Katlyn Mayer’s Advanced Biochemistry Blog

The Achilles Heel of the Mevalonate Pathway

 

The Mevalonate Pathway is one of the ways that the human body synthesizes its own cholesterol. Those who suffer from high cholesterol are typically prescribed statins which are inhibitors of the HMG CoA reductase enzyme in the pathway. Inhibiting other enzymes in the pathway such as Farnesyl Pyrophosphate Synthase (FPPS) can stop the prenylation of certain GTPases which can act as small oncogenic proteins thus indirectly inhibiting cancer. At the present time, the only inhibitor of FPPS on the market is a group of molecules called nitrogen-containing bisphosphonates (N-BPs) which are known allosteric regulators of the enzyme and are used for bone resorption disorders.

It was known that the enzyme preferred negatively charged hydrophobic substrates, but nucleotides and cholesterol metabolites were not effective in stopping catalysis. All of the N-BPs bound to the same pocket in the enzyme, raising the question of whether or not the allosteric pocket had a biological function for the enzyme. This directed the Park Group of Quebec Canada to investigate if one of the downstream products of the Mevalonate pathway might be an inhibitor. The research team chose to crystallize the enzyme with its product farnesyl pyrophosphate (FPP) to characterize this allosteric binding site. What they were shocked to find in their crystal structure was that FPP was bound in the allosteric pocket and not the active site.

They chose to investigate the thermodynamics of the enzyme using isothermal titration calorimetry, a technique we discussed in Experimental Biochemistry! The process was determined to be exothermic and driven both enthalpically and entropically. At first they were unsure if the product was binding in the active site or the allosteric site, as both would theoretically be able to stop catalytical activity but after crystallizing the complex, they were absolutely certain that FPP was bound in the allosteric pocket and not the active site.

This finding was astonishing to the researchers as there are very few enzymes in existence who use one of its products as an inhibitor. Their crystal structure was determined at 1.9 Å resolution proving their results to be very accurate. Their structure revealed that the negatively charged phosphate atoms of FPP formed salt bridges with basic residues, and the lipid portion of the molecule makes van der Waals interactions with hydrophobic portions of the molecule. These interactions generate a conformational change in the enzyme moving catalytic residues away from the active site. By superimposing crystal structures, the researchers determined that the mechanisms by which FPP binds the allosteric site in FPPS differs from N-BP’s binding in the same pocket. Without having trapped the product in the allosteric site, they would not have been able to predict how the enzyme would have been inhibited, even if they had been able to predict that the product would be an inhibitor at all!

This finding has important clinical applications. Because prenylation of G-proteins is important in certain cancer pathways, using a naturally synthesized product to inhibit the enzyme could be a safe, and promising anti-cancer drug. This finding could also be used to produce another type of anti-cholesterol drug that could be used in place of statins. Over time, statins can contribute to muscle pain and can cause irreversible liver damage (Mayo Clinic). Using a naturally produced substance such as FPP as a cholesterol lowering agent could be therapeutic.

Regardless of where this research goes, sometimes it is pretty cool to take a step back and appreciate some of the crazy things enzymes can do.

 

  1. Park, J., Zielinski, M., Magder, A., Tsantrizos, Y. S. & Berghuis, A. M. Human farnesyl pyrophosphate synthase is allosterically inhibited by its own product. Nat. Commun. 8, 14132 (2017).
  2. Statin side effects: Weigh the benefits and risks. Mayo Clinic Available at: http://www.mayoclinic.org/diseases-conditions/high-blood-cholesterol/in-depth/statin-side-effects/art-20046013. (Accessed: 13th February 2017)
  3. https://commons.wikimedia.org/wiki/File:Mevalonate_pathway_creation.jpg

 

 

12 comments

  1. Hi Katie! I completely agree with the enthusiasm that both you and the authors are expressing in response to this finding! Having a natural allosteric inhibitor to farnesyl pyrophosphate may be greater tolerated and have less side effects compared to statins. Since this paper was only published last month, I am excited to see what studies are going on currently introducing farnesyl pyrophosphate as an allosteric inhibitor to farnesyl pyrophosphate synthase. I wonder if the inhibition of this enzyme will have the same downstream effects as we mentioned in class (controlling cholesterol and cholesterol metabolites-27-HC and 25-HC, isoprenylation). My main question is whether the binding of FPP to the allosteric site would have different clinical implications compared to if the substrate were bound to the active site of FPPS. Great paper!

    1. Hi Melanie. I also wondered about what would happen if the FPP were bound to the active site. The authors mentioned initially believing that the substrate was bound to the active site. I think the reason that it isn’t is because it does have to dissociate away from the active site to continue downstream in the pathway. I’m under the impression that some of the product continues in the Mevalonate pathway and some stays near the enzyme to inhibit it.

