 All right. One more lecture. All right. So today we're going to be talking about amino acid metabolism. And if I could have your attention, I know it is so painful to be in here. It's so nice outside. So, but it isn't that painful. We're learning about amino acids. Okay. So today we're going to learn about amino acid metabolism. And so today's lecture is a bit painful because when you think about it, there's really 20 metabolic pathways for degradation and 20 anabolic pathways for building amino acid. So if we were actually to cover every pathway, I mean, we could spread this out over four or five lectures. So instead of covering hundreds of new enzymes and all these new pathways, we're going to sort of dip in a little bit to just a subset of the pathways, some of the easier pathways. And so if you want to have a comprehensive understanding, that would not be a requirement for an examination, but a lot of, even the textbook doesn't have all the pathways. Many of the steps are described as something magic happens here, and then something comes out of that. Okay. So we're going to start with catabolism. And then we're going to move to anabolism. We're going to look at both. We're going to sort of have a clinical slant today where we're going to be looking at a variety of metabolic diseases that come up with defects in amino acid catabolism. But then at the end of the lecture, we're going to be looking more holistically, you know, so where, how do we get some of the feedstocks of amino acids, these ammonia? Where does ammonia come from when all, there's all this nitrogen gas in the air? So we're going to be taking an ecological slant at the end of the lecture. Okay. So let's start with catabolism. So it's a two step process that is pretty separable. So first you remove the amino group, and that reaction forms alpha keto functionality on the, the amino acid without the amino group. And then we're going to process those two parts separately. The ammonia produced from the removal of this, that amino group is going to be ultimately processed, either recycled to be available for synthesis of new amino acids, or if we're burning amino acids, if you're eating tons and tons of, like an Atkins diet, you're eating tons of protein, well, you're going to have to get rid of some of those amino groups. And that comes out of a urea cycle. And the urea cycle is intertwined with the citric acid cycle by this so-called shunt. But then we have the carbon skeleton. And literally what we can do with that is just burn it for fuel. So we can feed it into a variety of pathways, either a Krebs cycle, or we can transfer some remnants of those carbon skeletons to other tissues from the liver. So liver is where the urea cycle is occurring. And we can, you know, transfer it as a ketone body and regenerate the acetyl-CoA, and then burn it that in the Krebs cycle. But ultimately, the idea here is burning fats, so fully oxidizing the carbon atoms in those, or burning amino acids, so fully oxidizing the carbon atoms in the amino acid to CO2 ultimately. Okay, so that's an overview of where we're going today. So let's look at the first part, this, the manipulation of the amino group. So there's lots of different sources for amino acids. So if you have, you know, a diet that is high in protein, well, you're going to have a lot of amino acids coming in. But also there's a certain amount of turnover. So you have cellular proteins that are degraded to amino acids, and those need to be processed. And there's different ways, but the thing of it is that the liver is the critical organ. So obviously you're going to have amino acid metabolism all over, but you need to transport those amino groups from whatever tissue they're produced in to the liver. And so there's two different molecules that are used to transport the amino groups to the liver. And so we have ingested protein or liver cellular protein gives you amino acids, and those amino groups are transaminated onto alpha ketoglutarate to make glutamate. Okay, and producing the alpha keto acid corresponding to the amino acid. So if you look at this, you see this is oxidized, right, and this is reduced. So this is not a redox process. It occurs very close to equilibrium. There's no ATP hydrolysis. One molecule is oxidized, the other molecule is reduced. So we're transferring electrons between the molecule by transporting this ammonia group and having the end product of these reactions be a keto group. Okay, and so alanine can come in here and from muscle predominantly, and that can be processed to pyruvate, right? So if you look at this, this is the alpha keto skeleton of a deaminated alanine, or you can have glutamine, right? And so you can shed this amido nitrogen and the amide functionality of this amino acid. You can give that up to make glutamate, and then glutamate can be deaminated to make ammonia. But what are we going to do with ammonia? So ammonia is toxic. We need some way to encapsulate the reactivity in the chemical properties. This is a very alkaline substance, right? So if you have a high level of ammonia, your pH is going to be really high. So you need to do something with that to safely remove it from the organism. So let's look at this transamination. So this is not a net redox process, but the molecules are changing oxidation states. So you have alpha-ketoglutarate, receives an amino group, right? So this is reduced, and then this carbon is oxidized, and so you're transferring from each of the 20 possible amino acids, the amino group, onto a glutamate molecule. So glutamate forms the pool of amino groups, the storage pool for amino groups in the cell. And so each of these amino transferases is able to remove the amino group, forming the alpha-keto form of that amino acid, and place that amino group on alpha-ketoglutarate to make glutamate. Okay, so there's no net gain. We're transferring amino groups. We're changing oxidation states, but there's no net change of amino groups or oxidation states. Okay, so this is a transamination. There's cofactor. So it's sort of a new type of chemistry. We need a cofactor for transamination reactions, and it's this periodoxal phosphate cofactor. So this cofactor is originally stored on the enzyme by forming this imine, or shift base, with a lysine. So the epsilon amino group of a lysine forms a shift base with this cofactor. But that's not a form of this cofactor that's going to be reacting with our amino acid. We have to remove this periodoxal group from the lysine, and then it can react and form a shift base with our amino acid. And so there's an aldehyde form of this, and then when it removes the amino group, ultimately you form periodoxamine phosphate. So you have the amino group