Hello. Settle down. Hello. Excuse me. Hello. Everybody say shh. Oh, that works. Thanks for helping. All right. So welcome back. You survived your first biochemistry midterm. So we had, you might have noticed, we had a little bit of a glitch with the email. So we typically email you guys exams. How many people got like tons of emails from me? Come on spam filters. So I actually sent out by mistake 70,000 emails. And I think I can no longer communicate with you guys by email. I've been permanently banned. So we actually had the exams scanned into PDF.  But because of the glitch, I got shut down. So option B, I guess I'll try to send the emails again, maybe under Sarah's email address. She's not here, so she won't know. But I've also put the graded exams on the third floor. Don't go anywhere. On the third floor in between McMillan and Geochem. You know where there's a stairwell there? So they're in the box, but you have to find them by banner ID. So I'll try to send them again by email. I got about, I think, 70,000 copies because I copied myself. And so my email box finally is empty now. So, but overall, you guys did really well. So in terms of averages, I actually didn't teach last year. So I don't know what they had going on. But in terms of when I've been teaching, so you're about 1% off from the typical average. One year we had really low average. And some of the questions were similar to the ones where on the exam where there was a really low average. So I actually thought I was pretty impressed. Four people got 100 points every single one. So you are now deities in here. Great, oh great, biochemistry students. They're actually, I think, four or five, 99%. So I think most of the questions were answerable. Now, there is a little bit of bump here with sapling. And I don't mean to show this to you like, you know, I'm adjusting the exams to very nefariously balance things out. I actually have surprisingly little control on the averages of the exam. We try to make them the same difficulty. But you guys are in pretty good shape. This is the first year we've had sapling and everybody's grade on average is raised 1%. So it takes a little bit of pressure off the exam and less time pressure. So I just said I'd use your email to send the exams. Yes. All right, so when you see your exam you might be interested in a regrade. So we've done this lots of different ways over the years. This year, Sarah. Just be aware that if you come in, if it's not something that's really straightforward, like mis-tapping, things happen, like things get separated, it's tallyed, etc. There is a possibility of losing points, right? So keep that in mind. You'll have one week for the regrade. You have about one week, I will be posting office hours for an available to me about regrades. I will be putting those up on the Canvas homepage, probably later in the South Intermediate. But I'll try my best not to have them available. And of course, for some reason, the time slots don't really work for you. Just shoot me an email and you can find them usually for any time to leave. All right, so we're going to move on. So yeah, geniuses. It's going to offer cash bonus, but I'll go poor. There's so many geniuses in here. Find them for the clicker questions. I didn't have the median. Oh, the average in Orgo? The mode. Oh, I don't know. Okay. Yeah, so, okay. So let's, I guess Orgo, the world is shifting and this class is now harder than Orgo, which I don't even understand. Orgo is hard. Okay, guys, let's practice again. Don't add anything to that. All right, so we're going to be looking at the Krebs cycle today. And so there's a variety of new cofactors and coenzymes that I'll be introducing. We'll be looking at the gatekeeper. So Gozer, the gatekeeper is pyruvate dehydrogenase and that controls basically the opening of a furnace door. And so before you put some sugar molecules in there, you're going to make sure it's right. So we'll look at the regulation of that. We'll look at the cycle. It's a carbon neutral cycle and that, you know, same number of carbons go into the cycle has come out. There's a beautiful illustration of the control of stereochemistry with the conitase. We'll be looking at that. Regulation overall. And then we'll look at the dual role of the Krebs cycle in both catabolic and anabolic pathways. And one of the reasons there's so many steps is not just to torture you to know more things. It's because there's basically more ways to lead out to other more complex anabolic processes. And so, and then finally we'll be looking at something special that you can't do, that plants can do in relation to this supporting anabolic processes. So one cofactor that you haven't seen before, they will be seeing today is thiamine pyrophosphate. And this cofactor, it holds aldehyde equivalents. And so for example, pyruvate decarboxylase, this is not an oxidation reduction reaction. Remember where did we see that one? Pyruvate decarboxylase. It makes acid aldehyde. Acid aldehyde then gets converted to ethanol. So fermentation. That's right. And so this is the beer, the beer bubbles, right, the CO2. This enzyme removes carbon dioxide and releases acid aldehyde. And there's not, the electrons are redistributed amongst the parts of this molecule, but no electrons