 Low, low, low, low, test, test. All right, we're gonna get started. Welcome back, if we could settle down. So just quick announcement tonight actually in Salomon 101. There's a great event, SACNAS, so y'all should come. Great speakers, panel discussion on women in STEM. So I'll be there. So okay, so today you're gonna maybe sense a little change in flavor of the lecture. We're gonna start to integrate lectures together. Think about bigger pictures. And so the fundamental thing that we're doing today is building sugar molecules from smaller molecules, three carbon sugars up to six carbon sugars. It's a process called gluconeogenesis. And we'll be looking at ways in which the enzymes involved in this process as well as regulation. And then we'll think a little bit about storage of glucose and how that's regulated, both the synthesis of starch as well as the degradation of starch. So we're gonna be sort of dancing in between enabilism and catabolism throughout today. And then the last part of the lecture to make the next lecture just slightly palatable, we're gonna put a little bit of that in here. So we're gonna do the catabolic pathways that process fats or fatty acids. And so this is a big picture of where we're at. So yesterday or in the last lecture, we looked at the Calvin cycle, remember three phosphoglycerate could be fed into these pathways. And so there was a little highlighting there of synthesis of larger sugars from this three carbon sugar. And so we know that in anaerobic cells you have conversion of pyruvate to lactate. But as that turns out, that enzyme's actually reversible depending on the true cellular state, delta G. So if lactate builds up, we can run that in reverse. And you'll see how the Krebs cycle is instrumental participant in these pathways receiving all kinds of catabolic degradation products and then feeding them into synthesis of glucose and glucose like molecules. And so just as a highlight in lecture 15 coming up in a little while, we're gonna bring everything together in terms of organ systems. And so there might be a little bit of lack of detail on that subject today and sort of the idea, why do we need to synthesize glucose? That'll become much more clear in a later lecture. But today we're just gonna focus on the process itself. So remember when we looked at glycolysis and there's a variety of steps that are rate limiting. Remember these are these valve functions. They're effectively irreversible. So we had hexokinase, PFK1, and pyruvate kinase. And so all of these had a very large delta G prime knot as well as a large standard chain or large cellular delta G. And so if we don't know yet why we need to make glucose but it is true that we do need to make glucose. So if we're going to do that, we obviously can't just go straight up the same list of enzymes that we used before because these steps are irreversible. So we're gonna need alternative pathways that are also thermodynamically favorable to get around these irreversible steps. And so here is the big picture. So you have the catabolic processing of sugars, glycolysis, synthesis of pyruvate. Remember pyruvate is then imported into the mitochondria. So today we're gonna be taking pyruvate and working our way back up to glucose. And these are these bypass reactions. So instead of going in the reverse direction in a very unfavorable enzyme, we're gonna go through a totally different enzyme and that's how we're gonna get around these troubles. Okay, and so we're gonna go through these steps one at a time and to understand them in a deeper level. So this slide is a little bit confusing. So the majority of gluconeogenesis does occur in the cytosol, but one of that first bypass reaction conversion to pyruvate to PEP, that involves both the cytosol and the mitochondria. And this is the forward direction in glycolysis. Remember the conversion of PEP to pyruvate with pyruvate kinase. This is effectively irreversible. So you'd think it would be just a one step, maybe a, so here we're going from this state up to this state. You would think that could be just a simple step to get you there. But like some of the rigmarole that we did with photorespiration, there's actually a lot of steps to get back from pyruvate to PEP. Now, this is confusing because there's two different ways to get from pyruvate to PEP and it depends on the type of tissue and whether lactic acid is available. When lactic acid is available, we can grab some of the electrons from lactic acid and make a reduced cofactor. So if you look at the previous slide, you'll see to go in the reverse direction, in the forward direction we made NADH, but in the reverse direction we need NADH. So we have to have some way to get some NADH in the cytosol. Typically, NADH concentration is much higher in the mitochondria than the cytosol. So there's two ways we can do that. If we have lactate, we can take the electrons from lactate, stick them on NAD to make NADH, okay? The other way to do it is to export NADH from the mitochondria using a carrier. So we transfer those electrons to oxalacetate, make malate, malate's transported out, and then we have the same enzyme but a cytosolic version working in the reverse direction to transfer those electrons back to NADH. Remember, this is towards the end, remember the crab cycle, this is towards the end where there's a pretty good equilibrium here. So it's definitely a reversible process. And so in muscle cells or other cells experiencing high levels of lactate, we can regenerate our NADH directly in the cytosol. But then we need to convert to PEP. So there's two enzymes involved. One of these we've already have seen before, and that's this pyruvate carboxylase. Remember, we're taking pyruvate as a three carbon molecule and oxalacetate as a four carbon molecule. And then we can then use this second enzyme, this carboxykinase. Remember, we need a phosphorylation there and we need a three carbon sugar to get to PEP. So there's transporters in the cell, both in animal cells, both for malate and PEP. Now, plant cells actually have transporters for oxalacetate, but we don't have that. So we need to convert the molecules into some other form to be able to get them back out of the mitochondria. And you can see these are the same enzymes here, right? So you've got this PEP carboxykinase. This is a cytosolic version. This is a mitochondrial version. Okay, so there's two different ways that we can do this first bypass reaction. So let's look at the individual steps. And this is what I said, you need some way to make NADH. So if you're doing anaerobic respiration, you're gonna have a high level of lactic acid. And so then we can just grab our electrons from lactate. But in other cells, you