that's been removed from whatever we're transferring amino group from, and put on the cofactor. And so I've added a slide at this point that describes the mechanism of this reaction. The whole strategy here is to avoid the formation of unfavorable carbanion. So instead of forming a carbanion, so if we just had a base pull off a proton, well you would get a carbanion. But instead we have this, you can see how this is highly conjugated, right? And so first we form a shift base between our periodoxal group and our amino acid. And then when we remove this proton, we can just rearrange the electrons, and so that there ends up being a double bond here. And then we can add a proton there and then get to this form of the shift base. So now instead of having a shift base here, you have it with your amino acid. So you've changed the orientation. You're about to release this amino group. And then you just reverse this, such that out the other end when you add water is the alpha keto acid. And our amino group is now placed on the cofactor. And we can do a similar reaction where we take an alpha keto acid and then place this amino group in lieu of that alpha keto acid. So we're transferring an amino group from one molecule to another. Does that make sense? It's a little crazy. Just hit a little mechanism today. Okay, so what we need to do is transport amino acids that come from proteins from all the tissues in your body to your liver. And the way this is done is you can imagine, well, glutamate is not a very good transport molecule. It's highly charged. Getting that across membranes is going to be difficult. So if we could first convert that into a less charged molecule, that would help us in our transport. So in the tissues, you have glutamate can be activated by forming a phospho-n hydride bond. And of course this could be hydrolyzed. That would release some energy. Or it's a great leaving group. So ammonia can react with this to make glutamine. That glutamine is less charged. It's great for transport of an ammonia. So we're taking ammonia from the tissue. Taking ammonia from the tissue. And then you see how we're releasing it in the liver. So the glutamine goes into the liver where we can hydrolyze off that ammonia. And then that ammonia can be processed in a type of tissue that has the enzymatic machinery to deal with the ammonia. And so this is just a simple transport. But in muscle, there's other ways, the other carriers for ammonia that we could use. So muscles have a high glycolytic rate. So there's a large amount of pyruvate in muscles. And so pyruvate is an alpha-keto acid. So it has alpha-keto group, carboxylate, and a methyl group. And so we can transfer from glutamate our storage form of ammonia. We can transfer that amino group to pyruvate to make alanine. So alanine, so pyruvate is the corresponding alpha-keto acid for alanine. Alanine is then less charged than pyruvate. Or it's about the same charge. When you think about it, pyruvate, but alanine is what's transported. Because alanine is wither ion. So alanine is transported anyway. Alanine is transported. It's received in the liver where the reverse reaction can occur. We can transfer those amino groups back to our storage molecule, glutamate, and we can convert an alpha-ketoglutarate to pyruvate. So transfer that, or we convert the alanine to pyruvate, alpha-ketoglutarate to glutamate. So the net effect is to take ammonia from the muscle and move it into the liver. These are hanging out of the liver, but it's actually occurring within the liver. So now we have glutamate. That's storing our ammonia. But to make urea, we have to release some of that ammonia back into solution so it can be fed into the urea cycle. And so glutamate, you can react or convert glutamate to the alpha-keto acid with release of ammonia. So this is changing the oxidation state here because here, this is reduced, this is oxidized. And so we're transferring those electrons from glutamate to NADP to form the alpha-ketoglutarate. So now we have ammonia in solution. That's good because we need that to process it as waste. So here's the overall picture. We came in either with alanine or glutamine. We made a bunch of ammonia molecules. So now we need to process those ammonia molecules. Currently we're in the liver. The urea cycle occurs in two organelles, in the liver and in the cytosol. But we're starting in the liver with the, or in the, I'm sorry, we're in the mitochondria and then with the ammonia. And that urea cycle occurs in the mitochondria and the cytosol. I'll get it eventually. And so there's different ways we can deal with this ammonia. If you're a fish, you can just release the ammonia into solution because you have all this water and as soon as you release the ammonia into solution, it's massively diluted. So we don't have to worry about the alkalinity of that molecule. If you're a vertebrate for us, we make molecules of urea. But urea is a denaturant. So if you had a really high concentration of urea, that would be bad. You would start to denature your proteins. So when we make urea, we put that in lots of water. We dilute it out so that it's less denaturing. But you know, that wastes water. You have to use a lot of water in order to dilute urea. And so the other option for birds is to make a molecule that looks a little similar to urea. It's uric acid. You can see, well, here's a urea. Here's a urea. And so this molecule is released as a slurry. Anybody that has received a gift from a bird has known about the slurry of uric acid. And that's just, it's doing the same thing we do but with less water. So it's a little more goopy. Okay. So let's look at us. You know, we're sort of focused here on vertebrates. And so we have, first, we need to begin to build our urea molecule. So we have ammonia, right? And so this ammonia is, or needs to be added to a carbon atom. Basically we need to have two ammonia and a carbon atom, right? That's the structure of urea. So let's start with bicarbonate. There's lots of bicarbonate in the cell. And the bicarbonate can be activated, right? And so this involves one high energy, phospho and hydride bond being broken. We've now activated the bicarbonate so that it can react with directly with ammonia. And so that synthesizes carbamate with release of inorganic phosphate. And you could see, man, we're so close. And you might think, well, you know, why not just do that again on the other side? Well, energetically, that's not very favorable. And we need a very complicated pathway. And all we're going to end up doing is just adding another amino group on the other side of this molecule. But first, we're going to activate, right? So this is our second high energy, phospho and hydride