have been transported to an electron carrier, right? Whereas there are, thiamine pyrophosphate is also involved in complexes of enzymes that do involve an oxidation state change. So sulfur is more, just barely slightly more electronegative than carbon. And so this, you can consider this as being an oxidation state here, because you can hydrolyze, this is called a thioester, and you can hydrolyze that to an ester ester. And so, yep, so we'll see two examples of that today. And there's other things, other reactions where you have an aldehyde functionality, which is held on this cofactor, and then transferred to other molecules. All right, so we've talked about anaerobic fermentation. So what regulates these decisions is the presence of oxygen. And you might get to the end of the lecture and say, Professor Solomon, there's no oxygen on these slides. Well, oxygen is needed in the reoxidation of NADH. And you'll actually see that in the lecture on Tuesday, on oxidative phosphorylation. That's how we're actually making ATP molecules. But NADH levels are regulated by this presence. So if you don't have any oxygen, reduced cofactors will tend to increase. Okay, and so we'll be looking at the minifates of pyruvate focusing on the Krebs cycle today. But we've already talked about this. So pyruvate, remember, this is the fermentation in microorganisms. And so you can have the decarboxylation. We've shifted electrons away from here to here, but no electrons have come out. We've just decarboxylated, leaving an aldehyde. And then we can transfer or reduce acid-aldehyde ethanol and oxidize NADH to NAD+. And so if we're in the absence of other ways to get rid of NADH, such as through oxidative phosphorylation, this is useful because it can allow glycolysis to proceed. So we've already talked about this. So here's the structure of thymine pyrophosphate. And so you can see the pyrophosphate is this two phosphates attached to each other in a phosphoenhydride bond. And then you have this thiazolium ring, which is the business ring. That's actually going to be what's carrying the active aldehyde. And then you have this other ring, nitrogenous ring here. And so pyruvate, the acid-aldehyde equivalent, comes on and makes a bond to this thiazolium ring to form this ethyl hydroxy substituent. But this can collapse to the aldehyde, acid-aldehyde. So this is TPP. It holds on to our aldehyde. And this one, we typically, so remember when we did kinetrophes, it's been a whole good part of a lecture going through that mechanism. Here you just get a brief view. And so if I just give you a brief view, you're really going to want to focus on the key features of this. And I'm not going to ask you to draw this whole thing out. But the trick this thing is doing is it's making a carbanion. And that carbanion is useful in this reaction. So you make a bond between your pyruvate and this ring. And you make a hydroxyethyl substituent. And that then collapses to the aldehyde. And so this is carrying. And so you can see when we remove the carbon dioxide that helps us to get to this point. And so this is just a way to hold and to decarboxylate a particular molecule. Okay. So I didn't mention it last time, but actually glycolysis occurs in the cytoplasm. And what you're going to see today occurs in the mitochondria. So the mitochondria, in order for us to provide substrates for the Krebs cycle, we have to get them into the mitochondria. And so pyruvate is actually shuttled in to the mitochondria. And that takes energy. We're concentrating pyruvate in the mitochondria. But it's important to realize the context of what's happening here. And the purpose of this is it literally looks like a furnace. The flames coming out of this are these reduced cofactors, NADH and FADH2. And the electrons that are pulled off the carbon atoms in formation of carbon dioxide are temporarily stored on this reduced cofactors, which are then reoxidized in oxidative phosphorylation that we'll look at in the next lecture. Okay. So let's look at this first step. So this is the organelle that we're talking about. Many of you have had cell biology. There's two membranes, right, to a mitochondria. They sort of look like primitive bacteria. And so the outer membrane is very porous. So molecules up to a 5,000 Daltons or five kilodaltons are able to just go right in. There's these little holes are called porins. Remember, we had aquaporin. These are not aquaporin. These are other molecule porins. And so about up to five kilodaltons. So definitely pyruvate is not as much less than five kilodaltons. So it goes through that outer membrane. But the meat of the Krebs cycle happens in the matrix of the mitochondria. And so the inner membrane is not permeable. And we use a simport shuttle to transport pyruvate into the mitochondria. So the concentration of protons in the mitochondria is very low compared to outside of this inner membrane. And so the simport operates such that the pyruvate and the protons come in, both come in. Protons are going down the electrochemical gradient and pyruvate is being concentrated. It goes up the electrochemical gradient. So it actually takes some energy. So when we release some of this gradient, what's going to happen in oxidative phosphorylation is that we create