can actually export NADH, effectively export without actually moving the molecule. So you hitch the ride of the electrons on a carrier and then re-deposit them on NAD to make NADH. Okay, so here's the first step. Where did we see this step before? Does this look familiar? So we have pyruvate, and we're synthesizing oxaloacetate. Do you remember? Anaplerotic reactions, right? So this is a three-carbon molecule getting converted to a four-carbon molecule. And so one of the reasons for anaplerotic reactions is anytime you siphon off a metabolite to an anabolic process, the level of all the Krebs cycle intermediates is decreased. But here we're sort of showing both sides. We're the filling up reaction. And then the next step, we're gonna be taking oxaloacetate equivalents out of the cell, okay? And so we're using these existing mechanisms, existing enzymes to feed into gluconeogenesis. So remember, this is biotin. It catalyzes the tautomerization of pyruvate to this form, which is much more reactive than carbon dioxide. This is the same exact slide, it's just a review. And so we're gonna make this four-carbon molecule oxaloacetate from pyruvate using this enzyme. And so this enzyme is regulated by acetyl-CoA. So now you have to put on your sort of integrative cap and think about why might we want to use what kind of reporting or indication is provided by a buildup of acetyl-CoA? What does that tell you about the rate of the Krebs cycle in terms of compared to the rate of feeding into the Krebs cycle? So that indicates the input is much higher than the generation of energy. So a high acetyl-CoA level indicates a high energy charge. Remember in the previous lecture, I gave you this idea of energy charge? So the concentration of ATP is very high relative to other molecules. So it's a surrogate marker of energy charge. So you could use ATP or you could use AMP in an inverse sort of way. Or you could use acetyl-CoA. It's telling you, you know, the cell is just stuffed full of energy. We don't need any more Krebs cycle. So it's a surrogate marker. And so what we're doing here is we're exporting again, reducing equivalents to the cytosol where we need them because we're gonna push glycolysis in reverse. So that, one of the dehydrogenase enzymes is gonna need as a substrate NADH. Okay, so this is the first step. And the second step in the first bypass reaction is this PEP-carboxykinase, right? And so we're gonna remove a CO2 going from a four-carbon oxalacetate to a three-carbon PEP. We're also gonna be adding a phosphorylation. That's what's necessary. Okay, and so this is a favorable process in thermodynamically speaking. So overall, if you look at both of these steps, you're converting pyruvate to PEP, but you're using both an ATP and a GTP equivalent. So it turns out that this PEP-carboxykinase prefers GTP. And ATP is preferred by this pyruvate carboxylase. So remember, pyruvate kinase is just making an ATP. So we're losing energy here by going in the reverse direction. We're using both an ATP and a GTP. Okay, so we're expending energy to go. And this, you know, it sort of makes sense that pyruvate is a less energetic molecule of phosphorylienal pyruvate. Does that make sense so far? So this is our first of three bypass reactions. Questions? Okay, so the second bypass reaction is this conversion, a fructose 1,6-bisphosphate to fructose 6-phosphate. So all the other enzymes up to this step are exactly the same as glycolysis. Remember, those are working right around delta G of zero. So you just feed molecules in at the bottom, and those will make it all the way up to fructose 1,6-bisphosphate. But now, this PFK1 step in the glycolysis direction was too exergonic to be reversible. So instead of transferring this phosphate back to an ADP, we just release it to solution. Right, and so that's exergonic because you have the increased resonance of the phosphate and you have less negative charges in this molecule, but it's not exergonic enough to synthesize an ATP molecule. So we're losing energy here as well, going in the reverse direction. Okay, and so this is just a simple hydrolysis catalyzed by this fructose 1,6-bisphosphatase 1. And so the forward direction is PFK1. And then this is our third bypass. So remember, hexokinase was converting glucose to glucose-6-phosphate. And so we're doing the same exact strategy again. There's just not enough energy to take this phosphate and put it on an ADP molecule. So, but there is enough energy to just hydrolyze this phosphate off, lowering the activation barrier with the enzyme. So it's delta G prime nod is exergonic. So these are, and you'll notice that these bypass reactions are very exergonic. They're effectively irreversible. You with me so far? These are three bypass reactions. Here you have those two enzymes and this interplay with the mitochondria, but these are all in the cytosol. These are all the exact same enzymes. You're just pushing things in the backwards direction by building up some PEP. And then you get to this step, the second bypass reaction and the third bypass reaction. So this is how we synthesize from pyruvate a molecule of glucose. And so we can either make glucose and export it from the cell. It's a great way to transport some sugar around, but we can also use the glucose-6-phosphate to make starch. So we can skip one of these bypass reactions if we're just making starch. Okay, so let's look at the overall process. Say, what would it take to take glucose all the way down to pyruvate then all the way back up to glucose? So let's look at, so this is the gluconeogenic, the upwards direction. So you have pyruvates going to glucose. You have ATPs, GTPs and NADHs being converted to ADPs, GDPs and NADs. So glucose, remember the summary of that is you're just synthesizing two ATP molecules and two NADHs, reduced cofactors. So if you sum these two equations together, you know, things on opposite sides get deleted. You end up with just hydrolyzing ATPs and GTPs. And so this overall, if you go from, obviously this is a bad thing. You don't wanna go from glucose to pyruvate back to glucose and around and around and around. You'll just be generating heat. So you'll just be hydrolyzing ATPs and GTPs. Each circuitous route that you make, you'll be wasting an ATP and a GTP molecule. And so these bypass reactions need to be co-ordinately regulated. So when one direction is on, the other direction has to be off to prevent this waste of energy. But what does that mean? We're typically regulated. There's some last minute order change in the slides if you didn't, it's just go a few slides ahead. So here you have catabolic processes such as glycolysis and you're making ATPs and NADHs and other reduced