 Hello, let's settle down please. Welcome back. You guys look really tan. Must have been a good break. All right. So welcome back. Today we have a lecture that is not on the exam. So, but it will be on the third midterm test. All right. So we do have a midterm coming up on Thursday. We have TA led review review session today. Sarah's, it's still at six to eight. Okay. In the science center. And then the midterms coming up and it's the same distribution of locations. So it's very, very, very important for you to go to the right room. Because if you don't, we'll run out of exams in one of the rooms. And your fellow students will not appreciate that. So we have a little bit to cover up. So this part will not be on the exam. So this was left over from the last lecture. Actually, I do have a question on insulin signaling, which was the very last part of the last lecture. And that is on the exam. But this will not be on the second midterm. It potentially could be on the third midterm. These are just sort of some odds and ends left over. So we talked about these G coupled proteins. And this is similar to those in that it makes a cyclic nucleotide. But this is has no, it doesn't have a bunch of subunits. It's just one receptor in that single receptor when bound to ligand transforms GTP to cyclic GMP. So instead of cyclizing, adenylate, you're cyclizing this GTP to cyclic GMP. And that's a second messenger. And so that can go around and activate other proteins. And so this is the structure very similar. It makes a three prime cyclic structure. So the phosphate, the five prime position, makes a second bond at the three prime position. So that's a class of receptors that you should be aware of. There's also another signaling molecule, this NO. And so the nitric oxide synthase makes NO from arginine. And this is a cool second messenger. And that is both a second messenger within a cell and between cells. This can actually escape from cells and then go into neighboring cells and activate them. This is a unique type of receptor. So many of you are interested in neuro. So these are important ligand gated iron channels in neuro. And so this binds cedicoline. And the binding of a cedicoline is an allosteric effector of the transporter protein, causing a rotation of helices that take a hydrophobic surface and convert it into a hydrophilic surface that's able to pass calcium ions. So that calcium ion signaling is important in how your neurons function. And this is one of the receptors that responds with a hormone, a cedicoline, and induces a signaling effect. We know that calcium can also be an important signaling molecule. And one of the ways in which calcium acts as a signaling molecule is through binding a molecule called cal modulins. Calcium modulin, cal modulin. And the binding of calcium to this protein induces a massive conformational change. So you see in this state, you have this linear configuration. And when you bind the calcium ions, you have a bent helix. So this one long helix is literally bent into two, and it can now bind to a certain domain within target proteins, and that binding can regulate those target proteins. And so this conformational change is necessary to expose the binding surface for the domain and the target proteins. So that's how one of the ways in which calcium can regulate signaling molecules. So we're going to talk about steroid hormones later in the class. And I already have mentioned that the first encounter of the steroid hormone with the receptor can either occur in the cytosol or in the nucleus. But no matter what, the receptor bound to the steroid hormone acts as a transcriptional regulator in binding elements in the DNA that induce expression of hormone responsive genes. And so that leads to transcription and the synthesis of new proteins. And so cortisol is an example. There's many steroid molecules that can do this kind of process. So it's a very general, wonderful world of signaling and receptors at a general sense. But you do need to know insulin signaling. It's on the exam, so try to work your way through that. If we have time at the end of the class, we could go over that some more if you want. It seems like you guys did pretty well on the clicker on that topic. So maybe you've got it. Okay, so these are all a summary of all the different signaling molecules that we've seen. Okay, so now to the main topic for today. We're going to be talking about the biosynthetic pathways involved in the formation of metabolized containing nitrogen. So a variety of biological amines. Again, for those interested in neuro neurotransmitters, a lot of those are going to be included in today's discussion. And then we're going to look at tetraparoles. So what's an example of a tetraparole that you guys can remember? Do you know what tetraparole is? It's not written there. Any idea? Anybody ever seen a tetraparole before? Did we maybe have like a whole lecture on tetraparole molecule? Remember heme? So that's a pretty scary looking molecule. And we're going to look at the pathways that make the heme group. And that's a similar pathways are also used. And the heme group is can be elaborated by different modifications to make other tetraparole. So you have four pearls, right? So we'll look at that. And then we're going to switch gears and look at nucleotide metabolism. This is setting the stage for the third part of the class where we're going to be looking at information pathways, how DNA is replicated, converted to RNA, RNA is converted into proteins. But we need to learn the words of that language, which is nucleotides and how they're made. And so that's today's topic. We'll look at the central role of PRPP and the synthesis and nucleotides that's going to be providing the ribose. And so there's different classes of purines and primitines. Many of you have, I'm sure, not shocked by this. And we're going to be learning the structures of those, as well as the pathways. And then some other related topics. Okay, so there's a variety of biological and mean molecules. Neurotransmitters are a large class of biological means. Polyamines often have important roles in association with DNA molecules and compaction of DNA. So tetraparoles, or the type molecules are the hemes, the chlorophylls, even member coenzyme B12 has a neat homolytic bond cleavage that we saw in that insanely complex ring that took like 30 steps for organic chemists to make, we're not going to go through that mechanism. But we are going to look at at heme synthesis. So here's a