bond broken, transferring phosphate from ATP to make this carbamoyl phosphate. And this reaction is catalyzed by carbamoyl phosphate synthetase, right? So in your original slides it said synthase, but this involves ATP. So we call those synthetases. That was a mistake. This occurs, as I've already mentioned, in the mitochondrial matrix. And so now we have carbamoyl phosphate. Our goal is to synthesize urea. How are we going to do this? So this is, I think, the last cycle in the whole semester. And that must make you very sad. But it's a pretty cool cycle. So here you have carbamoyl phosphate coming in, right? And so what we want to do is condense carbamoyl phosphate with ornithine. Now, ornithine is an amino acid. It's not one of the 20 naturally occurring amino acids in proteins, but you know, it has amino group. It sort of looks almost a little bit like, it's almost lysine, right? It's just a little bit shorter. And so ornithine, wherever that comes from, is combined with carbamoyl phosphate to make citrulline. Do you see how we've removed the phosphate? And we sort of color coded these atoms. You can see it sitting on the end of this amino group of ornithine to make citrulline. Now we're going to activate citrulline, right? And so we're going to make a new AMP-alated version right here, okay? And then we're going to condense that citrulline with aspartic acid to make arginosuccinate. And so you would say, well, where is this aspartic acid just came out of nowhere? This seems like, you know, it would be a bad thing to do. If you keep doing this cycle, every time you do this cycle, you'd be using aspartic acid. We need aspartic acid to make proteins. So we'll have to deal with that at some point. So here we have aspartic acid combining with citrulline to make this arginosuccinate. And if you can see here, if we were to break this bond, that would release fumarate, and say fumarate, I remember that from the TCA cycle. So that can feed into the TCA cycle, and it can make arginine. And then you have this guanidinium group in arginine, which can be cleaved to regenerate our ornithine and release our urea molecule. So all we've done, you know, in the big picture is taking that carbonyl phosphate and put an amino group there. And we've done it in sort of a roundabout way. But there's some problems here. This is not really a cycle. It's going to, if we just keep doing this, the amount of aspartate in the cell is going to go down, and the amount of fumarate in the cell is going to go up. So we need a way to interconvert fumarate to aspartate. Obviously, that way these two can feed into each other. And the way this is done is by this so-called shunt. So fumarate in the cytosol, remember we moved into the cytosol for part of the urea cycle, especially when we generated fumarate. Fumarate can be converted to malate, and either fumarate or malate can be transported back into the mitochondria, where it can be converted to acetylacetate. So we have this malate dehydrogenase. And that's sort of cool, because up to this point, we've spent some energy doing this cycle to make the carbonyl phosphate. It took some energy, and actually I should point out here, here we cleaved ATP to AMP. So we've used a total of four phospho- and hydride bonds to make this urea molecule. But in this shunt, when we're reconverting malate back to aspartate, we're generating NADH, one molecule, and that's equivalent of 2.5 ATPs. So this net process has only taken 1.5 ATPs. And the good thing is we've reconverted the output of the urea cycle into the input of the urea cycle. Fumarate has been converted back to aspartate. Aspartate has been exported out and is now ready for another revolution. So this is, again, a carbon-neutral pathway. The input is ultimately the ammonia stored on glutamate, our storage form of ammonia, is transferred to aspartate transfers that ammonia ultimately onto the urea molecule. Do you see how that works? So think about urea. So the carbonyl group came from bicarbonate. One of the amino groups came from ammonia, and the other one came from glutamate. Do you see how that comes together? And so we've accomplished our goal. We have a safe way to deal with excess amounts of ammonia. We can just take the urea, dilute it up, and release it. So that's the urea cycle. Any questions on that? Yes. Yeah, so what is the pathways that make uric acid? You might guess. Well, I don't know offhand, but I would guess maybe urea, maybe at some point there would be some urea that's combined together. But I don't know the biochemical pathways that make uric acid. Maybe the BIRD researchers, if there are any, can tell us. And you don't know, have to know either. We're studying humans right now. Okay, so we have acetyl-CoA. It can be combined with glutamate to make a regulatory molecule. Remember that fructose 2,6-bisphosphate? It's not really a metabolite. It's sort of a signaling molecule. We have a similar form of regulation on the carbonyl-phosphate synthetase, right? And so this signaling molecule in acetyl-glutamate is an allosteric activator of the carbonyl-phosphate synthetase. And this sort of makes sense, right? So if we have a large amount of ammonia, we're going to have a large pool of glutamate because that's where we're storing the ammonia. Arginine also is involved in the urea cycle, but it also contains lots of nitrogens, right? So in some sense, that's also a sensor for the amount of ammonia in the cell. And so if we're building up the amount of ammonia in the cell, we need to start to deal with that issue by activating the enzyme that feeds into the urea cycle. And so this is how this process is regulated. So overall, we've cleaved four phosphonohydride bonds, so we've used three ATPs to make two ADPs and one AMP. That's four ATP equivalents, and we fed in aspartate, and we got out a fumarate, right? And then, but we can convert mallet and oxidize mallet and reduce NAD to NADH. And so that gives us back 2.5. Remember, if we did from the oxidative phosphorylation lecture, it was about 2.5 ATPs. So the net cost to this cycle is less than it would be if we didn't have this cool way to shunt the fumarate back into aspartic acid. And so the cell is being resourceful here. All right, so we've only dealt with half of the problem. We've looked at really a lot of the fine details there. Now we're going to come out a little bit and look at some of the ways in which these alpha keto acids that are remaining, these carbon skeletons, are metabolized. But ultimately, what we want to do is use amino acids as fuel. So we're going to feed them in to the Krebs cycle or feed them into a formation of ketone bodies, which then can be transported to another tissue and then be fed