 Alright, we are ready. Please have a seat. So we made some headway on this lecture and the last lecture, so that's cool. We have a lot today, and please interrupt me. I mean, I felt like the last lecture was just sort of overwhelming, and it's a bit intimidating to ask questions, because there's so many concepts being integrated, but it's a real shame to end up with extra time and no questions asked. So I'll try to give time in the lecture to allow you to do that. So today, this looks really not hard, right? It's very short, but we're basically going to take that lecture six, where we just describe some molecules, and we're going to look at the pathways that make those molecules, and that was an early lecture. So there's just a lot of different pathways, and some of these we're going to come way, zoom way out in terms of detail, and we're just going to have like arrow, arrow, arrow, and like all the junk in between, who cares? So I'll try to point out along the way what's important. So this is a review, so remember beta-oxidation, that's how we take apart and catabolize these fats, so palm-tailed CoA, and so you have the alpha and the beta position, you have a dehydrogenase, you transfer these electrons up onto FADH2, and then you add water across the double bond, and then you oxidize the hydroxyl group to a carbonyl group, and then you actually cleave the molecule at that point. And so one of the things I mentioned in words, but then show a picture, was this pathway of transferring those electrons from that one step, remember where we put electrons on FAD, and moving those into the mitochondria. So remember this catabolism is occurring in the matrix of the mitochondria, and so we have a little mini electron transport change, so you have this dehydrogenase that initially takes the electrons, making that double bond, and then those electrons are transferred to another protein that also has an FAD turning it into FADH2, which is then transferred to a third protein, which also has an FAD converting into FADH2. So it's not exactly as simple as the succinate dehydrogenase mechanism that we saw in the Krebs cycle, where you have the enzyme doing the direct chemistry, actually passing electrons directly in, but the net output's the same. You have the quenol form, those two electrons end up there, and you only are moving a total of six protons across the bilayer, because you bypassing complex one. So what we're going to do here is those four steps about beta-oxidation. Remember that the product of each is a carbon skeleton that's two carbons less, so you have extract acetyl-CoA out at each step, and so you just keep repeating this until you get to the last step. But you should look at the molecule and come up in your mind of why it makes sense that you only get seven FADH2s and seven NADHs. It's because there's only seven bonds broken. The last one, the four carbon piece, you're only getting those one NADH and one FADH2 by breaking that into two. Yep, we're going to get to that today. And also, what do we do with double bonds and fats? What about trans fats? So that's where we're heading there. Okay, so let's do a little. We're going to do this again and again throughout the course, and let's sum up what we're doing here. So we're taking palmitil-CoA and converting that into some reduced cofactors. And then those acetyl-CoA is obviously, we're in the mitochondria already, so we can convert those through the Krebs cycle into reduced, more reduced cofactors. And you can sum these all together. Now remember the number of ATPs you generate depends on where you come in and electron transport. So if you come in at complex two, you get four fewer protons transported compared to coming in at complex one. So if you add all this up in total, you're generating 106 ATP molecules. So you have to count for the two ATP equivalents that you used. Remember we stuck AMP on there, so we went, that was going directly from ATP down to AMP to make palmitil-CoA, so we have to subtract that out. And so we get a total of 106 ATP molecule. But let's think about this in terms of the number of AT molecule equivalents per carbon atom. And so if you take that number, divide by the number of carbon atoms, so palmitil-Palmitate has 16 carbon atoms, you get about 6 ATP molecules generated per carbon atom that's fully oxidized as carbon dioxide. Whereas in the complete oxidation of glucose, you get a total of, if you do the calculation, about 5 ATP molecules per fully oxidized carbon. Because it makes sense, because this is a more reduced form of carbon and fats and so oxidation is what's releasing the energy here. And so if you do the comparison between the beta-oxidation or full-oxidation of fats compared to the full-oxidation of sugars, you get about 20 to 30 percent more energy when you oxidize sugar carbon atoms compared to, or when you oxidize lipid carbon atoms compared to sugar atoms. And so it makes sense that this is a great way to store energy. So there's two ways that we can store energy here. We can store it as fats, right? So we can store it as like palmitate. Or we can store it as glycogen. So palmitate, so when we store fats, those are typically stored in totally different cells. So you have this adipose tissue that has a large amount of fat. So getting the energy back out of the fats requires actually transport of the fats between cells. So it takes some time. So glycogen is within most cells. And so you can rapidly get the energy out. But glycogen is very hydrophilic. So actually if you weigh a glycogen particle, the majority of what you're weighing is just water, all that hydration that needs to happen from glycogen. Whereas fats are stored as these hydrophobic globules in a different type of cell. So you can pack more energy. You can store a larger form of energy. But it's less efficient, less quick to extract it out. So there's different reasons. So usually glycogen stores, say you just, everybody in the room stopped eating. So in about 24 hours, most of your glycogen would be used up. And then you would start working on those fat stores as well. Any questions so far? So we're going to get to these special cases that we're asked about. In terms of beta-oxidation. So what we're going to do here is deal with these situations. So here we have an unsaturated fatty acid. It's cis, remember the unsaturations in fatty acids, naturally occurring from fatty acids, are in the cis configuration. And so what we're going to do here is just do our beta-oxidation as we normally would until we get to the point where we arrive at this additional double bond. And then the general strategy is going to be to transform that molecule, whatever it ends up being, into something that looks like a beta-oxidation, one of the four steps in beta-oxidation. So the strategy here is to convert molecules into this enol form. So this is directly from the slide of beta-oxidation. In this enol form then we're in the right orientation to do our beta-oxidation. Remember that's a transform. So for example here you can do your beta-oxidation, remember each cycle, four