variety of biological amines. And so you probably have heard of some of these, we have dopamine, norepinephrine, epinephrine, GABA, serotonin and histamine. And so if you look at these molecules, you say, wow, this is going to be an easy class. They're very, very similar in structure to amino acids. So we had our last or two lectures ago, we learned about how amino acids are made. Those amino acids can be used as substrates to make these molecules. So you might see a tyrosine, for example, here, or what appears to be fragments of a tyrosine here, here and here. You might see another amino acid, glutamic acid, tryptophan and histidine. But in general, one common trend throughout all these biological amines is the removal of the carboxylate. So there's got to be some kind of decarboxylation reaction. And there's a cofactor that you've already been introduced to that moonlights as a deep carboxylation prosthetic group. So let's start and look at some of these pathways. So as I said, we start with tyrosine. The tyrosine can be hydroxylated. So we have a hydroxylase enzyme that hydroxylates the tyrosine. And then we just simply decarboxylate and we're done. So two step, bada-bing, bada-boom, you've got dopamine. So Dopa, some of you guys might have seen the movie Awakenings with Robin Williams. So he gave or that character gave L. Dopa, which is this molecule to patients with Parkinson's and it seemed to help in some of the effects. So it basically juices up the amount of dopamine in their cells, but then they all became catatonic again. So that was sad. So dopamine can be converted ultimately into epinephrine. So the first step, so you take your dopamine, it's got the hydroxyl group there, you add another second hydroxyl group at this position to make the norepinephrine. And then epinephrine, which is remember, that's adrenaline. So that is you methylate with Sam. Remember, Sam is our methyl group donor. And so that that's epinephrine. Okay, so this so far, very, very straightforward. You could guess the types of reactions going from amino acid. So these decarboxylation reactions are catalyzed by PLP. So remember when we talked about transamination chemistry with amino acids. And so remember, you can make the alpha keto acid. And then this is all reversible steps. So the alpha keto acid, another alpha keto acid can accept that amino group regenerating the pyridoxyl phosphate. So you can sort of transfer this alpha keto functionality and amino group between different substrates, so transferring amino groups. But the other so this reaction, remember, I mentioned that this cofactor helps in the formation of a carbanion at this position. It stabilizes that so without the cofactor, you'd have this unfavorable carbanion instead of having that charge focused on a single carbon, it's now spread throughout this long conjugated system. But you know, you could do a very similar chemistry instead of pulling off the proton, you can remove the carboxylate. And the intermediates are identical. So here, here you removed a proton, here you're removing a carboxylate. And so all you have to do is then release this molecule. Okay, and again, the this this reaction proceeds through a carbanion, intermediate, and that's stabilized by the conjugated double bonds here in this system. So decarboxylation, transamination, and also it can be involved in inversion of stereochemistry. So bacteria need D amino acids, right? And so you can invert the L amino acids to a D amino acids, as well with this cofactor. Any questions on that? Does that make a certain amount of sense? Sort of logical, carbanion, bad. Okay, so glutamate can be converted to GABA. And this is just straight up decarboxylation. Just take the amino acid, remove the carboxylate, you've got GABA. Again, with histidine, you have the Midazol group. And so you can remove the carboxylate and get histamine, even the name gives it away. Now tryptovan can be hydroxylated and then decarboxylated to make serotonin. So these are actually all neurotransmitters. This also is involved in your immune system mass cells release this and which initiates an immune response. Okay, so what about these polyamines? So here's an example of a polyamine. So polyamine has a polyamine has lots of amino groups. And it has lots of positive charges. And those positive charges make multi valent interactions with DNA molecules. Remember, DNA has a bunch of negative charges. And these are important in compaction of DNA. For example, in sperm cells, you need to compact all that DNA because basically all it is is just a DNA with a tail going around. And so the way this molecule is made is pretty cool. So we take this adomet. This is the member of the methyl, the prosthetic group involved in methyl transfer. But when you look at the structure of acid in a steel methionine, you can think conceptually, yeah, you can donate a methyl, but you could also donate this group, right? Either one of those removals or cleavages would result in removal of that positively charged positive charge on sulfur. So you can first decarboxylate the adomet, making a decarboxylated adomet. And now you have a propylamine group. Do you see that? And so if we cleave this bond, we then alleviate the positive charge on the sulfur. And so that's this is an activated form of propylamine. And so that propylamine can be donated to some other molecule. And so here we have ornithine. Do you remember where we saw ornithine? This one you might want to know for Thursday. It's been the urea cycle. Remember, it's sort of like lysine, small version of lysine, one less methylene group. So you can, you can decarboxylate ornithine to putresine. And so this is a smell that's not very pleasant. Things that are rotting make that smell. And you can see, okay, it's got a lot of amino groups. It's, it's not going to smell very good. So you have putresine. And now you have two positions in which you can add your propylamine from this weird decarboxylated adomet. So you can add a propylamine on one side, and then the other side, and then you've got spermine. Okay, so it's pretty cool molecule. And we're using our cofactor in unorthodox ways, unnatural ways. So you're left with an adenosine with a sulfur and a methyl group on it. Okay, so this is the actual decarboxylation reaction. So, so in this first step, you have a decarboxylation. And it turns out that a parasite, tropanosomes, or yeah, I think it's tropanosomes, African sleeping sickness, the ornithine decarboxylase in that organism doesn't turn