the gradient. So the reduction of the oxidize or the oxidation of the reduced NADH cofactors, that energy is used to drive protons out. So we're going to have to waste some of that energy to get the pyruvate into here. So this is a simport with protons. Any questions so far? So we're just beginning by describing where this occurs. So this is the gatekeeper. So this is the pyruvate dehydrogenase process. And so this is catalyzed by an amazing, truly amazing enzyme. It's the molecular weight of this complex is five million Dalton's. And so it has three different types of polypeptides, but many copies of each polypeptide. So there's an E1, E2, and E3. And each of these polypeptides is catalyzing a different part of the chemistry. And we'll see how that works in a moment. But what's driving this forward is the formation or the oxidation process here. And so we're oxidizing pyruvate has a pretty high affinity for electrons. So let's see. So the oxidation of pyruvate is providing a lot of this negative change in, get standard change in free energy. So we're de-carboxylating and oxidizing. So this is normally an aldehyde. It becomes a thioesterase. So it's more oxidized. Okay. And so this is effectively irreversible because the delta G is negative. And it's highly regulated. This is the decider, right? That's going to decide are we going to have the pyruvate converted into lactate or is the pyruvate going to be fed into the Krebs cycle? Okay. And so this is one of the cofactors there, coenzyme A. So here you have adenosine, right? So we've seen this before on NADP and FADH. And so this adenosine is actually modified in the three prime position. Where is NAD modified? I didn't remember either. It's actually on the two prime position, random factoid. And so adenosine, here's the pyrophosphate, right? And then that's bound to this vitamin, pentathenic acid. We need that from our diet. At the end of this, we have a reactive thiol. And so a thiol can make thiolester linkages to the substrate. And so this is the business end. But this is similar to other cofactors. So you might find it somewhat surprising that a lot of the cofactors are using this ADP basic thing. And the reason is because enzymes have evolved a particular domain that binds with high specificity to ADP. And that domain can be combined with the domains that regulate the chemistry here to provide highly selective binding of the substrate. And so we've already looked at NAD. And so again, you see an ADP functionality optionally phosphorylated at that position. And here this is an electron carrier. So you can actually think of coenzyme E as a carboxylate carrier, right? Because it's carrying the carbon it makes a bond to is in the oxidation state of a carboxylate. So this is an electron carrier, as we've already talked about. And FAD also has ADP functionality. And this is a two electron carrier. NAD is obviously also a two electron carrier, but it can be a one electron carrier, but not today. So today in the example of the Krebs cycle, it's going to receive two electrons. Okay. And all of these are structurally similar. So undoubtedly, the evolution, they probably had some original ancestor that evolved. So once you bind ADP, you can just take that ADP binding capability and mix it with different catalytic domains to be able to catalyze reactions. And so you can see the structural similarity. Here's the adenine in the ribose. So in some cases, it's phosphorylated. So remember coenzyme A can be phosphorylated. NADPH would be phosphorylated. NADH is not phosphorylated there. You have a two pyrophosphate for ATP. You just have another phosphate. Whereas on these other molecules, you have the business in, the thing that's going to be helping to catalyze the reaction. So on the other cofacts, this complex is just full of cofactors, lipoic acid. This is unusual. So up to this point, I've shown you cofactors that are non-covalently associated with the enzyme. But in this case, an Epsilon amino group of a lysine. Do you see it? So here's a lysine, right? Epsilon amino group. So that's a lysine side chain. I'm one of the amino acids in the E2 subunit polypeptide of this enzyme. And that makes an amide linkage to lipoic acid. Low-poic acid can be in an oxidized form or a reduced form. And so this can do two things. It can receive electrons, but it also, once it's received electrons, it can carry acetate equivalents, right, through this thioester linkage. So it has two functions. But because it's covalently attached to the enzyme, what this is going to be doing in the enzyme active site, is it's going to be taking our substrate and whipping it between different catalytic subunits of the complex. And it's going to be really torquing our substrate. So we need to, during the catalysis today, we need to first receive electrons from here. But we have to regenerate the oxidized form of this cofactor for us to proceed in the reaction. So this is lipoic acid. And again, we have thioester. As it turns out, thioester hydrolysis is very exergonic. And we'll see the molecular basis of that in a moment. Look at these things. The width of a mitochondria is 500 nanometers. This thing is one-tenth the width of a mitochondria. It's 50 nanometers wide. It has 24 of the E1 subunits, 24 