cofactors. And then the gluconeogenesis is an example of an anabolic pathway. We're taking ATP, NADH and GTP to synthesize a larger molecule, glucose molecule. And so the direction, you don't want this to go in a circle. Or you just needlessly hydrolyze ATPs. You just waste energy. And so you need with high energy charge. In other words, when the levels of ATP are much higher than ADP or AMP, you need to go in the anabolic direction. That means we don't need to burn any more glucose. Mine is well store some up in starch. Whereas if the levels of ATP are dropping, usually the typical average level is about 10-fold access of ATP over ADP and AMP. So if it drops much below that, then this pathway needs to turn on so that we can feed more pyruvate and acetyl-CoA into the Krebs cycle to make more energy. Okay, so there's a delicate balance here depending on the energy charge in the cell. So remember, this is the exact same slide I showed before. We calculate the energy charge by the summation of all adenylates with various phosphates. So we have ATP, the ADP form, and the AMP form. And then we have the useful forms of those molecules that can push anabolic processes forward. In other words, ATP or ADP, which you can think of as an ATP equivalent because two ADP molecules can be converted into one ATP. So really an ADP is just half of, or half an ADP is equivalent to an ATP. Or half an ATP is equivalent to an ADP stated correctly. So we're gonna come back to this concept of energy charge again and again and again. So there's really two things going on here. We need to sense the energy charge in the cell, but we also need to sense the levels of glucose. So energy charge has a lot to do with what you're doing. So if you're running around, you're gonna be using up energy. And the levels of glucose have to do with intake as well as utilization, right? So there's sort of independent parameters. So we don't wanna have any of these futile cycles, like so we only wanna make glucose if we need, if we're short on glucose, right? And we only wanna do glycolysis when we need some energy. Okay, so let's look at this. And so you might wonder, okay, so we need to respond to these two independent, they seem sort of connected, right? But in reality, if you just sit in the couch and drink sugary beverages, your energy charge is gonna be high and glucose is gonna be high, right? So we need to coordinately regulate these things to sense two different things, the energy charge, the levels of glucose. And so this regulation occurs at the irreversible steps. So remember, we already talked about PFK1. Remember, that's a good place. It's better than hexokinase because some of those feeder pathways feed in below hexokinase, but above PFK1, right? So this is a good place to regulate glycolysis. And here we have a molecule AMP. So you can see that obviously is a energy charge sensor. Okay, so if AMP levels are high, that means ATP levels are low, right? And so that means we need to make some more ATPs. We're gonna reduce the amount of AMP by converting it into ATP. Okay, but on the other hand, this reverse direction, this phosphatase that removes a phosphate from fructose 1,6-bisphosphate, well, if the level of AMP is high, we need to inhibit this reverse process. We don't wanna have this circle going on here. Okay, so this is just sensing energy charge. And we already talked about there's some redundancy here, right? So ATP is sensed, ADP and citrate, remember is to help us synchronize glycolysis and the Krebs cycle to have those be at a similar rate. But how do we sense glucose? Okay, so this is this mysterious molecule just appears out of nowhere, fructose 2,6-bisphosphate. So that's different than fructose 1,6-bisphosphate. So fructose 2,6-bisphosphate turns on glycolysis and turns off gluconeogenesis. And this is done by allosteric regulation of these two different enzymes. Okay, and so remember when we talked about allosteric regulators and how you could have these heterotrophic allosteric regulators, and these could be negative or positive. They could affect either the KM or the Vmax of the enzymes. So for example, here is PFK1. So in the presence of fructose 2,6-bisphosphate, wherever it came from, you have the KM being decreased, or you should say in terms of an allosteric regulator, the K.5 is decreased because their sigmoidal curves. Whereas in the absence of fructose 2,6-bisphosphate, this enzyme has a much lower apparent binding affinity. Remember there's an inverse to the KM or K.5 in terms of binding affinity. Okay, so PFK1 is turned on in the presence of fructose 2,6-bisphosphate and turned off in its absence. And here is this other enzyme, this phosphatase, fructose bisphosphatase one. And so in the presence of fructose 2,6-bisphosphate, that's turned off and in the absence it's turned on. It's the exact opposite of this other enzyme. So the allosteric regulator, fructose 2,6-bisphosphate, binds at a site other than the active site, changing the conformation in order to make the catalysis at the active site more or less favorable. Okay, so this is pretty straightforward so far. And here's a little picture of the molecule here, fructose 2,6-bisphosphate, similar to the glycolytic molecule. Okay, so here we have this molecule that's co-ordinately regulating. So when you have fructose 2,6-bisphosphate, PFK1 is turned on and fructose bisphosphatase is turned off. Okay, so at high levels of fructose 2,6-bisphosphate to recap, glycolysis is increased, gluconeogenesis is inhibited. At low levels of fructose 2,6-bisphosphate, glycolysis is inhibited and gluconeogenesis is induced. With me so far, but there's an obvious question. Where did this thing come from? It just came from above and indicated glucose. There's some, I've told you that it's a glucose sensor. But what does that mean? How is it sensing? So let's look at this a little more. This is the enzyme, confusing names here. These enzymes make an allosteric regulator. They're not part of glycolysis, they're part of regulation of glycolysis and gluconeogenesis. So fructose 2,6-bisphosphate is made by PFK2, different than PFK1. And fructose 2,6-bisphosphate is cleaved to fructose 6-phosphate for fructose bisphosphatase 2, different than fructose bisphosphatase 1. Same sort of chemistry, right? You've got ATP going in this direction and you've got just hydrolysis of inorganic phosphate in this direction, okay? Does it make sense? Isn't it annoying? It's like, actually the structures look nothing at all similar, okay? And so this PFK2 and FBPase 2 are actually one polypeptide, a protein with two domains. The other one, PFK1 and FBPase 1 are not just one polypeptide with two domains. So here you have one domain is