over that much. In other words, the protein once it's made is not really degraded. Whereas the human version of this enzyme, it's made degraded, made degraded, there's a certain amount of turnover. We'll talk about in our lecture 20, the actual biochemistry of how proteins are degraded. But so what we can do here is we can target this reaction with a suicide inhibitor. And that inhibitor would impact slightly the human version of the enzyme, but it will more aggressively inhibit the tropanosome version of the enzyme, because once that's inhibited, it can't get turned over. Okay, and so this is the treatment for that African sleeping sickness is treatment with DMF or DFMO. And so DMFO, you can see how this amino group, it's sort of similar to ornithine, except for it has this CHF2 substituent. And so this is going to bind to our paradoxal phosphate. And then instead of doing what it's supposed to do, you know, remember, we're supposed to decarboxylate this arrow supposed to go here. Instead, you get the electrons go up there. And then it gets worse and worse, you've got a cysteine on the enzyme. And that attacks this position, you actually make a covalent adduct with this inhibitor. And that's that's the end of the road. I mean, things might rearrange, but your cysteine is messed up. And therefore, your protein is no longer functional. And so this is a nice example of how we can selectively target a particular enzyme. Okay, any questions so far? We've been through the biological amines. So so far, not so bad, we're getting to the massive plumbing diagram that's sitting over there. Heems are a little bit more complex, the tetraperols. So the starting one of the starting molecules for this pathway is Delta Amino Levulinic acid. So I've drawn some of the structures here next to these. And so this molecule, two of these molecules is going to condense into a heterocycle nitrogen containing cyclic molecule. And then those heterocycles to make are going to be combined. Four of them are going to be combined to make preliminary heme like molecules, which will then be converted into heme. And so we can siphon off at various points in this pathway to make all a lot of different heterocyclic molecules. But we're just going to look at heme for now. So the first so here's the actual structure. So you have these vinyl substituents, propionate and methyl substituents. You've got your heterocycle for combine heterocycle into large heterocycle. And then remember, that's the molecule that can coordinate iron. And so to get things started, we need to make Delta Amino Levulinate or Levulinic acid. There's two different ways to do this. In animals, we can combine succinyl CoA and glycine. So this is this CoA group can get kicked out to make our Delta Amino Levulinate. Now actually plants, this pathway here was discovered at Brown University. You might notice in Sydney Frank Hall on the first floor that one of the offices says Center of Tetraperol Research. So this is Sam Beale. He used to teach this class a few years back. And he discovered that this is really amazing tRNA involved in this reaction. So you have glutamate. And you'll be learning about tRNAs and how you can add an amino acid that's called charging the tRNA to at the three prime position of the RNA molecule. And so this is the charged version. So you now have a ester linkage between glutamate and your tRNA. So this is charged tRNA. And now you just take that. And this is in the oxidation state of an ester. And so you can reduce that to an aldehyde releasing the tRNA. So you're not don't want to be using up your glutamic acid tRNAs. We need that for other purposes as well. And then you can rearrange this carbonyl in this amino group to get the delta amino levulinate. Okay, so there's a little piece of brown here as well. Okay, so levulinate acid can then be combined. So we can take two of those molecules and attach them together in this way. Sort of can imagine how that might work. We've color coded things to help you to see it. And so we need four of these heterocycles to make to begin to make our larger molecules. So those are combined. So you can see that at each of these combination places, we're losing an ammonia. So lose four ammonia. And then you close the ring. And now all we're going to do and remember these groups. So this blue group is appropriate. Do you see it? And the acetate is this ass AC. So we've abbreviated it here. And we can begin to decarboxylate. So free decarboxylate and acetate. We get a methyl group. Do you see it? And then you can decarboxylate the propionate group and get a vinyl group. So you can eliminate that's elimination reaction. And then we can charge the heme with an iron. Okay, so the key things. Am I going to ask you to draw these structures? No way. Am I going to ask you to have a sense of, you know, what the starting process is? Yes, probably. So you have to know levulinate. And you have to know that this molecule is formed by the combination of two of those levulinates. And then you just sort of do a variety. I might ask you what kinds of reactions occur? Well, you could say those decarboxylations, loss of ammonia, at the addition of iron. Okay, the details are just too much to actually draw these structures. I am evil, but not that evil. Okay, any questions on this? I know this one's a little bit overwhelming. Partially because they've started abbreviating things spontaneously. Okay. So you can make it. I mean, we've actually that's the whole pathway. We've made heme. And now you can break it down and you get into more sort of similar to putrosine, somewhat unpleasant things happen. So when you break heme, you can just open the ring up. And so, you know, you have the vinyl, the methyl, propionate, that's what those abbreviations stand for. Making things harder by abbreviation. And so this one, you can see it's a very highly conjugated molecule. So it's going to have a color to its green. So when you get punched, the green color is this bilverdin. Sometimes it's blue, and you might have different decompositions of this that have slightly different hues. Yellow color in your urine comes from bilirubin. It's a similar molecule, slightly modified. Okay, and so this is how we decompose this. So here we've reduced this double bond to give us bilirubin. Okay. All right. So that's all I'm going to cover on tetrapurals and biological amines. And now to something completely different, this is the main focus not only for this class, but for the remainder of the semester. Let's