of the E2, and 12 of the E3 subunits. Its molecular weight is five times the ribosome. So you might think of, wow, ribosome is pretty big. And the structure for that definitely gets a Nobel Prize. But this is five ribosomes. This thing's huge. And so you notice the arrangement. Do you see how they're sort of, you have little nooks and crannies here? And you have each subunit coming together. And you're going to have this lipoic acid whipping substrates between these subunits. And so here's the meaty slide. We'll spend a fair amount of time here. So this is actually the catalysis. So you have pyruvate. You form this bond to the active acid aldehyde, right? So you decarboxylate. You have not changed the oxidation state of this carbon. You've shifted some electrons away from the carbon dioxide over to here. But so far, no oxidation state changed. But then when we transfer things, so when we transfer this, do you see what's happened? Here you have a hydroxyl. Here you have a carbonyl. Do you see that? So this has become oxidized. So those electrons have been transferred. Remember, we can't just release them in a solution. They're transferred to the molecule that binds to the substrate. So this is oxidized. This is now reduced. It's received both the electrons and this acetate equivalent. And this acetyl group is now in a thioester linkage. So the release or the oxidation of this carbon releases a lot of energy. And some of that energy is stored in the reduction of the lipoic acid. But also some are stored in the formation of a new thioester, because thioester hydrolysis is more exergonic than ester hydrolysis. And so we've divvied up some of that energy. It's a lot of energy released at this point. And now we're going to have a transacetylation. So here's the E1 subunit. Each one has a name. It's confusing. I agree. Pyruvate dehydrogenase complex is the whole thing. Whereas this subunit is called pyruvate dehydrogenase. And then we have dihydrolipoyltransacetylase. So you can sort of see the names correspond to the activities. And then we have this dihydrolipoyldehydrogenase. And so here is the lipoic acid. It's attached to a lysine on amino acid in the E2 subunit. It's received a new bond, a thioester linkage to our substrate. And then it transfers that to coenzyme A. Remember the business end of coenzyme A has that SH group. And so that now forms a new thioester linkage to make a molecule of acetyl-CoA. So here the substrate is covalently attached to the enzyme. Here it's attached to a coenzyme which is then released. So the product of this enzyme is acetyl-CoA. But now look at, we have a little bit of a problem. If these are already reduced, then there's no way for us to accept the next substrate. So we need an oxidized form of lipoic acid because those electrons are transferred from the substrate to here. If you don't transfer those electrons, you cannot make a new thioester linkage. And so here we have a somewhat hard to predict and maybe not intuitive passing of electrons from one molecule to the next. So we have first the electrons are transferred from the substrate to lipoic acid, and then the electrons are transferred to FAD, two electrons at a time to make FADH2. And then the electrons are transferred from FADH2 to NADH. Why did this enzyme not, you know, just transfer the electrons directly to NAD? I cannot think of any speculative reason. It just, it seems to work. And so this is the overall process. So the products, the input is pyruvate, the output is acetyl-CoA and NADH. Okay, this slide is confusing. Please ask questions. Yes. That's right. There's only two electrons. So this is two electrons, this is two electrons. There's two hydrogens. But when you have the form of NAD that binds two electrons, there's only one, if you look back at that slide, there's only one hydrogen and then a proton. Right? So that's a good question. It acts a little bit differently than FAD. Any other questions? Does this make sense? This is the gatekeeper to the furnace. Okay, so here is the actual electron affinities here in corresponding standard changes in free energy. And so it's exergonic to reduce this molecule, the lipoic acid. But the transfer of electrons from FAD to NAD is endergonic. But these electron affinities were not these cofactors in an enzyme. They were the cofactors. You remember we had the half cell and just sprinkle in some cofactors. And just like we saw the manipulation of the pKa of histidine in our chymotrypsin lecture, you can also, the enzyme by placing side chains around a cofactor can modulate the electron affinity. Okay? And so this seems on the surface starting from the standard state to be quite endergonic. But overall, it's approaching close to equilibrium here. But it's a one-way of valve function coming into the next step. And so this is heavily regulated. This is the thing that's deciding what kind of, how we're going to be producing our energy. And so why does it have to be one-tenth the size of a mitochondria? Well, just think about our lecture on hemoglobin. Remember the elegant regulation of that? So you change one subunit and the other subunits change. We had the concerted and sequential models to think about this. We have the same kind of possibilities here. We can have coordinate