into the Krebs cycle in that other tissue because they come out as acetyl-CoA. So these different pathways, different amino acids give rise because they have different structures, give rise to different entry points into the pathway. And they can be called glucogenic or ketogenic. So glucogenic means that once you feed directly into Krebs cycle, you can take oxaloacetate and push that off to make some glucose and gluconeogenesis. So that's why we call it glucogenic. Ketogenic, well, we cannot use those to make glucose because there are these two carbon units here, right? And so there's no way to do that. But you can see that some of these amino acids show up in bulk. So as we're disassembling these carbon skeletons, oftentimes we can take one product when we maybe break an intermediate form of the amino acid into two pieces. We can take one of those pieces, feed it into ketone body formation. In the other piece, we feed in directly to the Krebs cycle. So let's look at some of these reactions. These are the cofactors that are involved. You've already seen pyridoxyl phosphate. Biotin, you've seen. Remember, carboxylation reactions. THF is new. And so this is important in one carbon transfers. You can transfer between molecules, either a single carbon or a single carbon that's in various oxidation sites, either being alcohol or an aldehyde. We'll see that in a moment. Atomet is only used for transfer of methyl groups between different molecules in THP, which we won't talk that much about today. It's very similar in structure to THF, but it's more involved in oxidation reduction reactions and enzymes. So there's a whole host of different cofactors in general that are used in the catabolism of those carbon skeletons of amino acids. So by the time we've seen it, like 15 zillion times, carbon dioxide binds, gets transported to the second active site where it is combined with another molecule to increase the number of carbons in that molecule by one. And so those are important. Atomet's new to us, so Atomet, so we have a methionine molecule attached to adenosine. Such that you might guess a highly-label methyl group. So here you have S plus. And so what we can do with this is we can transfer that methyl group to this some other substrate molecule that we need to increase by one methyl group generating this acidinosil homocysteine. And so there's a way that we're hitching a ride on this cofactor making this Atomet and then depositing that methyl group on some other molecule. So here's THF. So you have three parts. You have glutamate attached to amino-benzoate. But then the business end of this cofactor is this six-methyl-terrin, methyl-terrin, methyl-terrin, I think the P is silent. And so this cofactor, this part of the cofactor has these two nitrogens. And these two nitrogens can be involved in making new bonds to carbon atoms. And so this cofactor is only involved with transferring single carbon atoms between molecules, either as the fully reduced form or in these different oxidation states. And so if we bind with the two nitrogens, we get the equivalent of CH2OH. And if we make this bond, we get an aldehyde equivalent. So we can transfer those between substrates. Okay, so we have some cofactors. Let's do some catabolism. So we have a variety of amino acids that can be converted to pyruvate. And so we're just going to start scanning through some of these. Some of these are really easy. Yes, hi. Two slides ago. Right. So here, you can't really see. Is that an aldehyde? No, it looks like something. When it is released, it becomes an aldehyde. Or when this carbon here is released, it becomes an alcohol. So for example, serine. That last methyl group on serine is by taking this CH2OH equivalent off of here and putting it onto glycine. I think it's a glycine. We'll see in a moment. Yeah, it's a glycine. So that's the delivery form and the receiving form of carbon atoms for that cofactor. So we have three anine. And you'll see many of these pathways. You might guess, well, there's probably 20 different pathways, and they're all completely independent. But that wouldn't make much sense. We want to minimize the number of enzymes that we're using in these pathways. So in many steps along the way, we'll pass it to another, to another, to another until you get to something you can burn in a Krebs cycle, ultimately, or form a ketone body. And so here you have, for example, three anine. We can disassemble a three anine, right? And so first we're oxidizing this carbon to a carbonyl. We can now disassemble this to make an acetyl-CoA and a glycine molecule. So here's our glycine, and here's our acetyl-CoA. And so when we make glycine, we can take two glycine molecules. One of the glycine molecules, we can pick off that alpha carbon in an oxidation state of CH2OH and put it on THF. The other thing is we can react a second glycine molecule with that, you know, methyl from the methyl with the hydroxyl on it from the THF to make serine. So we've converted three anine to glycine to serine. But we need to feed into some pathway that generates energy here. And see, also, here's a similar set of reactions. Is this slide? I think eventually we deal with the serine later. So tryptophan, you can disassemble this aromatic ring. We're not going to look at that. It's really complicated. But you can sort of see that, oh, okay. That has this alanine equivalent in it. So you can break this bond, deal with this part of the tryptophan separately, and then you have alanine. Alanine can be converted directly to pyruvate. Do you see that? So the transamination, remember, transamination with that periodoxyl phosphate produces an alpha-keto acid. So you have alanine, and you've removed the amino group and put a keto group. That's a direct reaction. Serine, you can dehydrate to form pyruvate. And cysteine can be converted into pyruvate as well. In a lot of these, we've color-colored the atoms to get a sense, not for every single step what's going on, but generally what parts of the molecule end up in which of our metabolites. And so let's look at some aromatic amino acids. So a lot of metabolic diseases occur with the processing of aromatic amino acids. So if you're interested in medicine, pay attention here. So we have phenylalanine. And you can think, well, phenylalanine is very similar in structure to tyrosine. So it would be a simple matter to add that parahydroxyl group to phenylalanine to make tyrosine. So that's catalyzed by an enzyme. If that enzyme has a defect, if it's not functional, then we're going to begin to build up a phenylalanine. And the best we can really do with that is do an amino transferase reaction to make a keto group. But so if you have a deficiency in that