steps releases acetyl-CoA, so one, two, three steps gets you to over here. But now this is close to being this, but this is not alpha-beta unsaturated, it's beta-gamma, right? So it's just in the wrong place. And it's also in the wrong orientation. It's cis where it needs to be trans. So there's a handy-dandy enzyme, myosomerase, which takes this double bond, just moves it over here and changes from the cis configuration to trans. And now this is just a familiar substrate to the beta-oxidation enzymes and they're able to proceed to oxidize this molecule. Well what if you have two double bonds? So the same general strategy, so you clip away until you get to your problem. So for this linoleal-CoA molecule, when you remove a certain number of acetyl-CoAs, you get to this point, and again we need to be in this alpha-beta trans double bond configuration to get us into the pathway. There's no intermediate in beta-oxidation that looks anything like this. So the strategy is to summarize, you get to this point, we've already seen this chemistry where we just disomerize this over. So you go from cis to trans, cis-beta-gamma to trans-alpha-beta, and that feeds into the carb-cycle. And then you clip off another molecule and then you get to the step where you make the trans double bond on the subsequent cycle. And now you've got this weird sort of conjugated double bond. You might say, well, can't the enzyme deal with that? No, it can't. It doesn't like to have this, it changes the shape of the substrate so that you can't oxidize this. You need to deal with this problem. And the way that it deals with this is to reduce this conjugated double bond into this transform. So this is a weird substrate. You've got a cis double bond, you've got a trans double bond. You convert it into this form and then you isomerize. So you're going from the trans delta-3 to the trans delta-2. And then that looks familiar. That's a substrate. And so generally one of the thoughts that you might be thinking about at this point, especially the people with medical inclinations is what about trans fats? Well, generally trans fats can be metabolized. So the big problem with trans fats is in the transport. So when people have a lot of trans fats in their diet, the amount of LDL versus HDL is increased and that causes heart disease. So really in terms of the processing of these fuels, trans fats are a little bit less efficiently processed but they can be processed in these pathways. Because you can see and it makes sense some of these intermediates have trans double bonds. So it depends precisely where the trans double bond is and the flexibility of the enzymes that are manipulating like these isomerases, for example. So any questions on that? So we've dealt with various configurations of double bonds and fats and the strategy was to make it look familiar, make it look like this trans alpha-beta configuration. So what about odd number of fats? I think a question was asked about that. And so when you think about it, say you have 17 carbon atom fat. So there's nothing to stop you from just starting off and doing lots of beta-oxidation. But in the last step, instead of getting to a 4-carbon fat, you'll end up with a 3-carbon fat, right? And so at this point the cell says, hey, 3-carbon molecules, we know what to do with that. Let's convert those into a 4-carbon molecule. And so we can use a carboxylase enzyme, has biotin and you can carboxylate. This enzyme has a particular stereochemistry in which it carboxylates. So now we're up to a 4-carbon molecule and you might think, what might the strategy be here? So we're going to reconfigure this 4-carbon molecule to make it useful. Okay, we're in the mitochondria. Okay, can you guess where we're heading with this? Yeah, yeah, exactly, the Krebs cycle makes sense. We just have to move things around and make it look like any of those intermediates in the Krebs cycle. So you have this methyl-malonyl-CoA and then you can epimerize this. So it's sort of hard to see with this orientation, but you're just swapping the position of these two groups because that's the form of the substrate needed by the next enzyme for whatever reason. And then we're doing this really crazy chemistry here where we're breaking a carbon-carbon bond and shifting the group over by one position. And so this was actually a fascinating transformation when people were discovering these pathways. They said, how can an enzyme do that? I mean, the energy necessary to break this carbon-carbon bond is pretty high. So they found that there was a cofactor B12. But are that for the orgo-geniuses in the room? Do you remember, like, you know, what did you do when you had this kind of situation? We had a really strong bond and you wanted to break, you wanted to rearrange carbon atoms. What is one of the organic strategies that you remember? I'm sorry if I'm inducing post-traumatic stress. Can you remember? What type of chemistry? Okay, so I'll give you a little hint. So when we do this rearrangement, as it turns out, if you label a hydrogen, the hydrogen is never released to solving. So that same hydrogen atom here is going over here. And these two groups are swapping positions. Let's look at the cofactor. So this is this coenzyme B12. Look at this structure. So you're like, what is this sort of weird thing here? And whoa, dude, this goes all around, makes another ring. This is for the organic chemistry. They're like, wow, how do we make that? It actually takes 30 synthetic steps to make this molecule. So it's, wow, it's amazing. And it's got this cobalt. Like, what is a cobalt? Who put that there? Crazy, cobalt. It should be iron or a copper top or magnesium or something. Manganese. It's cobalt. And then they see this weird deoxy adenosine over here. And it turns out that this bond breaks. And what do you think happens to the electrons when this bond breaks? I'm sorry, it's just sort of fascinating orgo stuff. What do you think? Do you think do you have two electrons moving around when that bond breaks? Or another way of asking what does this word mean? Free radical chemistry. This is how we do this weird transformation. So what happens in this coenzyme is that first this coenzyme dissociates. So you have one electron goes up here. The other electron goes in the metal ion. This electron here, this thing doesn't just float away because it's held by the enzyme, but it's used to take the hydrogen atom off the molecule and then the enzyme switches the position of the other group, right, that CoA group, and then puts the hydrogen atom back on. So the reason it's cool is because it does free radical chemistry because that's what's necessary here. So it's a bit of a side. I'm a bit fascinated by that. It's okay. So the end product of beta-oxidation is acetyl CoA. And now we know we can feed acetyl CoA in the mitochondria already. So we can just feed that guy into Krebs cycle directly. But there's another option here, and it has to do with the state of the organism. So if you're starving, right, there's certain organs in your body that it's not optional to