regulation. So this can, we need this thing, like as soon as you stop running or starting, or you eat a big lunch, this thing has to immediately sense what's going on in the cell. You don't want to overshoot or undershoot. And so this, as soon as you have regulation at one subunit, the rest of the subunits also are responding to this process. So you have 24, 24 and 12 difference. And there's also the lipoic acid whip, which is taking your substrate. Instead of just tossing your substrate after each enzyme into solution and rebinding it, you could have an incorrect binding of it. You could have a problem with specificity. Instead, you're like next, next, next. There's no possibilities of a lack of specificity because you just don't let go. So you're just channeling the substrates between the enzyme. And we talked about the coordinate regulation. Okay, so this thing is amazing. It is regulated in all kinds of different ways. It's regulated by the abundance of the product, NADH and acetyl-CoA. So if those start to build up, well, that means that we're, NADH is building up and we have enough energy, right? We don't need more. But it's stimulated allosterically by oxidized NAD and free-CoA because that's an indicator that we need some more energy. Not only that, it's also modified by a covalent modification. So when the E1 subunit is phosphorylated, that phosphorylation is a negative allosteric regulation. So it turns the enzyme off. And that phosphorylation, so obviously you put a phosphate on amino acid, you need an enzyme to do that. And this kinase, PDH kinase, phosphoryzed that residue. And that kinase is turned on by the high levels of ATP and other signals that we have enough energy. Does that make sense to you? Think through it. So we're turning on, when we have signals that we don't need this process to occur, we're turning on the activator of the inhibitor, so we're turning off PDH. Do you see that? And so this kinase is regulating, they're all sort of, you might think, well, that's awfully redundant. We need this thing to stop on a dime. It has to be exactly in the zone, precisely regulating the amount of molecules entering the Krebs cycle. And these kinases and the associated phosphatase, which turns it back on, so these things are actually part of this huge complex regulating the process. You might say, why did it have to evolve that way? Why not just regulate E1, E2 and 3 directly? Perhaps it gives you a little bit more better responsiveness. So this has just got elegant allosteric regulation all over the place. So now, once we pass Gozer, the gatekeeper, pyruvate dehydrogenase complex, we're right into the furnace. And so this is where we're going to literally burn carbon atoms.  And so we're going to catch these electrons on NADH as they come off, as each carbon atom is oxidized, we catch those electrons, either on NADH or FADH2. So there's a total of eight electrons being caught per revolution of this cycle. And so these carbon dioxide is released here and here, and it's called the tricarboxylic acid cycle because some of these members or metabolites in the cycle have three carboxylic acids. So this is the overall process, and this is the central core of metabolism. All catabolism and anabolism funnels through glycolysis and Krebs cycles, so we really need to know this. It's very important that we know this pathway. And so in the first step, we have an aldol, not clasin, an aldol condensation, where we combine this acetyl group with our oxaloacetate. And so this step produces citrate. And do you see how it sort of looks like it's being bound, a new covalent bond is sort of backwards of where you might guess. Can you imagine what the mechanism of this step is? So we're going to want to make a bond, a new bond to this methyl group. So what might we do with this substrate to help that process, to make a new bond there? Rearranging, yes. Two, enol, enol, yeah. You're going to make an enol, right? So if you have a double bond, then you have the hydroxyl, put the electrons back down. Do you see how the electrons move? And so you can make a new bond. So this goes through an enol intermediate. It is highly exergonic here, primarily because hydrolysis of a thioester is so exergonic. The next slide will show why. And so this highly exergonic thioester hydrolysis is driving the combination of these two molecules. And this is very helpful because the enzyme that makes oxaloacetate is struggling. It's extremely slow. So the levels of oxaloacetate are pretty low. But this enzyme, as soon as oxaloacetate is made, it's converted into products, even starting from the standard state. And so why is thioester hydrolysis so exergonic? The answer's here, but what is the molecular feature of a thioester? So what are we comparing? Let's compare the oxygen ester hydrolysis to thioester hydrolysis. Now the product is the same, right? It's a carboxyl. You hydrolyze the either ester. You're going to get a carboxylate. So the free energy of the products is identical. But the reason it's so exergonic has to do with the substrates, the free energy of the substrates. And so what this is telling us is that the free energy for a thioester is much higher than for an oxygen ester. Why might that be? What can you tell me about sulfurs? Have