enzyme, you're going to build up early metabolites of phenylalanine. You're not going to really have a way to deal with those carbon skeletons. And that build-up causes the disease, causes the mental retardation in PKU. And each of these steps, you can see there's all these different steps. And at every step, there's a deficiency in an enzyme. You're not going to be able to fully metabolize that amino acid. So something unnatural is going to build up. So you have various tyrosinemias. So tyrosine, you can look at the way that we're disassembling the aromatic ring. You say, well, that's pretty hard to do to bust open an aromatic ring. That's a pretty stable structure. And so what we're doing is activating it to this form here, where we have these two hydroxyl groups. And in the next step, we pry the ring apart. And that requires some energy. So we have an oxygen molecule involved there. And now we have a linear form of the molecule, where these carbons that are highlighted here end up over here. And then through a few more steps, we can get to fumarate, which feeds directly into the TCA cycle, or acetoacetyl-CoA, which can be used to make ketone bodies. So this is ketogenic and glucogenic. Okay? So branch chain amino acids. I know it's sort of tedious. There's a lot of amino acids. We're not going to look at them all. We're looking at quite a few of them. So here's a branch chain amino acid. These things, first, you have the amino transferase. You see a common theme. In general, you deal with the amino group right away, and then you disassemble the carbon skeleton. So we transfer the amino group away, onto our storage molecule. And now we have the alpha-keto acids. But conveniently for all these amino acids, we have the alpha-keto acids, but you have this carboxylate. And so we can decarboxylate these molecules and add a CoA functionality. And so this is a dehydrogenase enzyme. So right, we're oxidizing this molecule. So we have an aldehyde equivalent that's oxidized to a carboxylic acid equivalent. This is the exact same enzyme that we saw with pyruvate dehydrogenase, right, when we converted pyruvate to S-til-CoA. The exact same set of cofactors are involved with that. And so then we have this CoA metabolite, which can be converted into a variety of in-products that can be used to make energy. Either ketone bodies are fed directly into the citric acid cycle, these two. Okay, and so here we have another disease. So if you have a deficiency in this enzyme, you begin to build up, especially this isoleucine metabolite, which is converted into this structure, which smells like maple syrup, right? And so the reason that people have a deficiency in this disease, they have this peculiar aroma. They smell like a maple tree walking around. And that's because they have this sotolone hanging out in their bodies, because their bodies cannot feed these carbon skeletons into energy-generating pathways. This is an aromatic volatile compound. Maple syrup, urine disease. Back in the old days, they used to taste and smell, just so you know. Luckily, doctors don't have to do that anymore. Okay, we can take asparagene or aspartate and move it into oxaloacetate. And so asparagene, you can say, oh, okay, yeah, I can just hydrolyze off that amido group to make the carboxylic acid. So we're at aspartate, and then the aspartate, we can deaminate here to make the alpha-keto acid. And as it turns out, because of the original structure of this molecule, we do these two simple steps, and we're already at oxaloacetate. We can feed that right into Krebs cycle. So that one's pretty easy straightforward. So what we're doing here is we're just feeding all these molecules. We're first dealing with the amino group that gets eventually put on urea, and the carbon skeletons are degraded so that they can feed into Krebs cycle, either in the liver tissue or being transferred as ketone bodies to other tissues where they feed into Krebs cycle. Okay, any questions so far on catabolism of amino acids? There's a lot of stuff I know. Painful. Okay, we're going to start building up again. So let's look at the biosynthetic pathways. And so ammonia is incorporated through glutamate and glutamate. So obviously we're making amino acids. So we're going to assemble the alpha-keto-carbon skeleton separately, and then we're going to add the amino group. And the amino group is being transferred from glutamate or glutamine. And so glutamate can be interconverted into glutamine. And so first you activate the carboxylic acid with the synthetase that uses ATP hydrolysis. And then you have this gamma-glutamyl phosphate, which can be used to react directly with an ammonia molecule to make glutamine. Okay, so both glutamate and glutamine are important in adding nitrogen atoms to amino acid structures. Okay? And so this is an amidation step here. Okay? Does that make sense? All right, and so this is an early step or an important step in the pathway, so it's heavily regulated. And you might think about it, well, this is a little bit dicey. We have 20 amino acids. And proteins sort of need, you know, all of those amino acids to make proteins. And so we need some way to have the regulation in these pathways since and correct for precisely the right amount of each amino acid. But here we have sort of a set of feedback inhibitions. So glutamine can be fed into incorporating nitrogen in things like AMP, CTP, tryptophan, histidine. So those are feeding back. But you might say, okay, well, red acts its digital, right? It feeds back. And if any one of those molecules is there, it cuts it off. But that wouldn't be good because there's other molecules that we need to make. And so this is a rheostat. Each of these inhibitors, allosteric inhibitors, is just slowing down the rate just a little bit. And so the combination, so if you begin to have all of these present, then the rate of this greatly diminishes. But with just one present, one of these products present, it just goes down a little bit. Okay, and so there's a variety of feedback inhibitors and you can see, ah, carbonyl phosphate. Why would the cell want to regulate this thing by carbonyl phosphate? Anybody have an idea, Trink? Where did we see that bad boy? You remember? It was way back 10 minutes ago. Yes. Yeah, right. So that was a catabolic pathway. And so why my carbonyl phosphate turned off this pathway? Yes. Say again? I'm sorry. Exactly. So this is a form of reciprocal regulate. Remember, I bring this up again and again. You don't want to necessarily just be, you know, building up, breaking down, building up, breaking down. And so by having carbonyl phosphate regulate the formation of the thing that's going to be adding amino groups to amino acids. Well, that's