a kinase and the other domain is a phosphatase, okay? Which one is which? Looking at the happy picture. And why? Should we turn on the clicker? Which one is which? Well, so one of them has something in there, right? One of them has something. Does that look like an inorganic phosphate? Oh, maybe. There's like an adenine hanging out here. So that's gotta be the kinase, okay? You with me? This is like layers here that we're building up. It's a little confusing. I'm gonna summarize the next slide. Okay, so we're making fructose 2,6 bisphosphate by these two enzymes are regulating the abundance of this molecule. We have a phosphatase and we have a kinase. It's a dual function single polypeptide with two domains. And here is everything. And we're gonna go through this and I'm gonna try not to reverse things. Okay, like I did last year. Okay, so you have one regulating molecule making two-domain enzyme, single polypeptide. We've got PFK2 and FBPase2. And now we're gonna add just one more layer onto this. So we're gonna have a phosphorylated version of this two-domain enzyme. And we're gonna have an unfosphorylated version. Now remember, this is actually phosphorylation of the polypeptide. And so when we have phosphorylation of this thing, the phosphatase activity is on and the kinase activity is off. And when it's dephosphorylated, the kinase activity is on and the phosphatase activity is off. Okay, and so in this state, we're making fructose 2.6 bisphosphate. And so in this state, we're turning on glycolysis, turning off gluconeogenesis. In this state, we're cleaning the phosphate off the bisphosphate molecule and we're removing fructose 2.6 bisphosphate, which has the effect of turning glycolysis off and turning gluconeogenesis on. And so the oscillation between the two activity states of this dual-domain enzyme is mediated by obviously a kinase and a phosphatase. In this case, we're talking about a protein kinase and a protein phosphatase. So the phosphatase removes the phosphate, favoring glycolysis, disfavoring gluconeogenesis, and the kinase adds the phosphate and this is turning off glycolysis and turning on gluconeogenesis. And this is where the sensing of glucose occurs. It occurs right here with this hormone insulin. So insulin is produced in your pancreas as a response to glucose being present. So if glucose is present, insulin is present. And if insulin is present, we shift this enzyme to this form, turning glycolysis on. We want glycolysis if glucose is present, turning gluconeogenesis off. Now in the absence of glucose, your pancreas makes glucagon. And glucagon shifts the enzyme to this form, removing the regulatory molecule fructose 2.6 bisphosphate, turning off glycolysis and turning on gluconeogenesis. If we don't have glucose, might as well make some more. We need to, you know, your body needs a certain level of glucose. And so if we're running low on glucose and perhaps we don't have, we're not drinking sugary beverages, we don't have any sugars coming into our diet while we need a certain level of glucose. Your brain dies without glucose. You have to have glucose in your body. Okay, does this make, this is a very confusing slide because there's layer upon layer upon layer of regulation. Okay, does it make any sense? Any questions on this? Yes. So that occurs in the pancreas. So there's receptors in the pancreas that cause the pancreas to produce insulins in these beta islet cells. So we'll look at that a little bit more in lecture 15 as well. Okay. You with me so far? I didn't, I explained it well. No, really? I did it this year? Cool. Okay, I'm gonna move on before you ask any questions. Okay. So we have a decision. Now it gets really, your mind is a little bit warped, but now I'm gonna take you to a whole new level. So we have pyruvate. And pyruvate is the decision point. You might say, what about acetyl-CoA? Could that be a decision point? So pyruvate can be through gluconeogenesis converted into glucose, or pyruvate in the mitochondria through pyruvate dehydrogenous complex, a irreversible step can be converted to acetyl-CoA which feeds into the Krebs cycle. And so acetyl-CoA, remember that's our happy surrogate marker of energy charge. And so if we have enough energy, no need for pyruvate to be converted to acetyl-CoA because we already have enough energy. Might as well store that, the energy in that pyruvate in a glucose molecule. So starch is just a beautiful way to store energy when you have enough. So acetyl-CoA, our surrogate marker of charge, is giving us a sense of how backed up the citric acid cycle is. And so this has one direction is, you can have them both going on, that would be confusing. You have one direction on, one direction off. But acetyl-CoA, can that be converted into glucose? Think about it. So acetyl-CoA, this pyruvate dehydrogenous complex is effectively irreversible. Remember we spent that beautiful slide, it's got this massive, what is it, five mega Dalton, mega complex thing. And that thing has got all kinds of regulation and that is effectively irreversible. So we're not gonna be going this way from acetyl-CoA, but maybe we could do some other more circuitous route. We'll think about that. Let's think about this. So one of the ways that you can make glucose and gluconeogenesis is by converting amino acids into glucose. So these are called glucogenic amino acids because they can be catabolized to these simple molecules that are involved in the Krebs cycle. So each of these amino acids listed can be converted into either pyruvate, alpha-ketoglutarate, oxyrate, fumarate, succinyl-CoA. And remember as long as, as soon as you convert any of these amino acids to one of these Krebs cycle intermediates, they're all interchanged, at least these guys, right? There's a cycle. So you make one, you've made them all and you feed into the Krebs cycle, every molecule you can feed in, you can feed out to make a glucose molecule. Okay, but let's think about this. So from pyruvate, you can take these amino acids, convert them to pyruvate and then up gluconeogenesis to glucose. And citr... And we can also siphon, we can have these amino acids coming in at very places in the citric acid cycle. And those can be converted to oxaloacetate. Remember, our bypass reaction involved oxaloacetate. And so that can feed into gluconeogenesis. But acetyl-CoA absolutely cannot be converted into glucose. Think about that for a minute. So it's obvious, you know, it's not just gonna go pyruvate dehydrogenase backwards, that's effectively irreversible. What about going forwards into Krebs cycle and being converted into oxaloacetate? Why wouldn't that work? Let me show you the picture. So why can't I just take acetyl-CoA, convert it