you guys had this in any other class? Maybe not. Now I don't trust anything they've taught you in orgo. They give everybody the ace now. There's no sugars, 100%. Unbelievable. Can you guess? The sulfur has more energy? Well, so if you think of it in terms of electronegativity, it makes no sense, right? What's more electronegative, oxygen or sulfur? Oxygen. So here they've given you a little hint. They show resonance. So oxygen that loves its electrons is going to be more likely to share electrons than this sulfur. What can you say about the size of a sulfur atom? Larger. And so how might that affect resonance? There you go. The orbitals are mismatched. There's no orbital overlap. Sulfur is a big Hawken atom. And it's just not possible. Yes, it's less electronegative, but it doesn't matter because there's no orbital overlap. So there's no resonance stabilization here. And so that's what makes this the hydrolysis of this so exergonic is because this is very unstable. It's stable enough to float around in solution. But many bacteria, you'll see if you ever take a microbiology class. There's a lot of thioesters in those bacteria. It's a primitive form of capture of energy, sort of similar to ATP phosphate hydrolysis. It's actually similar amount of energy is released in the thioester hydrolysis is ATP hydrolysis. So this is a primitive way also to store energy because the lack of orbital overlap and a lack of stabilization of the substrate. So this is important. Okay, so we made citrate, drove that forward by this insanely exergonic hydrolysis of a thioester. Now all we need to do is rearrange. So what can you tell me about the stereochemistry of citrate? To make this less boring, I'm going to just ask questions. So what can you tell me about the stereochemistry? Is it chiral? It's pro chiral. What does that mean? It can be chiral if one of these two identical substituents to this carbon were to change. Look what's going here. So we've actually highlighted to track the carbons and they did this experimentally by radio labeling. And they found that this enzyme made a eliminated a water molecule here but not here. It's pro chiral. How could it do that? It's the same. It can be rotated upon its mirror image.  So here you have a hydroxyl group and there's a proton here and there's a proton here. But this one is with 100% selectivity the one that's used in the elimination reaction and then you add back water. So where did you see this general strategy before in the class? Where you transform something and then put it back the other way. Galactose. So remember with galactose, that's an epimer. You want to invert the stereochemistry. So you oxidize it to carbonyl A chiral and then you stereoselectively put it back in the hydroxyl group in the right position. And so here we have a similar strategy. We eliminate a water and then add back. So here the elimination has stereoselectivity and that we add the water back. And so all the net reaction is we've moved a hydroxyl from here to here. Now this is now a chiral molecule, isocitrate. And so this rearrangement has a positive endogronic process, but it's pulled to the right by a rapid, the next step being a rapid step. Do you see this? So now we're just observing this curious selectivity and we'll come back to that later in the class. So we have isocitrate and now we're going to start dehydrogenasing. So that's an indicator that electrons are going to start flowing out of this molecule. Up to this point we've arranged the molecule in a certain way. And you might, as we're going through this pathway, you might say, why did it have to be this way? Why did the necessarily, the rearrangement of pyruvate into carbon dioxide have to go through these steps? And you'll see at the very end of the lecture it's extraordinarily efficient. It is conceivable that maybe you could change, manipulate these molecules in a different way, but probably evolution tried every possibility and this one was the most efficient. So we're going from now, isocitrate, oxidative decarboxylation, right? And then a second one, to go to alpha-ketoglutarate and then succinyl-CoA. So anytime we have energy releasing steps, I put a handy key, fire isocitrate. We can, so we just take this hydroxyl group, change it into a carbonyl group so we've oxidized. We can receive those electrons either on NAD or NADP. There's different isoforms of isocitrate dehydrogenase, slightly different sequences, but same reaction catalyzed. And those different isoforms can prefer one or the other. So for example, if anabolic processes are not using up our NADPH, we have the option of burning the leftovers. So this is like shredding papers, right? And so the electrons, this is a highly exergonic process, helps to pull that aconitase step forward. That was endergonic. So this releases a fair amount of energy because we're oxidizing a carbon atom. Remember we saw that it releases a tremendous amount of energy. And then we're taking this alpha-ketoglutarate in a almost nearly identical complex to pyruvate dehydrogenase complex. We are decarboxylating and we're ending up on our coenzyme A again. So this has the exact same list of cofactors, coenzyme A, NAD, lipoic acid, TPP, and FAD. It's a multi-meric complex just like pyruvate