helpful, right? Because then you won't be doing both at the same time. So there's all kinds of levels of activation. But wait, it gets better. This is a rheostat with each of these inhibitors turning up the rheostat, but you can adjust the global sensitivity of this rheostat, making the inhibitors more efficacious by another mechanism. So you have the adenylation of the glutamine synthetase. So you're adding an adenyl group to a tyrosine. That turns up the sensitivity of this allosteric regulation of this enzyme. So it becomes more sensitive to inhibitors. So in the presence of some of those inhibitors, we just saw if the enzyme is adenylated, then that's going to make it more, it's going to be more inhibited. So it's going to be decreased. But this enzyme is also activated or inhibited by a urodylation. So the urodylation of the adenylyl transferase turns off the adenylyl transferase. And this urodyl transferase is itself allosterically regulated. So we've sort of bumped, this is a cascade of inactivations, but the starting point is here, at this urodyl transferase. And so this is turned on by alpha-ketoglutarate and ATP. These are indicators of energy. There's lots of energy present. We have ATP, we have alpha-ketoglutarate, or it's inactivated by glutamine, right? Or inorganic phosphate, being a marker for low amount of energy in the cell. And so this enzyme is going to be more active when it's time to do an anabolic process, when there's high ATP or high alpha-ketoglutarate. And it's going to be less active with glutamine or inorganic phosphate. So there's layers of regulation giving us amplification of the regulatory pathways. But that is not the only form of regulation of synthesis of amino acids. That's a bulk regulation, right? We need something to uniquely regulate the formation of each amino acid, and we'll see how that's done in a moment. So now we have synthesized a glutamine, and we need some way to transfer that to a substrate. And this is catalyzed by glutamine ametotransferases. So we're transferring the ametonitrogen, not the amenonitrogen, by first releasing it from the glutamine, making a covalent bond to the enzyme. This ammonia then moves through a channel to a second active site, where it then reacts with some kind of activated molecule. And then we release the glutamate. So we loaded the ammonia on to glutamine, and then it's used to be transferred to some other molecule and regenerating the glutamate. So these are in the general class of enzymes. They allow us to utilize that glutamine that we've synthesized. So here's all the amino acids. We're going to go into even less detail on how we make each of these amino acids. Each of the amino acids are built from different building blocks. Alpha-ketoglutarate, for example, is used to make this group of amino acids. Three-phosphoglycerate is from what pathway? Glycolysis, yeah. So we're using bulk TCA intermediates and glycolytic intermediates. TCA, right? This is sort of both, because it feeds between the two pathways. PEP or erythros, you might say, or ribose 5-phosphate. Where do we see that bad boy? So remember the pentose phosphate pathway, that evil pathway. One of the evil outcomes of that pathway is to make histidine amino acids with the ribose 5-phosphate. Okay, so let's put this together. Let's look at the big picture. So you have both glycolytic intermediates and Krebs cycle intermediates feeding into the synthesis of these molecules. We know, well, so they sort of all attach, and glycolysis is attached to pentose phosphate, right? So we can make ribose phosphate, 5-phosphate and histidine. 3-phosphoglycerate can be used in this way, PEP and pyruvate. So there's all of these different ways to make molecules. You shouldn't be ultra concerned with memorizing which comes with which. If I show you some kind of, spend a little bit more time on it today, then those are the ones that you should focus more on. So pyruvate, we can also use Krebs cycle intermediates, pyruvate, oxaloacetate, and alpha-ketoglutarate to make a variety of amino acids. So this is the, you know, from 10,000-foot view of how we make amino acids. So we'll look at a few of these. So here's alpha-ketoglutarate. The same strategy is used as before. We overlap the pathways for efficiency, right? And so we, from glutamate, we can make glutamine, proline, and arginine. Okay, so we have overlap pathways, but we're not going to look at the individual reactions for that one, but we are going to look at this one because it's pretty straightforward. Phosphoglycerate is first converted to serine. That serine can then be converted to glycine or cysteine. And so here's three-phosphoglycerate. You might think about it. It's like, wow, we're pretty close. If we just turn this into an alpha-ketoacid, then we can transaminate that thing and make something very close to a serine. So first, we oxidize the hydroxyl group to a carbonyl. That's an alpha-ketoacid. So we can use an amino-transferase enzyme to transfer an amino group from glutamate to this three-phosphohydroxypyruvate to make our three-phosphoglycerate. And so then we just need a phosphatase. We clip off the phosphate off the end of this molecule. So this is pretty straightforward. You can see that these are, let's see, here you have three carbons. Here you have three carbons. You just had to rearrange stuff a little bit. The three-phosphoglycerate. Serine, okay. That can feed into other molecules. So we can take the serine and we can use the phosphatase to make a serine. And then we've already seen sort of a similar reaction here, but we can take the serine and use our THF cofactor to make glycine. So we can take that CH2OH group and stick it on the THF cofactor, or at glycine, okay? All right. The cysteine gets a little bit more complicated. So it turns out the cysteine is sort of conditionally essential. So methionine is essential amino acid, but methionine is used to make cysteine. So we make cysteine technically, but we make it out of an essential amino acid. And so you have homocysteine, right? And so homocysteine can be condensed with serine. And look at this thing. We're making a new bond where we can have, we bring a sulfur in between these two metabolites, and then we have options. We can break on either side, right? So we come together like this and then we cleave over here. So we're taking that sulfur off the homocysteine and sticking it on a, to make a cysteine molecule. And we've just bound the two together and then cleave the other bond releasing cysteine. So that was pretty straightforward. But where did the homocysteine come from? Well, that came from methionine. So here's homocysteine. Methionine can be reacted to make our SAM, this