Articles, Blog

Allostery and PFK FBPase

October 10, 2019


– [Instructor] So we’ve
been thinking a lot about how the cell can produce energy and how it can control
that energy production. We’ve introduced ideas like glycolysis and now we’ve seen gluconeogenesis. So these two pathways
are very much intertwined and they respond to the current status of energy of a cell. So they need to be able
to actually respond to the energetic needs of the cell. And this happens through a process known as feedback inhibition. So the idea is that the current
energy state of the cell is signaled by the presence of molecules. So for example, when the
cell has plenty of energy, there’s a really high concentration of ATP but when there’s not enough energy, the concentration of ATP is low but the concentration of
ADP and AMP are really high. So these two coupled pathways, glycolysis and gluconeogenesis
need to be able to respond to those concentrations of metabolites. And the way this works
is through a process known as allosteric regulation. So let’s first explore how this idea of allosteric regulation of enzymes works. There are two different types
of allosteric regulation: one is called allosteric inhibition and the other is allosteric activation and we will explore how
inhibition works first. So the idea is that we have
two different binding sites in an enzyme. One is the active site and one is a spatially
distinct binding site known as the allosteric site. The idea here is that when a molecule binds the allosteric site, it influences the overall structure or dynamics of an enzyme in a way that communicates
a signal to the active site changing that structure a little bit and making the enzyme not
as active or inhibited. So, the presence of what’s known as an allosteric effector can bind to that second site causing the enzyme to change structure and have a lower affinity, therefore more inhibited activity. So let’s explore how this works or and how this influences the
Michaelis-Menten kinetics of an enzyme. So what we’re looking at here is a Michaelis-Menten plot where I’ve converted the
X-axis to a log scale to make curvature a
little bit easier to see. When the inhibitor is added, we see a distinct change in the profile of the
Michaelis-Menten curve and that’s because we
have converted the enzyme to a structure that no longer
binds to the substrate as well so the enzyme is inhibited. Now let’s see how this changes when we think about allosteric activation. So the idea here is that we start off with a form of the enzyme
that is not active. Okay, so it does not bind
to the substrate very well. But in this case, when we add the allosteric
effector or allosteric activator, the signals communicated to
make the enzyme more active converting it into it’s active state. So if we compare Michaelis-Menten curves, in this case we actually see an increase in the activity of the enzyme, so we have a lower KM because then enzyme is binding to the substrate more tightly when the allosteric molecule is added. So let’s go ahead and see how these ideas apply to our energy-metabolism pathways. So we have these two coupled pathways, glycolysis and gluconeogenesis and we’ve said that the key step is the conversion of fructose-6 phosphate to fructose-1,6 bisphosphate. So the idea here is that when we are at a high energy state, we do not want glycolysis to be active because that produces more energy. Instead we want that
process to be inactive and we want gluconeogenesis to be active so we can store that energy, right and we’ve said that the signal for the high energy
state in a cell is ATP. So the way that this
allosteric regulation works is ATP is an allosteric effector that activates fructose bisphosphatase which activates gluconeogenesis but it allosterically
inhibits phosphofructokinase which inhibits glycolysis. The opposite is true for a
low energy state of a cell. In this case, the cell
needs to make more energy so it wants glycolysis to be active because glycolysis is the
pathway that creates energy. It wants gluconeogenesis to be inactive because that would try
to store more energy. We said that AMP and ADP
are the two molecules that signal the low energy state so the way that this
works is that both of them are activators of phosphofructokinase. So when they bind the PFK, it
activates the enzyme making glycolysis more active. AMP also is a potent inhibitor of FBPase which prevents the pathway
of gluconeogenesis. If we apply this idea to
a Michaelis-Menten plot, we see the expected outcome. When we add ATP to phosphofructokinase, it inhibits the activity of the enzyme. However, if we add AMP, it will increase the
activity of the enzyme. So let’s go ahead and look at an example of how this happens in the
context of an actual enzyme. So what we’re looking at here is the enzyme phosphofructokinase. And it’s a big, complicated enzyme but we’re gonna focus in
on just one active site and one allosteric site. So here we’re looking at the active site where we can see ATP bound as
well as fructose-6 phosphate. If we reorient our view, and now focus in on the allosteric site, what we can here is ADP bound. So the white structure
that I’m showing here has ADP bound and remember ADP should be an activator
of phosphofructokinase because it signals a low energy situation. So now this is when things
start getting pretty cool because we can now compare the structure of phosphofructokinase in the active form versus the inactive form. And doing this with the big picture, we see some subtle differences but those differences are most remarkable if we zoom into the active site. So when we do this, we see a couple of really
important hydrogen bonds that exist between the substrate and a glutamic acid in
the active structure. However, if we look at that glutamic acid in the inactive form of the enzyme, what we can see is a
very clear interaction between that glutamic acid and the substrate fructose-6 phosphate. And it’s not a good interaction, it’s actually that, that glutamic acid is now
occupying the same space as the fructose-6 phosphate wants to be. So, this enzyme will no longer bind to the substrate because that
glutamic acid is preventing the fructose-6 phosphate from binding. Hence we expect to see a
significantly lower affinity for the substrate. And what I’ve presented here is just one example of
how a molecule can bind at an allosteric site of an enzyme causing that enzyme
structure to suddenly change and influencing the
activity of the enzyme. This type of regulation strategy is incredibly prevalent
throughout your cells. In the next video, we’re gonna see how the
activity of these two pathways can be influenced by R2
hormones, glucagon and insulin.

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