dehydrogenase complex. And so these are producing a tremendous amount of energy and oxidizing or reducing some cofactors. So lipoic acid is used both by alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase. And this, you might have heard of arsenic poisoning. So if you give someone a lot of arsenic to get very pale, well it's because you're actually poisoning aerobic respiration. You're blocking both steps because the arsenate binds to these sulfhydryl irreversibly. And so it inactivates the enzyme. And so you do not want to stay away from arsenic for sure. Okay, so we have succinyl CoA and we're going to convert that to succinate. So we're going to cleave this thioester and that's obviously exergonic because we've learned that hydrolysis of a thioester is highly exergonic. In this case we're going to have substrate level phosphorylation of GDP to GTP. Does that make sense? Substrate. So where do we see substrate? What was happening when we saw substrate level phosphorylation before? You're transferring a phosphate from one metabolite to another. This actually turns out also to be substrate level phosphorylation but it's not, you're not transferring phosphate from the metabolite. So inorganic phosphate, the way the enzyme works is when you catalyze, so this thioester hydrolysis is driving a process whereby you put this inorganic phosphate on a histidine of the amino acid of this enzyme and then that phosphate is transferred from the enzyme histidine to the substrate. Okay, so you still could consider it a substrate level phosphorylation. This is called a synthetase because a nucleoside triphosphate is involved. So citrate synthase did not involve ATP, GTP, any of these things. This one does, so that's a bit of nomenclature. Anytime you see synthetase then we know that GTP or some nucleoside phosphate is being used in the process. Here it's not, it's sort of going in reverse, right? So we're not hydrolyzing GTP. We're using thioester hydrolysis, the chemical energy released from that to add a phosphate to GTP or to GDP to form GTP. And there's actually in different organisms, some use ADP here, some use GTP. It actually doesn't matter because they can be interconverted. There's an enzyme that can transfer phosphates from GTP to ADP. Okay, so when you make one you're able to make the other, but you'll see throughout the course there are some enzymes that for whatever reason use GTP instead of ATP. So we need to have some of that around. Does this make sense so far? We're going in a circle. It's dizzy. Okay, so we're almost to, now we've released a lot, we've removed a lot of electrons out of this molecule. And so, you know, there's not much left to do here. We need to release some more electrons and we need to get ourselves back into a configuration of the metabolite so we can make it a cyclic process. So we're going to have succinate, going to fumarate, right, dehydrogenating, that's releasing electrons. Then we're going to add a water molecule across that double bond and then we're going to oxidize that molecule. So there's a three steps. If you just boil it down to the meat of the chemistry that's happening here, you dehydrogenate. So this is sort of makes sense. You would call this a dehydrogenase. The other ones it's like a little bit less intuitive. So you're eliminating hydrogen. Those hydrogens are ending up on our reduced cofactor to make a new double bond. We're then adding water across the double bond with high stereoselectivity. These are chiral catalysts. We can do this with high selectivity. And then we're going to oxidize the hydroxyl group to carbonyl group. And this step actually is a transfer of electrons, but it's much less exergenic than the ones we've seen so far because we've already pulled so many electrons out of here. The electron affinities are less favorable for us to take one or two more electrons out. So let's go through each step. So succinate dehydrogenase. We can dehydrogenate or eliminate hydrogen, make a double bond. You would guess in terms if you just did this in solution with non-insomatic catalysts, you would expect a certain mixture by thermodynamics of the trans and the cis. You get exclusively the trans. It's an enzyme. It surrounds its substrate. Only allows the trans configuration. And so this molecule malonate, which is a confusing name because it's not maly. It's malonate. This is an inhibitor of this enzyme. Can you imagine how it might be an inhibitor? So what would happen if you eliminate hydrogen from this molecule? You're likely to set off a nuclear reaction. I mean, it's just not going to happen. How do you eliminate off of one atom? But do you see how it looks pretty darn similar? So the similarity in structure makes it so it binds. So this would be a competitive inhibitor. But as soon as it binds, it's like, dude, you can't have two, man. I only have one carbon atom. And so this is a competitive inhibitor of this process. And so you only get the trans product. And even though this is the transfer of electrons, a carbon is being oxidized here. It's not being oxidized by making a new bond of oxygen. It's being oxidized by making a new bond to carbon. You have a double bond here. But it's just close to equilibrium starting from the standard state because we've