prosthetic group. And so we can take the AMP, and the AMP is reacted with methionine, releasing pyrophosphate, which pushes the reaction forward. But that gives us this acidinacyl methionine, where we have this positively charged sulfur atom in our methyl group. We can then use that to transfer the methyl group to something else. And then all we have to do is release this adenosine group to make homocysteine. So this is basically homocysteine. Cysteine just has one methylene group, remember, not two. And so we can make that just by releasing the adenosine group. So this methionine is essential. We can't make it. And it's used to make the non-essential cysteine. So cysteine is sort of conditionally essential. Do you essentially get it? All right. That wasn't very good. I try. Okay. So oxaloacetate. Now we're getting very distanced. There's a lot of pathways underneath here. Oxaloacetate can be used to make aspartate, which then is fed into a synthesis of these four amino acids. Pyruvate can be used to make these amino acids. In general, the pathways that synthesize these amino acids require ATP, NADPH, and transamination reactions to put the amino group on. Many of them involve carbon group transfers on our carbon group transfer molecule. All right. This one is pretty straightforward. OAA to aspartic acid, because that's the alfaketo acid of aspartic acid. Yes. So aspartate. Say again. So aspartate. Yeah. There's options. You can... Right. Undoubtedly, there's a fine set of allosteric regulations. There's separate enzymes that lead to different fates for the aspartate molecule. Right? And so you can regulate each of those enzymes so that you get the right amount of each of these amino acids. And also you're able to do the urea... Urea cycle is aspartate neutral. Because remember, aspartate is always regenerated from fumarate. So in some sense, you could sort of ignore that. It's not depleting aspartate. It's just cycling aspartate to fumarate, back to aspartate to fumarate, because of the shunting reaction. So... But if we want to make molecules, we have to take aspartate molecules out and then make these other amino acids. I guess... Yeah. Makes sense. Okay. So in pyruvate, remember, that's the alfaketo acid of alanine. So you have carboxylate, carbonyl, methyl group, and pyruvate. And we just transaminate to make alanine, which just has a methyl group side chain. Okay. Pretty straightforward. We can stitch together these molecules to make the beginning molecules using the synthesis of aromatic amino acids. This is an important precursor in the synthesis of aromatic amino acids. Phenylalanine and tryptophan are essential. So the pathways that stitch these molecules together are found in plant cells. And so the plant cells make those amino acids, and then animals eat them up. Okay. Histidine is crazy. So you have this ribose 5-phosphate, right? One, two, three, four, five carbons. That ribose 5-phosphate gets in, ends up in the histamine molecule, all spread out. So you break that ring open and you lay it out like this, and then you attach some other groups to it. We're not going to talk about, you know, exactly how that's done, but it involves PRPP, which is not only important in the synthesis of histidine. It's also used in the synthesis of nucleotides. And so that's an important precursor. And yeah, so this pentose phosphate intermediate are in the product of pentose phosphate pathway. It can be used to make histidine. Okay. So general can sort of see how they're stitched together. All right. So here's a summary of everything from 10,000 feet. We have each of these amino acids is synthesized from different precursors, and some of the synthetic pathways are overlapping. So now we need to deal with this issue that we left unresolved, the regulation of this pathway. So we need just the right amount of each of the 20 amino acids. If we regulate upstream, way upstream, that's not going to give us the fine-tuning we need for regulating the exact amount of each of the amino acids. So we need to think about how this comes together. So there's all these different pathways that feed into amino acid synthesis, but many of them share common precursors and have overlapping pathways. So we need to deal with this problem. And the way that we deal with this is isoenzymes. And so here, for example, we have a stacked pathway. Aspartate leads to the synthesis of lysine, lead synthesis of methionine, and then isoleucine. So if we just turned off this enzyme here, we would turn off the synthesis of all of these amino acids. We need a way of dialing down the enzymes, not a digital switch, an analog switch. So by having different forms of the enzymes that are each subjectable to different allosteric regulation, we can dial things down, but not completely off. For example, you have these feedback regulators from threonine. So if those are inhibited, we still have some flux through this pathway in here to make our isoleucine, because we always need to, in the last step, convert threonine to isoleucine. So for lysine, that feedback's here, but some of those substrates can get through, get past that regulation by using the different forms of the enzyme. We have this fine tuning of all these different, very similar protein sequences that are catalyzing the same reaction, allowing us to get exactly the amount of flux through each part of the overlapping segments of the pathway. So does that make sense? Okay. That is the 10,000-foot view of amino acid synthesis with a little bit of details here and there. But now you think about amino acids. Where did the amino group come from? We don't have the capability to make ammonia. So there's lots of nitrogen. It's like 80% nitrogen or so in the air, into gas. So if you would just let things go to their thermodynamic endpoint, you would end up with just oxidized form of nitrogen. So ammonia is the most reduced form of nitrogen. So we need some way to synthesize ammonia. So there's this nitrogen cycle that goes through each of the oxidation, possible oxidation states of nitrogen. So there's a set of bacteria. Remember, fish are releasing ammonia directly. And then there's a set of bacteria that can oxidize that. So they literally are burning ammonia. Oxidation reactions are highly exergonic. So these organisms derive synthesis of ATP from burning ammonia to nitrate or nitrite to nitrate. But then there's other bacteria that do the opposite reaction. And so they take a fully oxidized molecule and convert it into a more reduced form of the molecule. Why would they do that? Let me give you a hint. These are necessarily anaerobic bacteria that do this. So what do we do? So we're aerobic organisms, right? What do we do with the oxygen? Why