Regulation of Krebs-TCA cycle

- [Speaker] To save us some time, I've went ahead and drawn out simplified version of the citric acid cycle here and if you remember, it begins with acetyl-CoA entering the cycle, and it combines with this molecule oxaloacetate to form citrate. And this citrate undergoes various conversions and oxidations, which eventually cause the two carbons that entered with acetyl-CoA, because remember acetyl-CoA is a two-carbon molecule. These two carbons exit as carbon dioxide. And as it's oxidized and loses it carbon dioxide, acetyl-CoA also allows us to produce the electron carrier molecules FADH and NADH, which if you recall, go to the electron transport chain to allow us to produce ATP. So to summarize, let's go ahead and write out the kind of overall chemical reaction of the citric acid cycle. So we have as our reactants, acetyl-CoA entering the cycle, we also have some co-enzymes like NAD+ and FAD, and we also have a freed GDP, remember GTP is formed in this cycle as well. And all of this eventually will produce carbon dioxide from the oxidation of acetyl-CoA. We reduce our electron carrier molecules. So NADH and FADH2, we also form a GTP. Now the reason I wanted to go ahead and write out this entire overall reaction of the citric acid cycle is because when I was first learning the cycle, I kind of often got stuck in the individual reactions that were taking place in this kind of merry-go-round. And getting confused by all these names like isocitrate and succinyl-CoA, and I think that when we're trying to understand in particular how this cycle is regulated, that is trying to figure out when essentially, this cycle is in full speed an when it's kind of slowing down, it's actually nice to kind of step back and look at the big picture. Now the first point I want to make is that in contrast to something like glycolysis, which we usually think about as a metabolic pathway that we really consider to be either on or off, the citric acid cycle is something that we usually consider always to be on, but to various degrees, depending on kind of the energy needs of the cell. And the reason it always has to be on, right is because it needs to be delivering these high-energy electron carriers to the electron transport chain, to allow at least some constant flow of ATP production, which is vital to a lot of the tissues in our body. In contrast, you can probably think of a case when, you know a theoretical case perhaps, where someone might not have any glucose in their diet. And of course glycolysis then could be off, but the body might be able to use fatty acids and other kind of sources of kind of fuel to enter the citric acid cycle. And indeed, kind of a clinical proof of this is that there are kind of disease states that involve mutations of enzymes in glycolysis, for example. But you would be hard pressed to find a living individual who has a mutation in their citric acid cycle because this cycle is so vital to life. So that brings me to my first point, which is that there is no hormonal control in the citric acid cycle, because it's on regardless of whether we're in the fed state or the fast state. Instead the major form of regulation of the citric acid cycle is through allosteric regulation. So I'll remind you that allosteric regulation is simply the ability of a molecule floating around in the cell, to bind to a specific enzyme that's part of this pathway, and it binds through a part of the enzyme that's not the active site, and essentially by doing this, it causes the enzyme to undergo a conformational change, and it can either make the enzyme work better, in which case we call it an allosteric activator. Or it can make the enzyme not work as good, in which case we call it an allosteric inhibitor. And the way that I remember that allosteric regulation is the major kind of form of regulation in the citric acid cycle, is I remind myself that since this cycle is always on, it still kind of wants to be able to adjust on a minute to minute, so rather fast way to the kind of energy needs of the cell. So I'm gonna go ahead and write that here, wants to respond to the energy needs of the cell. And they way that it can figure this out is by looking at the molecules it has floating around. And these molecules are often those that are involved in regulating this pathway allosterically. And finally, a third way that this cycle is regulated is by looking substrate availability. And this is exactly what it sounds like. Essentially, if the body doesn't have a lot of acetyl-CoA around for example, remember this is one of the major kind of substrates for this pathway. Then it makes sense of course that the speed by which this NADH and FADH2 is produced is gonna slow down because there's just not enough to enter the cycle. Now one high-yield example of this is when citrate, under conditions of high ATP generally shuttles a lot of it's acetyl-CoA into the cytoplasm for fatty acid synthesis. And of course when this happens, it's taking the citrate out of the cycle, and so it will slow down the overall cycle. On the flip side, let's say our body was in a very starving state, we haven't had food for quite some while. And in some cases amino acids can actually begin to break down from our muscles and enter in various places along the citric acid cycle. And one place they enter is, they actually are converted into alpha-ketoglutarate here. And suffice to say that