look at nucleotides. Nome and clature is confusing for nucleotides. And I am going to require a very high level of perfection on naming. So I'm going to ask you, you're going to have to know these names. Okay, so there's a nucleotide base. There's a nucleoside. That's where you add the ribose. And I'll show you structures in a minute. And then if you add a phosphate, the five prime position of the nucleoside, it then becomes a nucleotide. If you add a second phosphate, so a diphosphate, like ADP, it's still a nucleotide. Nucleotide is a general term. Adenolate is not just one molecule. It's any adenosine that has one or more phosphates attached to it. Confusing, isn't it? So that's adenolate. Now, of course, you can have a deoxy form of the molecule. So the two prime hydroxyl of the ribose ring can be converted from a hydroxyl to a hydrogen atom. And so that makes the deoxy versions of the nucleotides that are in DNA. Now, the non-deoxy forms are in RNA. And so these are easy, you're just adding deoxy to all of them. So you should know this slide. You should know it. You should know these names. And you should also know the structures associated with them. I'll show you in a moment. Now, of course, we have uracil. So uracil is only found in RNA. And when we slightly modify the uracil, it becomes thymine. And that's the version of that pyrimidine that's found in DNA. And so hopefully as you become familiar with these pathways, you can begin to sort of keep track of this ridiculous amount of information. There we go. So here's the basic structure. Now, luckily, all of the nucleotides have this general structure. So you have one or more phosphates making a nucleotide. This portion here is the nucleoside. When you add the phosphate, becomes a nucleotide. You have a ribose. Remember, ribose is an aldose of five carbon aldose that we've already studied in this class. Here's that critical two prime position. So the DNA versions of these nucleotides is going to have a hydrogen there instead of the hydroxyl group. All of them are attached to the five prime position to a phosphate and at the one prime position to our purine or pyrimidine base. So here is the structure of pyrimidine and purine. So and it's sort of one little trick that you can have to remember this is do you see how purine includes pyrimidine? It's built in there. You've added histidine to pyrimidine, basically. So here's the pyrimidine reg, exactly the same as here. And you've added an amidazole to that. So if you already know the structure of histamine, all you got to do is remember this guy, and then you've got this guy. Okay. And so the ribose is attached at the one prime position of the sugar to the one position of the pyrimidine or the nine position of the purine. That's just how nature set this thing up. Okay, so far. Okay, so here are the two purines and three pyrimidines. You need to know this. This slide just put a big star, asterisk, blinky lights, you need to know the structure. So you can remember which of these are purines, you've got the majority of the atoms, right? So these are just substituted purines and substituted pyrimidine. So in adenine, you'd added this amino group there, that's pretty straightforward. In guanine, you have a carbonyl functionality there and an amino group there. Those are the two purines. Remember, here's our embedded pyrimidine, here's our amidazole. Now the pyrimidines, you've got three types, you've got cytosine. So all you have to remember here is the base ring structure and then all these different substituent possibilities. And so cytosine, you've got amino group, a carbonyl group, thymine, you've got the two carbonyl groups and a methyl group. And uracil does not have the methyl group. So actually the way the biosynthetic pathways go, uracil is made first. And from that, we have a methylate to make thymine, but we'll see that in a moment. Okay, so I don't know what else to say beyond, this is just something that you need to know. It's the foundation of all life. Okay, ribonucleotides. Here is all of them. So I might say, for example, draw adenosine. Okay, so if you draw this molecule as shown here, that is wrong. Adenosine is not adenosine, that's adenylate. Adenosine is the version of the molecule without the phosphate. Okay, and we'll be stingy on partial credit there. So remember adenosine, guanosine, urodine, and cytodine are all the nucleoside portion. Nucleotide is adenylate, guanolate, urodilate, and cytodilate. Okay, but you should also know these names as well. So I might not tell you what I might say, just draw thymine. So if you put a ribose on that thymine, again, bad things will happen. Don't put ribose on your thymine. Okay, so here's all these ribonucleotides. And so you have the two prime position has a hydroxyl group. Okay, for the deoxyribonucleotides, it's the same exact molecules without the hydroxyl group except for this T, which is what U was, but you added a methyl group. Okay. So whatever you got to do flashcards, whatever for this, you need to know it. Okay, so looking at these more generally, so you have nucleoside monophosphate, don't say nucleotide monophosphate. These are nucleotides. That's like saying phosphate phosphate, right? So nucleoside is the unphosphorylated version, adding a monophosphate, adding a diphosphate, adding a triphosphate. So there's a nucleoside monophosphate. Nucleoside monophosphate is a nucleotide. Got that? I know it's painful and confusing. Okay, and so this is how this works. So you have NMPs, NDPs, and NTPs, right? And you can substitute the first letter for which base is attached. This is a generic variable when you say nucleoside. Okay. So that's that. So that's what they are. Now how do you make them? What are the pathways used? There's two general strategies. So we're going to look at purines first, and then look at pyrimidines. Purines, you start with the sugar, and you literally assemble the base one atom at a time, very slowly onto the sugar. For the pyrimidines, you make another heterocycle that's not a common base heterocycle and convert it, and then add that molecule to the sugar here, and then you convert it to the regular base, the regular pyrimidine that's actually in DNA and RNA. Okay, and so we're going to look at these pathways. Now we're going to start with the purines. And so PRPP, we've seen this. We didn't really, I just sort of had PRPP, and then I had some of the amino acids that use this molecule. So PRPP is formed by adding a pyrophosphate at this C1 position