to oxaloacetate and then pull that off and make some glucose? Why? I did not understand this as a student after taking biochemistry class. So I think maybe some of you might not understand this. Why not? Where are my friends that got 100 points on the exam? People are being punched. Think about it. Count the, look at it in terms of the number of carbon atoms. So you might be, do you got it? That's right. So you have two coming in, okay. Making this molecule, how many carbons in citrate? Six, six, five, four, four, four, four, four. Two come in, two come out. The thing that's removed from the Krebs cycle is oxaloacetate or actually malate is transported out of the mitochondria. And so if you put two in, you have two carbon dioxide's coming out, right? There's just, if you take an oxaloacetate, you're minus four, right? So you're sucking, and as soon as you remove oxaloacetate, all of the Krebs cycle intermediates decreased, right? But what about alpha-ketoglutarate? I said that was glucogenic. Why is that one glucogenic whereas acetate-CoA is not? Do you see it? How many carbons in alpha-ketoglutarate? Five. And so what happens if we're gonna take off like a malate? One of the carbons goes to carbon dioxide. Four of the carbons come out as malate. It's carbon neutral. Do you see it? So you can't take out acetate-CoA because it's a minus four when you pull that malate off. But if you come in as alpha-ketoglutarate, five carbons in, one carbon out of CO2, one out is the four carbons out as malate. You're not reducing the abundance of any of the intermediates. Does this make sense? And this is why, you know, plants are different than us. I mean, spinach sorta has a lot of similar types of biochemistry, but not this part. In plants, we saw the glyoxalate cycle where it could take two acetate-CoAs and make an oxaloacetate. But here, we can't do this. We can't go reverse through pyruvate dehydrogenase and we can't go forward through here because, you know, there's no net, there's a minus four carbons when you try that. So make sure this is something that's very, very critical. And so these are plants. Remember, plants, you could take your two acetate-CoA molecules and actually make a succinate molecule. And that succinate molecule, four carbon molecule, can be brought out and fed into the Krebs cycle, filling up the Krebs cycle to help replenish the capacity to do anabolic pathways that feed off of the Krebs cycle. Okay, so this is why plants have seeds, right? They're full of fats. And we'll learn at the later part of today's lecture how fats can be converted into acetate-CoA. But fats cannot be converted into glucose, period. End of discussion in animal cells. Glycerol can be fed into glycolysis, which is part of a fat, but the actual fatty acid itself is converted to acetate-CoA, can't go reverse through pyruvate dehydrogenase, can't go forward to malate through the Krebs cycle without causing damage to the Krebs cycle. I mean, it could, but, you know, every molecule you take out, you would reduce the efficiency of the Krebs cycle. You cannot productively feed it through that direction, okay? And so in plants, you can actually transport oxaloacetate. You don't need to do this malate shuttle that we saw in animal cells. And oxaloacetate can then be converted by this carboxykinase activity into PEP and then into various phosphorylated sugars. Okay, you with me so far? Little break in between. And actually I have a big announcement we're halfway done with the class with this slide. Woo-hoo! Halfway, lecture, whatever, 12. Halfway through lecture 12. It gets better. Let's finish up, cast your vote. All right, I wanna get going, throw up an answer. Guess, Hail Mary. Everybody all set? Is the number increasing? Please put something down. All right, enjoy your rest break. You're halfway through the class, rest break. This is an annoying question because there's just a lot of words. Right, so you spend half the time with figuring out which one it is. So there you go. Good job. The correct answer was C. All right, we're gonna dive back in. There's a lot coming up. So we've done gluconeogenesis. Woo-hoo! Now we need to think about where did this starch thing come from? So remember glycogen is animal starch and we have both alpha-1-4 and alpha-1-6 linkages. We have a bunch of non-reducing ends and a reducing end. But is it really a reducing end? You'll see today the exciting conclusion to that story. So this is how we store glucose. When we have too much glucose and our energy charge is good but the glucose keeps coming in, we need to store that away for a rainy day and so we're gonna make glycogen. So polymer has both these alpha-1-4 linkages, these linear parts and at each branch point, remember there's an alpha-1-6 linkage. So this is a review. We're gonna review a couple of these and then generally it's pretty branched. It's about every eight to 12 sugar residues is a branching residue. And so plants also have starches. They have amylose, which is this molecule here or amylopectin, which is very similar to glycogen but it's a little bit less lower frequency of branching. Okay, but we're gonna focus on the animal starch glycogen. So remember the structure is this helical and every time you have a branch, you have another helical segment coming off. And so as I said, amylopectin is a little bit less branched than glycogen. Okay, this is just review, just to freshen you up but how do we make this molecule? So the way we make this molecule is take a phosphorylated sugar. This is a very abstract slide. So I'm saying NTP. So this could be any of four bases and we'll see today in the case of glycogen, we're gonna use UDP. So we have some nucleoside triphosphate and that molecule is going to be literally attached to our sugar, right? So we're gonna make this new phosphoester bond and we're gonna release pyrophosphate. Now this is one of these strategies. So we're taking our molecule all the way down to the AMP level. And the reason that's beneficial because this product vanishes almost instantly, it's cleaved to phosphate, very, very fast process. And so this product is immediately whisked away. So Delta G is much more exergonic here because this other product is immediately removed. And so now we have this weird sugar. So in our case, we're gonna be thinking about glucose and we're gonna have a phosphorylation on that sugar and that's gonna be attached to an AMP or a UMP. So it's gonna be actually a UDP. But why, this is weird. Why, remember we saw this before with galactose where we were putting a UDP and we're inverting that stereocenter on galactose to make glucose. And so I mentioned at that time some of the benefits of doing that. One is the irreversibility