have fuel. So your heart needs to beat. Your brain needs to suck down, you know, sugar, suck down energy. And so when you're really, really starving, you need a way to convert these fatty acids into fuels for other organs. You know, or you'll die. This is a big, big thing here. And remember, we can't take this acetyl CoA and make sugar molecules and transport those around because we talked about that. If you go forward, it doesn't work. If it doesn't go backwards, pyruvate dehydrogenase is not reversible. So there's this pathway where you can take these acetyl CoAs. If you have extra ones of those from beta-oxidation in one tissue, convert them into a transportable form. You're not going to want a CoA sitting on your metabolite if you transport it out of the cell. It's a big CoA. And you'll start decreasing the amount of CoA in the cell. So you need to get the CoA off of there and transform the molecule into a form that can be passed into the bloodstream. So here are, this is called ketogenesis. And you form ketone bodies. Not all of these molecules are ketones. And none of them are bodies. So this is a traditional name. So this is not a particle, a body. I mean, it's like a planet or something. And the ketones, this molecule doesn't even have a ketone. It has a carboxylated and alcohol groups. These other ones have ketones. But look at this, acetone. Those squirt bottles you had in Kim Lab with acetone? Do you know that you can make that? And it's a fuel. You can actually fuel your brain on acetone. So don't try this at home. Let the pathways do the magic. Okay, so let's look at how do we make these things? These are some crazy transportable molecules. Did I skip one? I think I skipped one. So you can condense two acetyl CoAs. And I'm going to sort of step back from the Orgo-ish electron pushing thing, but for those that it might help you to remember some of these transformations if you think about the electron. But here you're putting things together like this. You have two acetyl CoAs. And then you're going to add one more acetyl CoAs. You have a total of six carbons here, making this HMG CoA. We're not to a ketone body yet. Not body, not ketone. And so here's this HMG CoA, and then we're going to cleave an acetyl CoA back off. So you put it on, take it off, and like make up your mind, man. But this molecule, because we took an acetyl CoA off, there's no CoA left, right? So this is transportable. This is just a small molecule, and we can transport it out of the mitochondria into the cytosol and then release it from the cell using transporter proteins. So we have an option. We could decarboxylate this, making acetone. Or we can take those acetyl CoA equivalents and also deposit some electrons on this mobile fuel. So if you put some electrons into this, it's going to be a better fuel, right? So you can, if you have some NADH in the tissue you're currently in, then you can deposit those electrons on here. And this can be transported into brain cells or in heart cells. So this is what generally is happening in the liver. That's one of the main producers of ketone bodies. And then this is a totally different tissue. So I don't think that this is a circle. We've now transported these ketone bodies. They've arrived at the brain and the brain's like, I'm going to die here. I need some fuel. And so we're going to reconvert this back into acetyl CoA. So the NADH product is you're moving acetyl CoA from the mitochondria of one type of cell into the mitochondria of another type of cell. And that acetyl CoA is raw fuel. That's the feedstock of the Krebs cycle. So here we're going to take our hydroxybutyrate. We can grab those electrons. We're in the mitochondria. These have been transported in the brain cell into the mitochondria. And now we can take those electrons back off. We need that right into electron transport. That's useful. And then we can take this molecule and transfer a CoA group from succinyl CoA. And doesn't that sort of make you wonder like, isn't that going to mess up Krebs cycle? No. It helps. That's the exact same reaction that happens in the Krebs cycle. It's a different enzyme. But that's what happens in the Krebs cycle. You convert succinyl CoA to succinate. That's not a problem at all. So now we've got our acetoacetyl CoA. And we can then convert that into two molecules of acetyl CoA. And so the net product, as you move those acetyl CoA equivalents from one cell into the other, and you can live a little bit longer. But actually when you're starving, when people are really starving, if you smell their breath, you'll smell acetone. That's from these ketone bodies moving around their body. Sounds like chem labs. Really tragic. So then you have, so this is what's happening in starvation. This is a hepatocyte or other name for a liver cell. And so one of the main types of cells that does gluconeogenesis is the liver cell. But the more gluconeogenesis you do, once you've burned up, remember those ketone or the amino acids can be converted into Krebs cycle intermediates and turn into sugar. Remember that? But at some point you're really starving so much that there's just not the proteins there anymore as well. And when that happens, the abundance of all the Krebs cycle intermediates keeps going down because gluconeogenesis just keeps going. We're in an emergency situation here. So if these Krebs cycle intermediates slows down, then that causes acetyl CoA to accumulate. So if you have more of these acetyl CoA coming in from ketone bodies, for example, you won't be able to process them anymore in the citric acid cycle. And so this leads to, I'm sorry, you make them from fats, from beta-acidation of fats. We're in the liver cell. And then these things can then, once Krebs cycle has slowed down, the acetyl CoA can be converted into ketone bodies and exported from the liver and where it can be used as fuel in other tissues. So there's this big sort of organismal view of the biochemistry. Okay, so we've looked at beta-acidation. We've described that that's in the mitochondria. The electron carriers that are used are the same ones used in electron transport. So this is furnace, right? So we're burning these fats and we're converting, ultimately, those fats into ATP molecules. The carrier that was used was the coenzyme A molecule. The product of beta-acidation was acetyl CoA, and we're using two carbon units. Each disassembly step, you know, we have four steps, but the net process is reduction in length by two carbon atoms. Now we're going to switch gears and look at catabolism of fats. So obviously we're going to need some reciprocal regulation here. We don't want to be making them and degrading them, but there's some differences. So these things occur in totally different places in the cell. So the biosynthetic pathways occur in the cytosol. They use a different reduced cofactor, NADPH. Instead of using coenzyme A, they use sort of a thing similar to coenzyme A. It has a pentothenate, but instead of being attached to ADP, it's attached to a serine on a polypeptide. So we'll look at that in a second. And the