do we need oxygen? We're bringing all the pathways together. Exactly. So if you're an anaerobic bacteria, you don't have anything to pass your electrons to. You don't have the oxygen to pass your electrons through. But you could just pass it to some other oxidized molecule. So this is how you deposit those electrons that have come through the same similar electron transport chains. And then you just, in the last step, instead of depositing the electrons on oxygen, you deposit them on nitrate to make nitrogen gas. And so we have all this nitrogen gas around. But then there's this other conversion. Conversion of nitrogen gas to ammonia. In other words, fertilizer synthesis. And so this reaction, you might think, well, surely exergonic, right? Is it? Would you guess that the reduction here is exergonic? Okay. Well, this is, let's see, the full oxidation. That's this part here. And so that's exergonic. The other way, could that be exergonic? This nitrogen fixing? It is. And that's very surprising. Nitrogen gas converted to ammonia releases 33 kilojoules per mole. So that's an easy reaction, right? Well, there's this little problem. That bond is about a thousand kilojoules per mole in strength. The bond between those two nitrogen atoms. It is exergonic. At equilibrium, there would be 100,000-fold excess of ammonia compared to nitrogen gas. But there's this little problem in between. A thousand kilojoules per mole is necessary to crack those two nitrogen atoms apart. So although it's exergonic, delta G double dagger is huge. It's a thousand kilojoules per mole. There's a huge activation barrier to this reaction. So it's thermodynamically very favorable, but kinetically impossible. Do you see that? So you can read it, too. So you might think, well, you can do this chemically. You can take your nitrogen and a billion people on Earth are dependent on this reaction. If this reaction didn't occur, billions of people would die. This is how we make fertilizers. We take nitrogen and we put tremendous amount of energy into that molecule. We would bake it at 400 degrees and put it under hundreds of pounds of pressure because we're trying to pry those two nitrogen atoms apart. That's a tremendous amount of energy that's necessary chemically to transform nitrogen gas to ammonia. But if we want to do this with enzymes, these enzymes have been evolved such that they can do this reaction at atmospheric pressure at room temperature. So you think about it. It's what an amazing enzyme. You take something that requires tremendous amounts of energy and you are able to do it under standard conditions. So this occurs in these denitrogenase enzymes and there's a little electron transport going on here. So here's a complex between the two enzymes that are involved in this reaction. So you have the dinitrogenase reductase, which is transferring electrons here to our dinitrogenase in the center, I believe. So here's our dinitrogenase in the center. And inside of the dinitrogenase are all these very ornate and elaborate metal clusters. Remember that? Remember that oxygen-evolving complex that we saw before? These are batteries. See the batteries? They're right here. And we're taking these electrons and charging the batteries one electron at a time, transferring from some original source down into this reductase. The reductase transfers electrons one at a time. It begins to charge up the batteries in these metal centers to the point where we've invested a tremendous amount of energy in here and in one step it takes into and makes ammonia. We don't want to get stuck somewhere in between for the same reason that we talked about with the oxygen-evolving complex. We don't want any free radicals going on. Okay, so let's look at each of these steps. The dinitrogenase reductase is a, literally, it's just a one-electron carrier. So it accepts an electron from ferrodoxin. Where did we see ferrodoxin before? There's a lot of questions today. Zorro. Yep, yep, photosystem one. Very good. And what was it used to do there in photosystem one, that ferrodoxin? NADP, oxido, reductase enzyme. It's making NADPH molecules, transferring electrons to the NADPH synthesizing enzyme. But here we can also use those electrons on ferrodoxin to transfer them to the dinitrogenase enzyme by first transferring them to the reductase. So the ferrodoxin binds to the dinitrogenase reductase, transfers one electron at a time. This reductase then cleaves two ATP molecules, which allows to jam those electrons into the dinitrogenase and also give some energy into the dinitrogenase. So for every electron we jam into the dinitrogenase, we cleave two ATP molecules. So what do we have here? We have a six electron transformation of our N2 to make NH3. So now those electrons are received on the dinitrogenase one at a time and stored in these elaborate metal clusters. So there's a molybdenum metal ion here, and that's also involved in holding this thing together. So we charge it up. We put a tremendous amount of energy, hundreds and hundreds of kilojoules promote energy into this dinitrogenase. So then when it binds nitrogen in one bolt, it transfers those electrons to its final product. And so here we derive those electrons from metabolism so we take pyruvate dehydrogenase, for example. We can make some NADH, which then eventually you can transfer those electrons onto ferrodoxin one at a time to make the reduced form. Transfer from one electron at a time from ferrodoxin to the dinitrogenase reductase, which when ATP is hydrolyzed, it allows you to transfer electrons from this enzyme to this enzyme, the dinitrogenase. So you have this conformational change of the dinitrogenase reductase, which is catalyzed by ATP hydrolysis. And that allows you to have enough energy to insert these electrons where they need to go in the dinitrogenase when they're transferred. And you say, well, wait a minute. The reaction we're doing here involves six electrons. But because of the mechanism of this enzyme, we're not only reducing nitrogen to ammonia, we're also reducing protons to hydrogen gas. That's a necessary part. So actually it takes eight electrons in this process. So, and that means 16 ATP molecules needed to be used up to make this NH4, this ammonia molecule. So that's a tremendous amount of energy. Any questions so far? Any of that stuff? All right. That's a tough one, huh? Hey, test, test, test. Okay, turn off the clicker. Turn off the clicker. Turn off the clicker. So why was I chuckling? And people were struggling up here, you know? So one of the answers was reductive deamination. Why is that wrong? Yes, it's going the other way. And what is NADP, a cofactor? Yes. She's sure. Okay, so the correct answer was D. D, good for you.