of the ribose. Okay, so it's phosphoribosil 1 pyrophosphate, okay, PRPP. And so you can see, okay, this is activated. So what we're going to do is we're going to attack this position with the enzymes going to help us in this effort. We're going to add our nitrogenous base at that position. And when we do that, we invert stereochemistry. So this is in the alpha configuration. And all of the DNA molecules, if you look back on the previous side, they're in the beta configuration, cis with the highest numbered carbon substituent. Okay, little detail. So that's PRPP. So and we also saw that in synthesis of tryptophan, histidine. And now we're seeing it for nucleotides. Okay, so we're going to take that. And remember the differentiator, the purines, you start with PRPP and build the base onto that. For the primidines, you're going to start building the base, get to an intermediate form of the primidine, add that to PRPP, and then convert it into the actual primidines that are used in DNA and RNA. Okay, so for purines, the bases are built stepwise on the PRPP for primidines. You first make this intermediate base, it's called oritate. And that's added to PRPP. And then it's converted to the primidine. Okay, so let's start with purines. So we have PRPP. This is the actual, this is in another very important slide that tells you where everything is coming from. Okay, and so these nitrogens, for example, are the ametoneitrogens of glutamine. So remember, we have the alpha amino groups on glutamine, and then we have the ametoneitrogens, those are on the side chain. So when you add those ametoneitrogens, glutamine becomes glutamate because it's given up that side chain nitrogen, you've got glycine in here, formate. So you know that that probably is using THF, a cofactor to be added into here. You've got carbon dioxide. And we have one nitrogen from espartate. Okay. Okay, so how do we put this together? So the first step is very important, because it's the regulated, it's one of the regulated steps. There's feedback inhibition here. So the things at the branch point, and at the end of the pathway inhibit this. So this is glutamine, PRPP, ametotransferase. So here we're doing the first steps. We have the alpha configuration here and the pyrophosphate. And we've then taken a ametoneitrogen off of glutamine and put it here in this beta configuration. So this is the actual first nitrogen, remember, attaching to the ribose to the base at this nine position. And then we're just going to start building up the molecule. We've now abbreviated this group becomes R. And we're just showing here and we slowly build up this first ring, right? So this is the imidazole like functionality. And we built this up by adding glycine. And then we added a formate. So this aldehyde functionality. We added glutamine again. This nitrogen here getting ready for that second ring. And so these are all lots of intermediates here in this pathway. We then add, what are we doing here? We're adding the bicarbonate, which first must be activated, makes a lot of sense. We always see that like in the synthesis of carbonyl phosphate, we then add our aspartate, but we're only taking the little tiny nitrogen here, making the corresponding alpha keto acid there. And so and then we keep building this up another formal group, close the ring and now we've made an acetal monophosphate. So this is the actual branch point. So up to this point, the synthesis of both purines has gone through this one common intermediate. So we wouldn't want to regulate this whole pathway just by the levels of IMP. The big lesson from today, you want every base there and about, you know, even concentrations. You want to have a balanced pool of within the primitines within the purines and also between those two families. Because DNA is a polymer that uses all of them. You can't have, you know, one be less abundant. So this is IMP. And so at IMP, we branch out and there's some amazing and just I think highly creative regulation going on here, the nature has come up with. So we have the the easy stuff feedback inhibition. So you just take your the end of the pathway, either GMP or AMP and inhibit the first enzyme at the branch point, right? So this is discriminating the synthesis of AMP or GMP. And so if we regulate those steps, then we have just the right amount of those two. But then we also need to regulate between them. And there's this non coincidental choice of activating nucleotides in these reactions. So in the synthesis of AMP, GTP is used. And the synthesis of GMP, ATP is used, right? So if GTP levels go down, AMP levels track with it. Okay, so if GTP levels go up, AMP levels go up. And vice versa. And the same for this. So this is creating the crosstalk between these pathways so that we we need both AMP and GMP at the same amounts. Okay, so we're making a polymer that has both of them. Okay, does that make sense so far? So we've now synthesized AMP and GMP. Any questions? Yes. Okay, so this is a summary of all of the pathway. So remember we have ribose 5 phosphate converted to PRPP. That's our activated form of the ribose sugar. Then we're going to build up the purine. And in the first step is the highly regulated steps. So these are not digital switches. Remember we've seen this before. Each one contributes just a bit to the regulation. It's not like if you make one molecule of AMP, this enzyme is completely off. It would just be half reduced activity. So this is controlling the flux through this pathway. But down here you have the more elegant balancing of nucleotides. I haven't even drawn so you can draw the arrows in between these because they have the shared substrates creating the crosstalk that we just saw. Okay, so this is lots of regulation in these purines. So that wasn't so bad. So we've now covered that whole middle section going up to GDP and ADP. It's really helpful to sort of keep track over here. Okay, so what about the other molecules? The pyrimidines. Any questions on purines? Okay, here's the pyrimidines. Pyrimidines are even easier to make, right? So you've got just aspartate and carbonyl phosphate. So you're like, oh, wait a minute. By the way, where does this pathway occur? Well, it occurs in the cytosol. When we saw carbonyl phosphate before, where were we? Do you remember? The mitochondria. Well, we saw it in the urea cycle, that's right. And then we saw it in the mitochondria. So this is not your grandmother's carbonyl phosphate. This is a cytosolic version made by a completely different enzyme