product or the donor for each of these two carbon units is going to be malonyl CoA. So this is a three-carbon CoA. And so this is not the simple reverse of beta-acidation. Any questions so far? Pause a bit. Okay. So this figure is a bit confusing. Circle the word matrix in cytosol, because our general trend was to have it be the opposite way when we've drawn this in the past. So here, what we need to do in the first step is take our acetyl CoA and feed it over into the cytosol. That's where we're going to be building fats. And so we need a transporter. And so the way this works is, you know, I suppose it could have evolved an actual transporter for the acetyl CoA, but then you would have net efflux of coenzymes, and that would be bad. And so another way to do this is sort of how we transported NADH into the mitochondria from glycolysis. You hitch a ride on some existing metabolite, whatever's in your environment. So we, for example, we already have a molecule that can react with an acetyl CoA. It's citrate synthase. That's the first step in Krebs cycle, which occurs in the mitochondrial matrix. And so this four-carbon oxaloacetate has converted into a six-carbon acetyl CoA equivalent containing molecule called citrate. This six-carbon molecule has been transported into the cytosol through a transporter where we can then cleave off the acetyl CoA molecule. That requires ATP hydrolysis. And that regenerates oxaloacetate. And some of you might say, aha, I found the kink in the armor. This is how we're going to do gluconeogenesis. We've got oxaloacetate in the right place, right? We can do gluconeogenesis. Well, if you don't put it back, all subsequent transport will grind to a halt. So no, it doesn't work. You cannot do gluconeogenesis with this oxaloacetate because it's got to go back in to be able to shuttle another acetyl CoA out of the mitochondria. So we can then take our oxaloacetate, convert to malate. We've seen this enzyme. This is a cytosolic version of malate dehydrogenase. And then we have two options. So the synthesis of fats is going to require initially acetyl CoA and also NADPH. So where did we make NADPH in this class? Do you remember? Yeah. What about animal cells? Where did we make NADPH? Do you remember? Is it post-traumatic stress inducing? To think about that slide again. Remember that pentose phosphate pathway? Induces a lot of stress in me. That thing was scary. But the non-scary part of that pathway is actually what made the NADPH, that oxidative wing. And then you have the tinker toy part that regenerates the sugar molecule. But NADPH is just that oxidation phase. So the NADPH could come from pentose phosphate. But if we don't have enough, we can convert NADH into NADPH by this alternate pathway. So we can either convert or transport malate back into the mitochondria so that we're not leaching the Krebs cycle intermediates. Or we can convert to pyruvate and put those electrons on NADPH if we don't have enough from pentose phosphate pathway. So pyruvate can then be transported back in the cell. So do you understand here? So we're hitching a ride on whatever there's, you know, Krebs cycle intermediates or the mitochondria is chock-full of those. So we just picked one. We hooked up with it. We put a 4-carbon. We convert it into a 6-carbon, move that guy out, and then drop the acetyl-CoA again. So the benefit is we're not moving coenzyme A here. It's staying in its own compartment. But now we're ready to do some synthesis of fats because we're in the side of salt. Does this make sense so far? Questions? No one to rush. Any questions? Okay. Well, so now we need to commit. We have to make a commitment. It's the step of commitment. So we take our acetyl-CoA and we convert it into a 2-carbon molecule into a 3-carbon molecule. And again and again and again, the way we generally do this is a carboxylation reaction involving a biotin cofactor. So biotin, remember, activates or puts our carbon dioxide in the right compartment, converts the substrate to be in this alternate form where it's more reactive, and then we're going to condense the carbon dioxide on the tail end of the acetyl-CoA and this carbon-2 to make malonyl-CoA. And so this is the highly regulated step in the synthesis of fats. And malonyl-CoA helps to regulate beta-oxidation. Remember that carnitine transporter? So acetyl-CoA is using different shuttles in these two different pathways. Do you see that? So here we hitched a ride on this citrate molecule, whereas when we were going the other way for beta-oxidation moving from the cytosol in the mitochondria, we made a bond to a carnitine molecule. So a carnitine transporter is actually red-related by malonyl-CoA. So when there's malonyl-CoA, we don't want to be doing that. Okay, so here's the regulation of this. Again, it's important that we have this reciprocal regulation. So if we have malonyl-CoA, that molecule is absolutely marked and committed for a synthesis of a palmitate-CoA. And so at that point, if malonyl-CoA is building up, we want to turn off the carnitine acyl-transphrase. That was the molecule that got the acetyl-CoA into the right compartment for beta-oxidation. And then we have our usual cohort of hormonal regulation going on here. So generally, anabolic processes occur in the presence of insulin. And when glucagon comes up, that means you're starving, it's not a good time to be synthesizing fats when you're starving. So we have the general hormonal regulation. We also can regulate these same hormones, can regulate the extraction of fats out of triacylglycerols, these lipase enzymes. And this picture is a really huge complex, this acetyl-CoA carboxylase ACC. Okay, does that regulation make sense? So it's the same, biochemistry is boring. It's just like the same thing again and again. Reciprocal regulation generally towards the beginning of when you're getting things going. So here you're regulating the transport step by one of the products after the transport for synthesis. So this is fatty acid synthase. It's a huge single polypeptide with count them. One, two, three, four, five, six, seven active sites. One polypeptide. That's sick. Look at this. So you have this KS and the ACP. Those are going to be involved in holding substrates. So we're going to have malonyl-CoA's coming in in one position, and we're going to have our growing chain on the other position. Then these other four enzymes are going to catalyze a four-step process. The product of each four-step process is to extend the fatty acid by two carbon atoms. And we're going to see the same sort of chemistry we saw in beta-oxidation, but in reverse. So obviously we need different enzymes here. And so there's ACP molecule. It's sort of like coenzyme covalently attached. So what is the structure of coenzyme? It's this plus adenosine residue. So there's two phosphates there. But instead of having adenosine, we're actually going to covalently attach this pantothenate onto the ACP protein. And what is this? This is a whip. We've got four active sites. We need to have our substrate visit each active site. Okay, time