  2. This paper is certainly an interesting one. Nothing like a little mystery and surprises to spice up the science world! Seeing as we learned in experimental biochemistry that it can be very difficult to produced a crystal structure, it is quite fascinating and lucky the research team was able to have multiple crystal structures to determine a mechanism for the binding. Even with this method inhibition, would it still be worth researching an inhibitor that binds to the active site rather than the allosteric site? It might be effective in instances where mutations change the shape of an enzyme but maintains function. Either way, the potential harmful side effect of long term statin use is a great reason to try and use a natural substrate.

    1. Nick, I agree that ideally an inhibitor that binds to the active site would be more effective than one that binds to an allosteric site, but I think for the time being, this naturally occurring product that binds at the allosteric site is one of the best options for long term use in cholesterol management.

  3. Hi Katy! This research piece is so interesting! It is amazing to think about how much effort, money, time, etc. goes into cancer research (many other diseases as well) when our bodies might be producing therapies that are better for our health (don’t have these deleterious statin effects that you mention). I have never heard of an enzyme that catalyzes the formation of a product that is also an allosteric inhibitor of this enzyme. I believe I have only ever learned about enzymes that have allosteric inhibitors that are well downstream of the direct product. Because of this, I wonder what implementing the usage of FPP as a drug would really look like. I would imagine that one would not want to just dump FPP in cells since it is the enzyme substrate and this might enhance the reaction we are trying to inhibit (prenylation of GTPases). So do you think this therapy might be some FPP-like molecule that specifically will not bind the active site of FPPS but still maintains affinity to this allosteric site? Thanks for a great synopsis! Really enjoyed your post.

  4. The work the authors present here is clearly exciting and frankly really cool. While I’m sure that producing natural allosteric inhibitors to this enzyme will undoubtedly effectively inhibit the enzyme, I’m curious as to how these inhibitors might interact with the mevalonate pathway as a whole. Do you think that natural allosteric inhibitors might be so similar to the product molecule of the reaction that it could react with other enzymes in the pathway and produce unwanted side effects? While it’s exciting that this research might lead to alternative to statins, as many people do not tolerate these drugs well, I’m not sure if allosteric inhibitors to FPPS will be the panacea that some people suggest that it may be. Although, I certainly hope to be proven wrong!

    1. Brock, I agree that using a natural substrate as an inhibitor might cause problems especially since many of the enzymes in the mevalonate pathway can work on each other given that the molecules in the pathway are so similar to one another. One can hope that it would be a viable option as a medication only because it wouldn’t cause the issues that statins are known to cause in long-term cholesterol patients.

  5. Hi Katy! I found this research piece so interesting. It is amazing to think about how many scientists are slaving over finding cancer therapies, meanwhile, our bodies might simultaneously be producing a really efficient one.

    I don’t think I have ever heard of or learned about an enzyme that makes a product that also acts as an allosteric inhibitor. I have only heard of products much further downstream acting as allosteric inhibitors. This makes me wonder what using FPP as a therapy would actually look like. It seems that dumping FPP in cells would not be the best idea because it does bind the active site of FPPS and causes the reaction we want to inhibit (prenylation of GTPases). So, do you think this drug would just be an FPP-like molecule that is synthesized such that it binds the allosteric site but avoids the active site?

    1. Gianna, you make a good point. Using the product of an enzyme in a pathway might work as an inhibitor, but it might also continue down the pathway as it is now in higher concentrations for the next enzyme to utilize as a substrate. Drug companies might consider using a molecule similar in structure to FPP as an inhibitor for FPPS.

  6. Hi Katy,
    This paper is really cool. We’ve seen allosteric inhibition of enzymes by downstream products or side products, but it’s really interesting to see the inhibition be so direct. The authors cite that the enzyme that comes next in the pathway, geranylgeranyl pyrophosphate synthase, is also inhibited by its own product, although not allosterically. I wonder if drug companies will focus on creating an inhibitor, based on the binding of FPP, that specifically binds allosterically and or if they will attempt to make a drug with the specific goal of it binding both allosterically and to the active site. Thanks for the post!

  7. I thought this article was really interesting; we tend to fall into a rut of thinking that all enzymes are alike but there are always exceptions and those are often the most exciting. The fact that the FPP was bound to the allosteric site and not the binding site goes to show that there are still new things to discover in the world of enzymes. I also think it’s really fascinating that there are new ways of treating cancer that involve less invasive and harmful measures, just by using information we already know and using enzymes the body already produces itself. It’ll be interesting to see whether the authors choose to proceed developing treatments and potentially giving them to mouse models. One question I have is that if the binding site remains open, does it also leave it more open for attack by a possible antagonistic substrate?

  8. Hi Katy! This was a phenomenal paper, and is a rather exciting step forward in the field of cancer research. Finding a molecule that binds to the allosteric site rather than the binding site was a surprise find being that the inhibition went through a direct process rather than going through the usual cascading manner. This new information can serve as a cancer treatment target to look into.Scientists should consider a treatment in which a molecule structured like FPP can be used as an inhibitor that binds allosterically. A downside to this possibility is that using natural allosteric inhibitors could create harmful side reactions/products.

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