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 research team to investigate if one of the downstream products of the Mevalonate pathway might be an inhibitor. The researchers 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 agent.
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.
- 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).
- 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)
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