for that chemistry. Time for that chemistry. And it's going to whip around our substrate on a covalent whip between four active sites. And so the business end of this is a sulfhydryl. And so this is going to make a thioester linkage. Just like we had a thioester linkage with coenzyme A. So here's how it works, four-step process. So first step, you're going to load... In the first cycle, you're going to load malonyl-CoA on ACP. We've got our long whip ready to go. And we're going to have acetyl group, acetyl-CoA equivalent, be bound to this other position. So we have two molecules. And that goal here is to condense these molecules together. And when you think about it, malonyl-CoA is an activated form of a two-carbon molecule. We've added a carboxylate. And carboxylate is a great product to an enzymatic reaction because it's rapidly whisked away. So delta G is very negative. So in this first step, we're going to take advantage of this activation of acetyl-CoA into malonyl group by a decarboxylation reaction. So you move... You can see how the electrons flow makes a lot of sense. This is not exactly right. You go up into the carbonyl back down and kick out. But the net product is release of CO2. And where did the CO2 come from? Do you remember how we synthesized this? Do you remember where it came from? It came from a metabolite. It came from CO2. That's the exact same carbon dioxide that we put on there to activate. It's now falling back off. And so we've now extended our carbon chain or two-carbon molecule into a four-carbon molecule. But now we need to reconfigure this. And so we have to get it into a different form. So what we're going to do is we're going to reduce. So we're in the cytosol. We've got lots of NADPH from pentose phosphate pathway. Or from the transport, we've converted NADH to NADPH. We're going to then reduce the beta-position carbonyl group to a hydroxyl group. And then we're going to dehydrate. This is the same chemistry we did in the oxidation, the catabolism of fats, but in reverse. So we're then dehydrating into a membrane in the reverse direction. We're adding water molecules across the double bond here. We're dehydrating or eliminating to make a double bond. And then we're going to reduce again to the fully reduced form. And so now we're ready for another cycle. And so what's going to happen now is that this thylester linkage is going to be swapped for a bond here. So we're going to move this growing metabolite onto the KS domain. And that gets this ACP domain ready to receive the next malonyl CoA. And so we're feeding the molecule. So here's a picture over here. Look at the colors and the way things are growing. So we're not adding to the very end of the fat. We're adding to the beginning. See, and it's extending by two carbon units with each cycle. Do you see that? So here's our first step that we started with. And then we ended up moving that four carbon molecule to this KS position. ACP received another malonyl CoA. We condensed that in. And you can see by the colors that's ending up over here. After the four step process again, we've got that acetyl unit injected at that position. Take another malonyl CoA, stick it there. Another one. Just keep repeating this until you get to palmitate. So fatty acid synthase makes only palmitate. It does not make a C18 molecule. It doesn't add double bonds anywhere. This molecule, or this amazing polypaptide enzyme, makes palmitate the C16 molecule. So obviously the very last step, when this is attached at this KS position, there's a thioester linkage to the enzyme. So you have to release the palmitate. And you do that just with this last activity in here, this TE or thioesterase activity on the enzyme. So that enzymatic activity is not used in the building up cycles. It's just used in the cleavage of the final product off of the enzyme. And so this is an amazing factory of synthesis. Does that make sense so far? I'm not rushing. Any questions? So we're going to sum it up. Let's see what we've done here. So we've taken acetyl-CoA. And we've mixed, remember that first step, we had one acetyl, one malonyl-CoA. And the subsequent steps, it was just the extending molecule in that KS position. We're just adding malonyl-CoA. So we start with one acetyl-CoA, and then we add seven malonyl-CoAs. Use 14 NADPHs. Remember, there's two steps that involve reducing equivalents to make palmitate in carbon dioxide. And you say, wow, that didn't take, you know, that wasn't so bad. It's not that much energy. Well, how did we make the malonyl-CoA? So we had to use seven ATP molecules to convert our acetyl-CoA, to activate the acetyl-CoA for these condensation reactions. So we actually, the true starting point was acetyl-CoA, making malonyl-CoA. So if you sum them together, you've actually used seven ATP molecules. And if you look at beta-oxidation, it's not the simple reverse. It takes more energy to synthesize fats than is harvested when you degrade them. So the number of total reducing equivalents is the same, right? You've got seven of that type, seven of that. Here you've got 14 of NADPH. But here you're only using ATP in the first step to make the coenzyme A derivative. And whereas here, every single malonyl-CoA that came in, you needed to use an ATP molecule. So you do not want these to be going in circles. That reciprocal regulation is important. If it was going in a circle, you'd just be warming up. Your cell would get hot. So okay, now we've made the 16-carbon palmitate molecule. And now we're going to do these decoration enzymes. And so you can use palmitate directly, but you can make the chain longer. You can add double bonds. And we're totally not going to look at all the details here. So you can elongate how? I don't know. You do. You can desaturate, probably with an enzyme. You can elongate again. You can just keep going if you want. You can desaturate the longer molecule. And that's where animals stop in terms of desaturation in plants. You can make really fancy unsaturated fatty acids by adding a second and a third. And these are important. These are referred to as essential fatty acids because we cannot make them, but they provide helpful properties. Remember, we want to have a constant fluidity in our membrane. So we need a wide assortment of lipids in our toolbox to be able to regulate the fluidity. And so these are important fatty acids from plants that are necessary in our diet. So the details, don't worry about it. Okay, here's a cool enzyme. So if you want to add a double bond to a fatty acid, it's going to take a certain amount of energy to add that double bond. So the way this actually happens is by these mixed oxidases. So you're actually going to need the electron affinity of an oxygen molecule. I think it had a ton of electron affinity. You've got to pull these two hydrogen atoms off of this long alkyl chain. So the way that we're going to do this is use an oxygen molecule to pull those electrons off. That has a very high affinity. But wait a minute, this is a four electron transformation. So oxygen going to two water molecules