of this. So as soon as we make that pyrophosphate, that thing is immediately cleaved. That product is gone so the reverse direction is disfavored starting from the cellular state. There's a larger thing to grab. There's this big UDP or NTP to grab. And so that helps in the binding, if any, helps the catalytic activity to have something large to bind to like that. It's relatively still reactive. So we're preserving its reactivity because hydrolysis of that or transfer of the NTP or NDP to another molecule is still relatively energetically favorable. And the big thing is it allows us to mark molecules. So you're gonna see two processes take the buildup of glycogen and the degradation of glycogen. And the buildup reactions, we're gonna have these UDP tagged glucose molecules. But in the breakdown process, there will be no UDP tagging going on. So as soon as you put a UDP tag on glucose, that marks it for synthesis of glycogen. It helps us to regulate these processes. And so here, remember glucose-6-phosphate, that's the product of hexokinase. But you might imagine, if I'm gonna wanna make an alpha-1-4 linkage, I don't wanna make an alpha-6-4 linkage. So really what I need to do first is move this phosphate to that hemiacetal position. I need that to be in the one position because I desire to make one-four linkages, not six-four linkages. So we take that glucose-6-phosphate either from hexokinase or coming up from gluconeogenesis and I mutigenize it or I convert it to a glucose-1-phosphate. And then when I have the glucose-1-phosphate, I'm going to put a UDP group on there. So I'm gonna add the uridine monophosphate to the existing phosphate that's already there to make a total of UDP. So now we have UDP glucose and it's UDP-related at the one position. And this is catalyzed by this UDP-glucose pyrophosphorylase with me so far, so we've tagged our molecule. And now we have glycogen synthase, very creative naming here. And so we have glycogen, or we have our tagged glucose and we're going to add that through this enzyme at the four position. So we're gonna make a new acetal linkage between the one position and the four position dropping off our UDP. And so this is a chain extending capability. We're not adding it to the one position, right? We're adding it to this four, this non-reducing in. We're extending it, each cycle of this glycogen synthase extends that in by one residue. Okay, and so we're gonna keep doing that. But to be able to start to do that, this enzyme glycogen synthase does not know how to take just one, two mono saccharides and combine them together. Just hasn't evolved that capability. It can only extend pre-existing non-reducing ends that are at least eight residues long. So we need something else, it's just how it evolved, to make this seed so we can then extend with the synthase. And that seed is made by an enzyme called glycogenin. So glycogenin is not technically an enzyme. In that it is not possessive. So when it makes one, there's a single glycogenin protein per starch granule. And it never leaves the starch granule. So when you have glycogen starch, it's actually a mixture of protein and carbohydrate. And so let's look at this. So there's a tyrosine residue on the glycogenin protein. That tyrosine residue is attached to the first glucose. Again, we're using the same feedstock, this UDP glucose, and we're making this one linkage, the one position of the first sugar attaches to the hydroxyl group of a tyrosine amino acid within the protein. And then the same, so this is called gucosal transferase activity of the glycogenin protein. And that glycogenin protein has a second activity, which is a chain extending activity. So the first activity puts the first sugar on to the tyrosine. The second activity makes alpha-1,4 linkages. So it adds the second, the third, the fourth protein and stops right around eight total glucose molecules on there. But then it just stays. So you still starch granules have a glycogenin, every single one at the center, is glycogenin attached through a tyrosine to that reducing end. So there's actually technically no reducing ends, right? Cause this is an acetal, right? Looks weird. Yeah, looks funny cause it's coming out straight. So that's not reactive at all. So we've primed it and all we do is take our glycogenin synthase. Once we've primed it with glycogenin, so glycogenin is weird. It's binding and it has two different activities but it's not processive. So it doesn't like let go at the end and then prime another starch granule. The only way it's gonna be released is if we completely degrade this starch granule. So here's the glycogenin protein sitting in the sea of a starch of glucagon or glycogen. And so we also need branches. So if we have just glycogenin synthase, we just have alpha one four linkages. We need alpha one six linkages to make branching. And so this enzyme is creatively called glycogen branching enzyme. So glycogen branching enzyme is weird. Look at what it's doing. So it takes a preexisting stretch of alpha one four linked glucose molecules. It cleaves off such that this length is in the ballpark about a seven sugar residue is long and it transfers this stretch to the sixth position. And you might wonder, well, why does it pick the sixth position? I don't know for sure, but I could speculate. It probably has something to do with this thing hanging out on a methyl group. Remember the sixth position? You have that methyl group, you have the hydroxyl and so it's very accessible. We're gonna try to pack in as much as possible but we don't wanna make branches here and here because it's just, you wouldn't be able to stuff as much a starch into a granule. Okay, and so this branching enzyme is able to do this again and again and again. So glycogen synthase just keeps cranking along, extending here and when they get to a certain length we can branch and then we branch and branch and branch. So this is not precise science. It's not like if you took a ruler to every stretch of glycogen that it would be exactly a certain number of amino acids long. It has to do with when the branching enzyme comes about, oh, that's getting long, I better cut that and move it over there. So it's a general, the general average length. It's about eight, okay? So this is it. This is the anabolic process that makes glycogen. We've now stored it. We're gonna go backwards. We're gonna go the other direction and this one's pretty easy, right? So you just take your glycogen at your non-reducing end. You have an enzyme called phosphorylase which is going to cleave that glycosidic linkage in a replace with a phosphate, right? And so this is handy to have a phosphate because we could just take a mutase, move