that's an isoenzyme. Okay, so here's pyrimidine. So here's our carbonyl phosphate. This is made by cytoplasmic variant of carbonyl phosphate synthase. It has the same exact mechanism. Remember, you have the bicarbonate, you have a biotin cofactor, I believe, I think. Then you add the phosphate, and then you add the ammonia, and then you add actually, I don't think it has a biotin cofactor. That's an error. You add the ammonia, and then you add that second phosphate, right? So there's two ATP molecules being used to make this. And so this carbonyl is combined with aspartate to make in carbonyl aspartate, very creatively named. And you can look at how the aspartate is added on here. So do you see how it's added? So this is the side chain. This is the alpha-carbon carboxyline amino group, right? Added to carbamate. So remember carbamate, carbonyl phosphate had a phosphate here, and that has now been substituted by aspartic acid. Do you see it? Okay. And then we go to oratate. And so this is our precursor base. And remember I said we're going to add to that precursor base the ribose in the form of PRPP. We're adding it here at this one position of this heterocycle. And so this has a carboxyl at this position and a carbonyl group. So oratidylate. Okay. And so this is the transfer. This is carbonyl phosphate synthetase 2. You have the glutamine coming in, providing that amido nitrogen. And that nitrogen is not just released into solution where it would be protonated. It's being sequestered here in this channel where it can combine with activated bicarbonate, phosphorylated bicarbonate to make our carbonyl phosphate. So we have a total of three active sites here. One to remove the amido nitrogen. Here we're reacting our bicarbonate ATP and ammonia. And here we're adding that second phosphate. So remember it's carbonyl phosphate. Okay. So it's one enzyme and the substrates pass through this channel. This little gopher tunnel going through this enzyme. Okay. So we have a rhododylate. Now we're going to convert it into bases that are more primitines that were more familiar to us. We're going to make UMP. Okay. So all we did here is to decarboxylate. Okay. The rhododylate to make uridylate. So that's UMP. Remember uridylate means that there's a phospho group on that uracil. Okay. But we didn't add uracil to ribose. We added oritate to ribose. Important distinction. And now we can just phosphorylate this. You have monophosphate, diphosphate, and triphosphate. Okay. And so this last step can go back and regulate the process. So you can have UTP. I'm sorry. UTP gets converted to CTP by converting the carbonyl group to an amino group. Okay. And so this reaction, remember the first step in permitting biosynthesis was a combination of aspartic acid and carbonyl phosphate. That's regulated by CTP. So remember CTP is at the very bottom of the pathway. So it goes back to regulate the very first step in the pathway. But then you can see this curve shows us that ATP inhibits the inhibitor. So they competed in an allosteric regulatory site. CTP increases the amount of substrate for the enzyme to reach its half-maximal rate. But ATP puts it back to where it was. So CTP, primidine or purine. Primidine. ATP, primidine or purine. Why? That doesn't make any sense, does it? How can it make sense? The purine is regulating the perimidine. TNA have more primidines than purines? Right? So remember this is the same motif again and again. This is a balanced pool of purines and perimidines. The allosteric regulation of the enzyme that initiates perimidine biosynthesis is balancing the pools. So the more purines we have, we have to ramp up the synthesis of perimidines so we can balance that out. So now it's all simple. We can move phosphates around and ATP is what's providing the phosphates. The way in nature, I actually lied, ATP is a little bit higher concentration because ATP is masquerading this other small role of transferring energy around in a large number of biochemical reactions. It's also in DNA. So ATP is providing the phosphate to take the nucleoside monophosphate to the diphosphate and the diphosphate to the triphosphate. So all that phosphorylation which is up in here. So the arrows going up to the GTP, ATP, and UTP, it's all coming from ATP, that phosphate, because it's left over from metabolism. So now we've covered pretty much the whole middle section of that chart. So we've covered all of this so far. So we've made the ribonucleotides. So now we need to look at the deoxyribonucleotides. Do you understand so far everything? Any questions? Okay, are you guys doing? You need more time? Yeah, you need more time. Double check your work. Okay, I think we should wrap it up. Okay, everybody vote. Guessing time. Throw some answer in. Okay, is this a dangerous experiment? And why is it dangerous? That's not your question. Okay, so what's release? What is, where is the label at the end? So gaseous C1402, that sounds dangerous. Yeah, that's right. Okay, did that question suck? Yeah. Will you get that kind of thing on exam? Nope. So the answer is E, not C02 would not be in CTP. Okay, all right, let's collect ourselves. Okay, here's why. Because it was here and then it was gone. So I sort of put everything together on one slide. Okay, let's focus. Come back together now. Come back together. Okay, so we need to make the deoxy forms of these nucleotides. And so let's get to that. So there's one enzyme that converts all four ribonucleotides to deoxyribonucleotides. So this one amazing enzyme takes, doesn't matter what the base is, it converts it to the deoxy form. And it's simple chemistry, right? We're just reducing this position, right? And oxidizing our cofactors. Kali's not directly by NADPH, actually those electrons get into a little electron transport chain and then are eventually delivered at this position. But the overall reaction is any nucleoside diphosphate being converted to a deoxy nucleoside diphosphate. Okay, so here's the actual transfer of electrons. So here's our enzyme ribonucleotide reductase. This enzyme converts the 2' hydroxy form of the ribose to the deoxy form of the ribose. Those electrons come from here, but ultimately they start up here. And there's two different ways we can do this. So we can take electrons from NADPH, put them on a molecule called glutathione. So you can have a disulfide link between two of these glutathione molecules to make the oxidized form. Okay, and so when we transfer those electrons from NADPH, we reduce this to the single molecule