need a total of four electrons. So if we just make a single double bond, we're just pulled two electrons out of our metabolite. So we've got to get the other two electrons from somewhere else. So we're going to waste a molecule of NADPH to be able to go all the way from oxygen to the four electron reduced water. And the reason that we have to do this silliness seems sort of wasteful. You have oxygen and NADPH. It's because it's hard to do this chemistry. You're trying to dehydrogenate a fully saturated molecule. And so that's what was necessary through evolution. Does this make sense? So we've now made a fatty acid with a monounsaturation. And we can use that in our bilayers. Okay, let's finish up, please. Everybody vote. All right, it looks like most everybody's voted. Any other last minute voting? All set. Okay, you want to turn it off. And the answer is D. You guys rock. You get so smart. Okay, I'm not kidding when I say there's a lot left. So let's go through it. Now we're going to go into exotic land. So we're going to start making some of these other lipids. We're going to make trisoglycerals and glycerophospholipids. And so obviously these molecules involve a molecule of ultimately glycerol. And glycerophospholipids are even trisoglycerals. We're going to have to involve glycerol here. And so where do we get the glycerol from? Well, we can get that from glycolysis. So in glycolysis, one of the products is D, glyceraldehyde 3-phosphate. And if you remember the stereochemistry of lipids, that hydroxyl group is in the L configuration. So again, we're going to see a similar strategy. Convert the molecule into something that has no chirality. And then stereoselectively reduce it. And so in this case, the strategy is take the deglyceraldehyde with triosisomerase. This is actually also a glycolytic intermediate, dihydroxyacetone phosphate. But now we're going to do this stereoselective reduction of the carbonyl group into the L configuration, because we want to have our fatty acids sort of pointing in different directions. But we already have the phosphate, because that was already sitting there on the metabolite. The other way we can do this is from a fructose. Remember the catabolism of fructose, you make glyceraldehyde. Remember that get converted into glycerol, if you remember that one slide. Glycerol is close, but we need to add a phosphate. It turns out that fructose has that particular stereocenter already in the right configuration. So we don't need to do this stereoselective reductive strategy. We can just take our glycerol molecule and phosphorylate it with the kinase to make L-glycerol-3-phosphate. You're with me so far? We're beginning to build up these glycerol-phospholipids and trisoglycerol from this glycerol starting point. Now it's obvious what we do next. We've made fatty acids. We can just stick those fatty acids onto our hydroxyl groups on the glycerol so we can add one, and then a second. There's an enzyme acyl transferase that makes new ester-linked fatty acids at these two positions. So that's cool. So this is actually phosphatidic acid. This is the metabolic starting point for elaboration into all different types of glycerol-phospholipids. And so the general strategy here is, well, one thing you can do is you can remove the phosphate, my trisoglycerol, and then you have another hydroxyl group or a diisoglycerol. You have another hydroxyl group. You can make a new ester-linked fatty acid to make the trisoglycerol. So phosphatidic acid is also the feedstock of synthesis of trisoglycerols. But then the other thing you could do is add a variety of different head groups. So this is very close to our glycerol-phospholipid. It is the founding member, but we can just attach different head groups. And the way this is done, there's two different strategies. The basic chemistry is you want to make a phosphodiester linkage to your head group. So you already had the phosphate on the alcohol, because that was a phosphatidic acid. But now you want to add, make a new phosphoester linkage to all the different head groups. I should remember that table, we had all the head groups. We need to attach those. And I'm not going to tell you specifically the type of chemistry that's used for each individual head group. It's too much to remember. This is the general strategy. Either you activate the lipid or you activate the head group. And activation, remember this is a strategy we've seen again and again. We've taken in this case a CDP. So we make the CDP linkage. So we take a CMP, add it to our phosphatidic acid to make this activated form. And then that's a great leaving group. It's a much better leaving group than an O-minus. So the same exact strategy we've seen before. We come in, nucleophilic attack of our head group onto the phosphate leads to cleavage of this phosphoanhydride bond. And then the other way we can do this is to activate the head group. And so we can take our diacylglycerol, remember phosphatidic acid we can cleave off the phosphate to make diacylglycerol. And then that was ready for a nucleophilic attack on our activated head group. So in either process we're releasing CMP and we're attaching our new head group. So the same strategy is used again and again. If you want to do some chemistry it's helpful often to activate either the lipid or the head group. And so this is just the table as a reminder. This slide has not changed since you saw it last. These are the variety of head groups. And you're not responsible for knowing whether the head group is activated or the lipid is activated for each of these. But you can attach all kinds of fancy lipids or head groups to the lipid. What about sphingolipid? How in the world are we going to put this together? And just think about, think through all the structures that you've learned in the last semester and you're like, oh man, how are we going to do this? So this sort of looks like a fatty acid, right? You know, it's got a carbon-carbon link instead of a ester link. What does this look like here? Sort of close to this one part here. I heard it. It begins with an S. Is that hell? Second letter an E. Yeah, there you go. Okay, so this is a serine but sort of not a decarboxylated serine. Do you see it? And that has an amide linkage to a fatty acid. So the way these things are put together is you condense a serine with palmitil CoA. Remember serine is an amino acid. Acid is carboxylates, great leaving group here. And so when we condense these two molecules together, you release CO2 and you're getting sort of close. You now have a carbon-carbon bond. You can reduce the carbonyl group to hydroxyl group. You can take this amino functionality and react it with a fatty acyl CoA. Remember acyl CoA is a great thioester leaving group. And we can instead of making an ester linkage, we're going to make an amide linkage but it's the same general idea. You got this nucleophilic attack on the fatty acid to make this molecule. So, cool. So that's how we make the beginnings of a Sphinx-like molecule. And then we can take this and we can add, remember the Sphinx