the general idea here is that if you have of this substrate around, it's gonna push the cycle to go faster which makes sense in this case right, because the body is alerted to the fact that it's starving, and so it wants to be able to produce more NADH and FADH2 to produce more ATP. Alright, so that's kind of a general overview of substrate availability, but now I wanna talk more in depth about this allosteric regulation. Now you wouldn't notice if I had told you, but it turns out that there are three reactions in the citric acid cycle that have a very large negative delta G. And remember large negative delta G means that these reactions are largely irreversible, which means that they are good targets for regulation because once these reactions essentially occur, it's usually like a ball rolling down a hill and will allow everything else to occur. And these three reactions are the conversion of oxaloacetate and acetyl-CoA into citrate, as well as the conversion from isocitrate to alpha-ketoglutarate, and alpha-ketoglutarate to succinyl-CoA. Now to go ahead and keep this kind of diagram as clear as possible, I'm gonna go ahead and kind of abbreviate the names of these enzymes, but of course you can always go to Wikipedia or a textbook and remind yourself what these enzymes are called. But of course this enzyme from octyl acetate and acetyl-CoA to citrate, is citrate synthase, and isocitrate to alpha-ketoglutarate is isocitrate dehydrogenase, so I'm gonna say ID. And then alpha-ketoglutarate to succinyl-CoA is alpha-ketoglutarate dehydrogenase. Now the allosteric regulators that these three enzymes can end up being kind of a long list, but my hope in this video is to just be a resource for you to come back to and to kind of justify why certain things are allosterically inhibiting or activating these enzymes. So why don't we start off with the allosteric inhibitors, and one kind of easy allosteric inhibitor to remember is NADH because it allosterically inhibits all three of these enzymes. And the reason it does this, and it should be apparent to you why this is so, is that, notice that NADH is a product of the overall citric acid cycle, right? So it's a product right here. And so if we're building up NADH, it's essentially a sign to the body that the citric acid cycle is going faster than the, essentially the electron transport chain, which is using up these NADH, can consume those NADHs. And so it's time for the citric acid cycle to slow down. Now another allosteric inhibitor that should make some sense to you is ATP. So if we have a lot of ATP in the body, it makes sense that this would be an allosteric inhibitor of processes that produce energy right, Because we want to conserve energy and not kind of make more energy than our body needs. So in this case, it turns out experimentally for some reason, only two of these enzymes have been shown to be inhibited by ATP and those are citrate synthase and isocitrate dehydrogenase. Now the final allosteric inhibitors to talk about are actually products that form in the citric acid cycle and these products, if they accumulate in excess amounts, can actually negatively feed back by allosterically inhibiting some of these enzymes. And the two notable products that do that are first, citrate, which can allosterically negative feed back on it's enzyme citrate synthase, as well as succinyl-CoA, which not only negatively allosterically feed backs onto the alpha-ketoglutarate dehydrogenase, but it also can actually negatively feed back on the citrate synthase enzyme as well. And one way that I kind of remember why succinyl-CoA might wanna kind of feed back all the way back to the citrate synthase is to recognize that the citrate synthase is the kind of first kind of point of entry into the citric acid cycle. And so, if it can stop the citric acid cycle sooner, it will essentially waste less energy so to say. Alright so that kind of sums up the allosteric inhibitors, but what about the allosteric activators? The first allosteric activator that always comes to my mind is ADP. Remember that ATP is hydrolyzed by a water molecule into ADP, and a free phosphate group. So if ADP levels accumulate in excess of ATP, then it's basically a sign that the cell is running out of it's power, it's running out of it's ATP, and it will therefore need to produce more ATP. And so, if it needs to produce more ATP, it makes sense that it would want to activate the enzymes in the citric acid cycle. And it's easy to remember because it activates the same enzymes that ATP inhibits. So those are citrate synthase and isocitrate dehydrogenase. Alright, so we're hitting the home stretch here and there's actually only one more allosteric activator that we need to talk about, and that is calcium. So why might calcium be an allosteric activator? Well remember that our muscle cells require an influx of calcium to contract. So presumably you know, if we're exercising really hard, of course our energy needs go up, but our calcium levels inside of our cell are also going up, because of all that muscle contraction. So, this is essentially a way for the body, especially in skeletal muscles to essentially couple muscle contraction with producing more ATP to meet the needs of those contracting muscles. And again, this is experimental evidence, but calcium has been shown to allosterically activate isocitrate dehydrogenase, as well as alpha-ketoglutarate dehydrogenase.