glutathione, make two of those. Glutathione, those electrons can then be transferred to a glutaridoxin, which is a protein, regenerating the disulfide-lengthed version of glutathione. So this glutaridoxin has a cysteine disulfide, which is reduced. And then those electrons can be transferred from glutaridoxin to our ribonucleotide reductase, taking a cysteine disulfide linkage and reducing it. And from here, the electrons are going to go into the 2' position of the ribose sugar. Okay, but you can also do this in a similar pathway. Instead of using these two molecules, glutathione and glutaridoxin, you can use FAD and thioridoxin, but it's the same thing. When the molecules are oscillating between a oxidized and a reduced form, and then back again. Okay, so this is how we get the electrons, and this is now the activated picture of our ribonucleotide reductase. We have, instead of a disulfide link here, we have sulfhydryl free cysteines. And this has a regulatory subunit and a catalytic subunit. The catalytic subunit has these really bizarro free radical containing amino acids. So tyrosine amino acid, tyrosyl radicals are formed. But actually, when we start interacting with the substrate, this tyrosine is regenerated and we make a cysteine free radical on the sulfur atom. Okay, so we just have XH. Something free radical is going to be providing some catalysis here. And there's two different types of regulatory sites. There's a primary regulation site that's responding to energy charge and overall feedback. Do we have enough in general deoxy nucleotide or nucleoside triphosphates or diphosphates? But then there's also a substrate specificity site. And we have a whole slide, very, very complicated slide, describing what's going on at this one regulatory site. But the big picture is it's providing balanced pools and feedback regulation. And then this site is responding to feedback regulation and to energy charge, the amount of ATP in the cell, whether it's time to do anabolic processes. So here's the actual reaction. We have the three prime position, not the position where we're moving the hydroxyl, but we're going to take a hydrogen atoms. We have some amino acid free radical that takes our hydrogen atom off here, leaving a free radicals, the homolytic bond cleavage. So we have those single headed arrows, if you remember from Orgo. And then we're going to protonate the hydroxyl group, making a wonderful leaving group. So now we have a H2O leaving group. And so we sort of activated this hydroxyl group to leave the enzyme. And so then we lose the water molecule, giving us this really amazing weird, you got a carbocation and a free radical. And then you add the hydrogen back, confusingly, after you take the, from the, remember you had your disulfide-linked ribonucleotide reductase, that was reduced to that electron transport chain, making those sulfhydyls. And that hydrogen is being added back here. So the actual hydrogen being added at the two prime position was from the enzyme itself. And then we add our hydrogen atom back. Why do we do it this way? I have not come up with a good answer. It's not, initially it's like, okay, maybe I have something to do with disfavoring and elimination side reaction, but we can have homolytic elimination reactions as well. So if you guys can think of something, that would be cool. But I cannot, but it's something is going on here where there's got to be a reason why evolution made us take this hydrogen off and then put it back on and making a free radical. Okay, so that's the actual chemistry, really seemingly unnecessarily complex. So it's sort of important all DNA comes from this one enzyme. This makes all the deoxy nucleotide. So you're going to want to have everything to be absolutely perfect there. Okay, we've already looked at this. Yep, so moving on. So this is the regulation. So we have those two sites. We have the first site, the primary regulatory site. So that's generally turning the whole enzymatic activity on or off. It's sort of like a dial. It's a rheostat, but the specificity allosteric site is more regulating the abundance of each individual nucleotide, making sure it's a balanced pool. So this one's a little bit easier to understand. So we have ATP, indicator of energy charge in ourself. We have high ATP. It's time to do anabolic processes. That turns everything up, increases the activity of synthesis of everything. We have some feedback, deoxy ATP. So as that tends to get high, we're going to want to make infinite amounts of deoxy. We don't have any ribonucleotides left. We need to have some RNA molecules as well as DNA. So this turns it off, but you might wonder, well, why don't they all come in here? Well, the balancing is occurring over here. So once balanced, you can take any one of them to regulate the activity of everything. So the balancing is here, and a lot of this is feedback regulation, right? But a lot of it is also crosstalk. So here we have DCTP and DTTP being regulated by either deoxy or not deoxy ATP, right? So if we have more of these purines, we need to make more perimidines. So this regulation is balancing the pools of perimidines and purines, okay? And then we have a variety of feedback regulators going on here. DTTP comes back and turns things down or decreases the amount of that molecule made. DGTP can affect DATP. DATP comes back and turns everything off. So it's all sort of, it's like a football playbook, right? It's very complicated. But the big picture here is balanced pools. Is it time to do anabolic processes and making sure we have both DNA, multiple precursors for DNA and RNA, okay? So definitely try to understand this slide, read the textbook about this slide. I know it's complicated, but it's important because like I said, all DNA comes from this one enzyme. So we want to regulate that. So now we just have one little bit left. So over here, we have DCTP and then we need to make, we made DTTP, but the DTTP, I'm sorry, we made DUNP and then DTTP from that. So what is DUNP? What the heck is that? It's not in DNA, DUNP, dump? No, there's no dump in my DNA. So CTP, yes, there is deoxy, CTP, that's fine. But we also have this DUNP. And so remember, this is the difference between RNA and DNA. So this is in RNA and this is in DNA. So we need to convert the DUTP to ultimately DTMP. So we need to add, change that methyl substituent was the only differentiator between U and T. And so this is the enzymes that carry this out, thymidylate synthase. And so what we need to do here