had a double bond? Is a trans double bond? Whereas other lipids resist. So there's a, again, if you need a double bond in a fat, generally you're going to use the mixed function oxidase because that uses the electron affinity of oxygen to pull those electrons off and to make the double bond. So now you've got ceramide. So ceramide is sort of like phosphatidic acid. It's the founding member of a class of lipids. And then you can continue to modify this in various ways. You can add a glucose, you might remember UDP glucose, or you can add different head groups. So you can take a choline, aqua-phosphatidylcholine, and bring that all the way to diacylglycerol. So take the phosphate with it and stick it on this molecule, this ceramide molecule. So that's how you make them. Just remember, serine and palmityl-CoA is the starting point. Okay, and so the medically-acclined amongst us might be interested that these Sphinx-like molecules are also turned out to be blood groups. So this O antigen, these A antigen, B antigen are more fully glycosylated types of sphingocene or ceramides. You have the ceramide base, you have a glucose, and then each shape is a different type of sugar. So the types of sugars that are attached there actually determine the type of blood antigen. So the ceramide molecule is a constant across all blood antigens, okay? But that's just an aside. So now, what about this guy? How do we make cholesterol? Four rings fuse together. What kind of crazy chemistry do we have to do to make this? So we have a very greasy molecule. We've got four rings. We have an alkyl chain here, and we have a hydroxyl group. And if you look at it, you can sort of look around in this molecule, and if you do some labeling studies, and you have labeled acetate, you'll see that this cholesterol molecule, the most fundamental basic building block of cholesterol, is acetate. Acetate is used to make those isoprene units, which then are stitched together into cholesterol. But ultimately, the original source of everything is acetate. So let's look at this. This is a general schematic, an overview slide, and then we'll dive into the detail in each step, because it's important, especially for medical-interested people. You want to know how cholesterol is made, because a lot of people die from problems with cholesterol. And so we have three acetates that can be combined into mevalonate, sort of funny-sounding name. Mevalonate looks almost like isoprene. Do you see? Remember the structure of isoprene from the exam key? So here you have... It is sort of funny. Maybe not from the exam, unfortunately. Some of you tried to draw cholesterol, and that was amazing, because it was sort of true, that answer. I was just very courageous to draw a cholesterol. But let's look at this. Let's not remember, okay, exam be gone. Okay, it's gone. So now what we're going to do is decarboxylate and add a double bond, and there is isoprene. But it's a fancy form of isoprene that has a pyrophosphate. That's a fantastic leaving group. When that's cleaved off, any time that's cleaved off, you make pyrophosphate, which is immediately converted into inorganic phosphate, driving the reaction for it, making delta G more negative, starting from the cellular state. So here is isoprene, and here I've drawn... This turns out to be a molecule called squalene, and I've drawn lines here. So you can see how the isoprene units are stitched together. And there's a single enzyme that goes from here to here. Whoa, that's the best. How many bonds can you make in a single enzyme? Okay, so let's bring you through the details. Acetyl-CoA, two of them are combined to make acetoacetyl-CoA. Familiar? And then you add another one using HMG-CoA synthase to make this molecule. Where do we see it? This one you don't even have to remember back to pentose phosphate. Try the beginning of the lecture. These are ketone bodies. This is the initial step in the exact same enzymes in making ketone bodies. So we have HMG-CoA, and then the commitment step, the highly regulated step, is the HMG-CoA reductase. And so this enzyme converts your HMG-CoA to mevalone. And so this is the highly involved conversion of two NADPs to NADP. NADVHs to NADP. Can you release your coenzyme A? Okay, cool. We're committed. We're going to make some cholesterol. The mevalone A is activated. And then curiously, another enzyme does the second. Why didn't they just put two on there to start from ATP to AMP? This is not how it evolved. So here we have two phosphates attached. This is highly activated. What we're going to have to do here is stitch together isoprines, making carbon-carbon bonds. So we need some pretty good activation to be able to do that. And so then we're going to actually add even one more phosphate in this step. And so this enzyme actually capitalizes both steps, you know, both of these transformations. And so you're going to decarboxylate creating your double bond. It's helpful to put the phosphate there. It's a great leaving group. Helps you to decarboxylate and make your double bond. And then we can isomerize this isoprene-ish looking thing into this isomer of an isoprene-ish looking thing. So we make an allele pyrophosphate. We just move the double bond over by one. And now what we're going to do is start stitching isoprines. So the building blocks are isoprines. We're going to stitch them together. So we're going to take two isoprines. We have the allele form and the not-allele form of isoprene. Stitch them together. Here I'm showing the bond that's being made. I put a dashed line between them. So each stitching step releases a pyrophosphate driving the reaction forward. So we make geromeal pyrophosphate. And then we add another one. And we make pharnaseal pyrophosphate. So if you count the carbons, plus five equals 10, plus five equals 15. So now we're going to take two of these C15 units and stitch them together in the other orientation. So these were head-to-tail condensations. And then in this last step, we're going to have a head-to-head condensation. And it turns out that we need to say reduce cofactor to do that as well. So now we've made this really, really greasy squaling molecule. So now we need to convert this into our four fused rings. How are we going to do this? This is magical. Now, excuse me while I get very excited, because I spent two years of my life studying this enzyme. So this is squaling. Squaling can be activated to an epoxide form. That's pretty cool. So this is called oxido-squaling, or squaling to three epoxide. And then in one step, you're going to fuse all four of these rings together to make the building block of cholesterol. So that's called lanostral. And then you do some more small modifications to convert lanostral into cholesterol. So this cyclase enzyme is totally insane. So here is the structure of it. And there's actually aromatic amino acids that stack. So in each position, you have a partial positive charge form each time you make a ring, and that's stabilized by the stacking of the pie aromatic groups. And so not only does this thing make four rings when none