that to the sixth position and off we are going to a glycolysis. So glucose one phosphate, it's like, what's that? We need to move that first to the sixth position but we're preserving some energy here by in this single reaction both cleaving the glycosidic linkage and adding the phosphate molecule. So this is a phosphorylase, phosphorylases. And so here we have a bunch of non-reducing ends. This enzyme can work on them all simultaneously clipping off one monosaccharide at a time from each non-reducing end. And then you get to a certain point and this glycogen phosphorylase just cannot deal with how packed things are near the branch points. So we need a de-branching enzyme and it looks almost like the opposite of the branching enzyme. So it takes a certain stretch, right? And moves it over to a reducing end. But in this it's a little different because it leaves one glucose. And this single glucose needs its own little enzyme, this glycosidase activity of the de-branching enzyme to cleave that off. So we have the transferase activity transferacing large stretches of sugar near a branch point. And then we have the glucosidase activity cutting off a glucose. So that's how we disassemble the thing. Obviously we don't wanna be making it, tearing it down, making it, you don't want this to be a circle. You would waste energy. So we need again, coordinate regulation. Thank God it's a lot more simple than the last one we saw. So we have two key enzymes here, this phosphorylase and we have the phosphorylase that we saw previously. And then we have the glycogen synthase. And then again, these are regulated by conformational change induced by addition of a phosphate to amino acid within the enzyme. So we have the less active form that's de-phosphorylated for the phosphorylase and the active form that is phosphorylated. And the same with glycogen synthase, but it's the opposite. In the glycogen synthase, the phosphorylated form is inactive and the de-phosphorylated form is active. And it makes a whole heck of a lot of sense that glucagon turns one on and turns the other off. So what is gluca? Glucagon is a signal from the pancreas saying we're running low on glucose. Stop making glycogen, start degrading glycogen so we can bring our glucose levels back to a steady state. And we have insulin here, also regulating this process. Okay, so this one's a lot more simple. We have a kinase and a phosphatase, right? And so the kinase is adding the phosphate to the phosphorylase and the phosphatase is removing it. And the same thing here, we have a phosphatase removing the phosphate and kinase is adding the phosphate. In this case, this one kinase activates another kinase that then adds the phosphate. Okay, so it's a lot more simple, but the same goal is achieved. Coordinate reciprocal regulation of the degradation and the buildup of glycogen. With me so far, we're gonna do something entirely different now if there isn't any questions. So I wanted to cover a little bit on the next lecture, the lipid metabolism. There's just a lot, remember the lipid lecture? Just describing the structures, took a whole lecture. We're gonna look at some of the pathways to make all those structures. So we need more time. So instead of talking really fast next time, we're gonna start with this. Now we're gonna talk about the catabolic processes that degrade fats into simpler molecules like acetyl-CoA. Remember triacylglycerols? You have ester-link fatty acids attached to a glycerol molecule. And so there's a lipase that cleaves these fatty acids off, so that's the first step in the process. We have a lipase. And now we have fatty acids in solution. The glycerol molecule that's produced one of the two products of the lipase. We have fatty acids produced and glycerol. Glycerol is sort of good, but it's backwards. So if we wanna feed this into glycolysis, this is the wrong stereochemistry. So when we cleave off those fatty acids, we have the L-prochiral stereosopharm. So here this is L when we add a different substituent. So we have L-glycerol-3-phosphate. And remember we had a previous example where we had to invert a stereocenter. Do you remember where we did this before? And do you remember how we did it? Does this look familiar? Nature has the same trick here. So first you oxidize the same as in galactose. That's an epimer of glucose. We inverted that one stereocenter by first bringing it to carbonyl, which is sort of achiral, and then stereoselectively adding the water back. Okay, and so we first transfer some electrons from this molecule to NAD to make NADH. We have a carbonyl group, and then we stereoselectively add the hydroxy group in that D position. And so now we have D-glycerol-3-phosphate. We can feed that into glycolysis. We wouldn't have been able to feed in L-glycerol-3-phosphate. The next enzymes wouldn't know what to do with that. Okay, so same synthetic strategy that we saw with the galactose catabolism. Okay, and so what we need to do here is activate. So we dealt with glycerol, just feed that right into glycolysis, and then we're good to go with that. But the fatty acids, we need to first activate them. And so the end output of this is to make a new thioester-linked fatty acid. And you'll see throughout the remaining slides, we have an R-group. The R-group is the fat, whatever it is, right? So if it's palmitic acid, there's 16 carbons total in that fatty acid represented, abbreviated as an R-group. So we're gonna do the same exact strategy, very similar to what we saw just previously with the UDP glucose, but here we're gonna have an AMP group attached to the fatty acid. So we have a phosphoester linkage to this, in this ester here. And then we're just gonna swap in a coenzyme A, remember that has a free thiol, and that can make a thioester linkage. And so this reaction, because we're going from ATP all the way down to an AMP equivalent, that produces a pyrophosphate. Delta G is very negative because pyrophosphate is immediately whisked away and converted to two inorganic phosphates. And so both of these steps are exergonic starting from the standard state. Okay, so all we've done so far, so we have the same exact alkyl chain, fatty acid, but we've stuck it on to this large coenzyme A. So the net process, we've used an ATP and we've converted that all the way down to the AMP form to activate the fatty acid. So fatty acid contabilism starts in the cytosol, but think about what we're gonna do. Fatty acids are a reduced form of carbon relative to sugar. And what we're gonna do is feed this into the Krebs cycle. So we gotta radically decrease our oxidation state here. And so why not do that, where we can very productively utilize that energy, catch those electrons? We could