is add a 2DUNP, just add a methyl group. That's the only difference between a U and a T is a methyl group at that position. And it's a really weird reaction. Again, if anybody can figure out why nature did it this way, please let me know. So we have our THF. That's cool for, you know, adding a methyl group to stuff. Remember, THF is a cofactor both from methyl, but also at the alcohol, oxidation state, aldehyde, oxidation state as well. So this is the methylene form of this. So we take from serine, right? We can add that side chain methyl group with the hydroxy group and that forms this. So this is the wrong oxidation state. And so this enzyme is combining a carbon in the wrong oxidation state with an actual hydrogen that's not typically used on this cofactor to make a methyl substituent. But then you've screwed up your cofactor. It's not THF. Now it's DHF, right? It's dihydrofolate. Okay, so to, if we want to catalyze this reaction more than once, we have to regenerate our THF. And that's done by this hydrofolate reductase. Okay, and so we're adding that electron back on there and that from NADPH. Okay, so you have thymidylate synthase, which is catalyzing the addition of a methyl group to DUNP to make DTMP. And then we regenerate our cofactor with dihydrofolate reductase. Okay, and then we reach, this is an uncharged cofactor. And then we charge it again with the serine side chain in the wrong oxidation state. So that's how nature set this up. So let's, and this is important because if you want to, as a chemotherapeutic agent, cancer cells grow very rapidly. So the amount of DNA synthesis and cancer cells is much higher than in normal cells. So you can poison this enzyme or this regeneration of the cofactor. And that will slow down all DNA synthesis. So for DNA, we need all nucleotides. But so, you know, that's why people have such bad side effects with some of these drugs because their DNA synthesis is inhibited as well as the cancer cell DNA synthesis. But the cancer cells impacted more. And so we can put Fdump in here. Fdump, it's a funky sounding thing. And that can inhibit thymidylate synthase. And then a variety of other molecules, you guys might have heard of methotrexate, that can inhibit the regeneration of the cofactor from this DHF to the THF form. Okay, and so Fdump comes from fluoroyuricil. So fluoroyuricil is here. But this is fluoroyuricil with a DUNP, right? So you add the ribose to this. So this actual, this inhibitor that you add or drug that you add to cells is metabolized into its active form. So it hijacks the necessary enzymes to add the PRPP group there, making this Fdump. And then the Fdump is similar in structure to DUNP, and it inhibits this step. Okay, so let's look at this. And then there's this methotrexate, looks a lot like our THF cofactor. It also inhibits this process. You can also inhibit the addition of amino nitrogens to any nitrogenous base by adding glutamine analog. So here's our glutamine amino acid. Remember the amino nitrogen is what's being added at all these circled positions. And so if we add something that looks like glutamine, that can also inhibit the addition or the synthesis of these rings. Remember we're building these rings up one piece at a time. Now again, these are going to have side effects. I mean these things look like glutamine. Glutamine is not only used to transfer amino groups between molecules, it's also used in proteins. And so again, you're leveraging the fact that the cancer cells grow so quickly and that's why these inhibitors are efficacious with horrible side effects. So there's the different chemotherapeutics. So degradation, not quite as important. We don't want our genetic material degraded in general, but sometimes there is turnover of cells and you have a nice juicy steak. You're bringing in a bunch of nucleotides and all that meat. So you need, you know, in your diet, you need to be able to process these things. And so the way these are generally deconstructed is different for purines and pyrimidines, purines. You can go AMP to Inicine to Hypoxanthine, ultimately ending up in uric acid. Remember uric acid looks a lot like pyrimidine, or I'm sorry, purines. It's got those two fused heterocycles. Okay, remember it's sort of like two urea groups if you look at the structure. And this uric acid, no, if you're a bird you just squirt it out in an emulsion. But if you're a person you put it in your urea and it's excreted in the, or you put it in your urine and it's excreted as urine. So the pyrimidines are actually can be recycled. So CMP, UMP can be converted into uracil and the DTMP can be converted back into thymine. Okay, so you can release the ribose. Okay. And so there's a certain amount of recycling, but we're not going to go in all in the details here. Just one, I think one slide on the details. Okay, so let's look at this formation of uric acids. So you have GMP, AMP. You remove the ribose in both cases. So you're left with the nitrogenous base, guanosine, or guanine I should say and hypoxanthine. These can be converted to xanthine, which is very close to a uric acid. So hydroxylase can add a hydroxyl group here and oxidase can add the hydroxyl group and that's uric acid. So uric acid can be released from the cell or from the organism in the urine. And so if you have a buildup, if you eat too much red meat or if you have genetic predisposition, some problems in some of these enzymes, you can have sometimes a buildup of uric acid that causes arthritis like symptoms in some of your joints. Okay, so that's purines. And the last step, so you can add in, you can salvage these. Remember, when we made these originally, we built them up on the sugar. Either we made an intermediate base and then converted it to the right, added that to a sugar and converted it to the normal form of the base or we just built it up atom by atom onto the PRPP. So we need different enzymes to take these decomposition products and put them back, put the ribose back in. So we have these phosphoribosyl transferases, APARTs and so forth. And these can regenerate the AMP, GMP, the ribosylated versions of them. All right, so I think this is the overview. And yeah, it's up there. So study guide, this isn't the third midterm. You need to know the structures. Probably we'll ask both on the midterm and on the final structures of some of those nucleotides. This is a good figure to study and also the figures that color code the atoms and the nitrogenous bases are also very helpful. That could be asked as well.