existed to start, it also has a syringe and injects the cholesterol into the membrane. So the effluent of this enzyme gets released into the membrane. So cholesterol, that's a good place. If it's where we need to have cholesterol in that environment. Okay, so that's how cholesterol is made. I'll calm down now. I love it. It's beautiful. So cholesterol can be converted into a cholesterol ester, and this is the transport form of cholesterol. So as you're probably well aware, it's important for us to be able to transport cholesterol around our body, either being produced by this biosynthetic pathway or coming in from our diet, and the safe form of transport of cholesterol is in this ester form. But we also talked previously about how cholesterol can be converted into other hormones. It's useful to use cholesterol to feed these other pathways because the abundance of cholesterol is very high compared to these hormones. These hormones are very low abundance. But I'm not going to torture you with all the pathways. I don't know if I remember, these are the collection of hormones that can be made from cholesterol. Okay, so let's think about the regulation of cholesterol synthesis. So we have two inputs. So it's not like anabolic, catabolic. It's anabolic lunch, right? So you have anabolic pathway making cholesterol, and you have lunch, whatever you ate. Everybody is processing their meal, their lunch, and that food, both of these make cholesterol. So we need some way to have both processes regulated, both the uptake and the synthesis. And it turns out that cholesterol can be degraded into these oxysterols, and these oxysterols, once cholesterol levels get relatively high, the oxysterols turn off HMG CoA reductase. Remember, that's the biosynthetic pathway. But they also turn off uptake of the cholesterol. But they turn on storage of cholesterol. So if you have too much free cholesterol floating around, that's bad. You want to stimulate the safe storage of that cholesterol in this ester form. And we also have our hormones. Again, we want to stimulate anabolic processes, the presence of insulin. And when you're starving, not a good time to be making cholesterol. We want to be using the cholesterol we already have. Does that make sense? Any questions? I was stretching aloud when I asked for questions. All right. So many of you are going to be helping patients someday. And the HMG CoA reductase is the target for a large number of these cholesterol-reducing drugs, these so-called statins. And the reason that they're good drugs for HMG CoA reductase is because part of these statin molecules looks exactly like this mevalonate. So that was a substrate for that enzyme. So these are competitive inhibitors. These statin drugs inhibit HMG CoA reductase. So that is important for you to realize. So now we're going to switch gears. We've talked about the synthesis of cholesterol. What about the transport? So there's lots of different particles that are involved in this transport. And so there's a plasma lipoprotein. And so these are particles that have a little protein on the surface. But mostly it's just a flat globule. It sort of looks like a mice cell. So it's not a bilayer of these particles. It's just a monolayer. And inside of the particles is a varying amount of proteins and lipids and triacylglycerals. So the chylomicrons, if I'm pronouncing that wrong. Is that right? Anybody know? Haven't had that class yet? Chylomicrons are mostly triacylglycerals. They're actually really big, greasy-looking things. VLDLs are a little bit more dense. So you can see as the density increases, the amount of protein increases. So you might have heard of bad and good cholesterol. So you have LDL and HDL. And the bad one is the VLDL and the LDL. The good one is HDL. So let's look at the structure of these things. So in these lipoproteins, so we have these four different types. And they have different sizes. So chylomicrons, we actually had to change the magnification to zoom out because these things are massive orbs. And so VLDL is smaller. LDL is smaller. Yeah, and HDL is just tiny. And so in these, we have a lipid layer. But instead of having water inside of this particle, we just have fats, grease. So if we don't need a bilayer, it would be counterproductive. If we had a bilayer, you'd have that hydrophilic surface on the inside, like in a cell, you have water on the inside. But instead, we want to have just one layer, one leaflet here. But you might imagine these are going to be less stable than cells. They just have one layer of lipids. So as especially these larger ones are floating around, cholesterol is just going to be sort of dropping off around. Little macrophages come along and gobble up the cholesterol that's falling out of these. So these are somewhat unstable, especially the VLDL and the chylomicrons. So they're going to be leaching cholesterol. And so this is how it works. So the liver plays a central role. So you have cholesterol coming in from your diet. And you have fatty acids. Those are processed and into smaller parts. So we have bile salts coming from the gallbladder. And those are helping to break down the fats, the various lipids in your diet. And these are then packaged into these largest of particles, chylomicrons. These are then transported to the liver, where they are converted into asterified forms of cholesterol. So C is cholesterol, C is the asterified form of cholesterol. And so then these are transported in VLDL and the cholesterol and fats are delivered to your periphery, the tissues. But as you go around in this circuit, you're sort of dropping cholesterol along the way. So you have macrophage cells that are harvesting up. Think of HDL as a vacuum. It's going around in your capillaries and your blood vessels and sucking up the fats that are dropped off. So this returns cholesterol from your tissues and from your circulatory system back into the liver. So it's very important that there's a balance between the amount of the things that are delivering fats and cholesterol and the things that are picking them up. So this is why we think about this. So this is a physiological view of what we do after we do the biochemistry. So I think the last slide is the LDL particles have on their surface an apoprotein. That apoprotein is recognized by a receptor causing the endocytosis or the engulfment of the particle. The particles are digested in lysosomes where the fats and cholesterol is released and stored in the cell and then the receptor is recycled after you've degraded the fats in the various particles that you might be injecting. But the LDL is the carrier, generally the carrier, the delivery and the zip code on the LDL particles, this apoprotein, so that zip code is read by these receptors saying this is where you need to drop the cholesterol. So obviously all cells need lipids and cholesterol and so they need to be distributed. All right. I made it. That's it. No more fats. Next we have amino acids. It's even worse. We'll get through it. The third part of this class is a lot less painful. We'll be there soon.