The presence of the fluorine prevents $5$-methylation of the uracil core by a folate cofactor, which otherwise would turn it into a thymine moiety.
  3. This inability of the enzyme to convert the $5$-fluorouracil moiety into a thymine moiety (which has a much lower affinity for the active site) means that the enzyme has no way to reject the $5$-fluorouracil or the methylating folate species, once bound.

The particulars of this interaction are nicely spelled out in Mishanina et al.$^\dagger$, conveniently available in preprint:

[5-fluorodeoxyuridylate] reacts [as normal] with the active site cysteine and undergoes condensation with [N,N-methylene-tetrahydrofolate] but prevents proton abstraction and elimination of the tetrahydrofolate [product], as a consequence stalling the reaction at the covalent ternary complex [of enzyme, inhibitor, and folate species].

Scheme 4A from the preprint linked above illustrates well the specifics of the chemistry. (I refrain from reproducing it here due to uncertainty regarding its copyright/license status.)

As well, Figure 2 from the Mechanism Description section of the Wikipedia page on thymidylate synthase shows very nicely the enzyme interactions with the normal deoxyuridylate substrate (click image for larger version):

$\hspace{20pt}$ dUMP in thymidylate synthase active site
Image by CJerc (Own work) [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0 )], via Wikimedia Commons

$^\dagger$ Mishanina et al. Bioorganic Chemistry 43: 37 (2012). doi:10.1016/j.bioorg.2011.11.005

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