have done it in the cytosol, but then we'd have to ferry all those NADHs in and that would be wasteful. Why not take the whole fat, stick it on a carrier and whisk it in here? Okay, and the other advantage we have here is see we have coenzyme A here. If we would just move, there's different ways we could have transported it. We could have just transported this thing in, but there would be this net movement of coenzyme A into the mitochondria. The alternative way is first transfer this fatty acid to some other molecule such as a carnitine. So here we have a thioester linkage. We transfer it to make a new ester linkage to carnitine. So carnitine is just a molecule that has some charges and that can be bound to these transporters. Also coenzyme A is a bit big for being transported. And so this is a bit smaller than coenzyme A. It helps in the transport process. So we then take our fatty acyl carnitine, transfer it into the mitochondria, and then release it, regenerating our thioester linkage. So now we have the full length of the fatty acid, and it's the same molecule. There has been some chemical transformations, but the end product here is the same molecule. We have our fatty acyl coenzyme A. And we'll learn in the next lecture that this is the point of regulation of this process. We'll come back to that. It's regulated by malonyl coenzyme A, which is involved in the synthesis of fats. So we don't wanna be synthesizing fats when we're degrading them. Reciprocal regulation is a happy good thing. Okay, so here is our oxidation. So here we're in the furnace. Like the match, start transferring electrons. Do it in a controlled manner. We don't wanna just have a big puff of smoke. Have this thing converted into acetyl coenzyme A. We want this thing to be gradually and systematically taken apart. And the way we're gonna do this is by cleaving the bonds indicated in these blue lines. So this is beta-oxidation. So we're gonna take each of these bonds and cleave them. Is there any questions so far? We have a little pause. We're about to light some matches. It's gonna get exciting. Any other questions? We're in the furnace. No? Wow. So it's pretty simple. There's only four steps. So this is a process called beta-oxidation. So the beta, you know, alpha, beta, gamma, delta, delta, delta, delta. So beta is the second position. So the way that's why we call it beta-oxidation. And you're gonna look at this synthetic strategy. You're gonna say, nature's lazy. I've seen this before. So see if you remember, we first have a dehydrogenase transferring to FADH2, some electrons making a double bond, a true dehydrogenase. So we're eliminating hydrogen, making a double bond, this beta position. And then we're gonna add water across that double bond. Does that sound familiar? And it's gonna only be in the L configuration. Does that sound familiar? Then we're going to oxidize. So here's the flame is ignited. We've transferred some electrons to NAD to make NADH, right? And so we know what we're gonna do with those. We're gonna feed them into electron transport, complex one. So now we have a carbonyl group. And then in this next step, we're gonna cleave at that beta position, releasing an acetyl-CoA and at the same time simultaneously adding a new thioester linkage to repeat the process. So all we've done here is we've taken out an acetyl-CoA and we've reduced the length of the fatty acid by two carbons. And we're just gonna do this again, and again, and again, right? So we're gonna take, so if we start depends on how many fatty acids or how many carbons in the fatty acid molecule you start with, but you're just gonna take these out two carbons at a time, making acetyl-CoA. So where did we see this? Before. Look at this, I think I have a summary slide. Look at the synthetic strategy. Eliminate a hydrogen. It's okay, I don't want, somebody else. Yes, ding-ding-ding, citric acid. Remember that regeneration part where I had, I just remember I showed you some slides where I just show you the carbons doing the chemistry. First you make a double bond, then you add water making L-malate, right? And then you oxidize this to a carbonyl group, right? And then you convert it, or here you're doing something new, you're cleaving this bond off. Okay, so it's the same synthetic strategy and you've now converted these carbons to the oxidation state that's compatible with the Krebs cycle. This is where, so we're at the sugar, we've removed these four electrons. One ended up on FADH2. Now how might we regenerate the FAD? Can you imagine how that might happen? Do you remember an enzyme called succinate dehydrogenase? That's also called, see we're integrating, man, come on. We're integrating. What lecture did we learn about? What happens to FADH2 in the name? Is it complex four, two? Good, complex two. And that one, where did those electrons go? Immediately succinate dehydrogenase right onto the ubiquinone to make ubiquinol. It's a two electrons are transferred there. Here it's almost the same. It could have been that this enzyme was sitting in the intermembrane, receiving FADH2, it was generally not released. It just sits there as a tightly held prosthetic group. But in this case, we have a little mini electron transport chain. So there's actually three enzymes. So this particular enzyme is floating around with this FADH2. It docks with another enzyme, transfers those electrons to that enzyme, which also has FAD making FADH2. And then it docks with the third enzyme, which is in the mitochondrial membrane, trig in with an FAD, converting it to FADH2. And then it goes into ubiquinol. So the net product is six protons move, not 10, because we've short circuited, we skipped complex one. For the NADH is made, those are going through complex one. And so we get the full 10 protons moved across the bilayer. Does it make sense? And so we repeat this step. It would say we start with palmitil CoA. So you have a total of 16 carbons. At the end of the day, you're repeated taking two carbons off, two carbons off, two carbons off, until you get to eight acetyl CoAs, seven FADH2s. Because the last one you have four left, you cleave from those four carbons, you just get one FADH2 and one NADH. And you have seven NADHs. So now we've brought this thing down to a manageable oxidation state, but fatty acids come in to acetyl CoA. So there's no going up to glucose. It's absolutely impossible. You can't go backwards, pyruvate dehydrogenase cannot go through the Krebs cycle and pull off some malate that just doesn't work. So we cannot live on these fats. They can only be used to make energy in something else called ketone bodies that we'll hear about in a moment. All right, does that make sense? Halfway done.