already released so much energy from this molecule. And so then you're going to take fumarate, and you're going to stereoselectively make L-malate and not D-malate. So we can control the addition of water. We can position the water molecule, position the fumarate, and have this new bond to the hydroxyl group be stereoselectively formed. And so we have now L-malate. We've added water. We've not changed an oxidation state. Now we do have a new bond of oxygen, but it's the same oxidation state. It's close to equilibrium starting from the standard state. And so we don't have malleate, cis-double bond, and we don't have D-malate. Are you with me so far? So we're getting ourself back to oxalacetate. Yes? Yeah, you'd have to look at the crystal structure. It must be, I would imagine, it has a lot to do with holding the water molecule and the fumarate molecule in a very precise orientation so we can make bonds, hydrogen bonds perhaps, to our water molecule to hold it exactly at one side of this molecule. But in reality, does it matter what happens next? It's destroyed. The stereochemistry is destroyed because we're going to oxidize this bond. But the next enzyme prefers its substrate in the L form. So it matters for the next enzyme to bind the substrate, but we're going to eliminate this stereochemistry at the next site. It's just beautiful elegance control of stereochemistry. And now, this step's a bit surprising. Look at this. So you have l-malate. You're going to oxidize that carbon. But look at this. It's pretty endergonic. So starting from the standard state, this would be a very hard reaction to do. Thank goodness we have a cellular state that citrate synthase is providing a lot of the energy, which is the next step. That's providing the energy to get us through this. But the concentration of acetyl acetate is very low. So the affinity for electrons is very high. It does not want to give up these electrons at this point. And so we do pass some electrons here, but very grudgingly passing those electrons. We've made it all the way around. All right. Any questions so far? Citric acid cycle. We've just been through the furnace. We've burned. So let's look at the input and the output. Two carbons in, how many come out? Two out. So the concentration of these molecules is unaffected by this because it's carbon nutrient in terms of carbon atoms. Two carbons come in and every time you turn around, two carbons come in, two carbons come out. We've stored a lot of energy. We haven't made one GTP now, but mostly all the energy is being stored on these reduced cofactors. This is only about 2% of the energy of the whole aerobic respiration process. The large amount of the energy is still here. Okay, so we have two carbons coming in, two carbons coming out. And yes, we've already covered that. And so we can do a summation here. Look at this. So if we take pyruvate already all the way to carbon dioxide, and we don't have any cofactors present, we're released tremendous amounts of energy, 2200 kilojoules per mole. But when we store those electrons on the electron carriers, this process is just slightly exergonic starting from the standard state if you sum everything up. So our energy is hiding out now. 2% is on GTP, and 95% is left on these reduced cofactors. So what is that? Almost 98% total is captured. So it's extremely efficient. This has evolved to be very, very efficient. In the next step, oxidative phosphorylation, that's not quite this perfect. So we're going to lose some energy in the form of heat in that process. But up to this point, we've captured a lot of the energy. We've moved the energy around and it's now sitting on these reduced cofactors. Okay? So this is odd. So I mentioned that the TCA cycle is used for this anabolic processes. And so what does that mean? So that means that some of these substrates are pulled out of this reaction and assembled into more complex molecules. What's going to happen if you pull a molecule out of here? And it's carbon neutral. So if you pull one molecule out, they all decrease in abundance.  So what happens if you take too much out? The thing stops. You can't make any more energy. It is useful for this thing to be involved in anabolic processes, but there's a price to be paid that anytime you remove a molecule out, you're going to have to have some other way to put a molecule back in. We don't want to decrease. These are all inter-converting. Removal of any of them will reduce the abundance of all of them because they're inter-converting. And so how does this happen? You might wonder. Okay, so we'll come back to a connotase. Okay, so let's come back to this finish-up connotase. So remember we had the red coloring on some of the substrates on top. So that's from this experiment where you have C14 labeled acetate. And it's an aldol condensation to make a labeled citrate molecule. And then a connotase is going to take that citrate, convert it to isocitrate, and what they discovered. So you would expect that you could plausibly eliminate this or this. But they saw exclusively elimination here and then adding the water back on this side. And they didn't see the other molecule with the label down there. And that's like, well, how could it, this is achiral. Or it's not achiral, it's prochiral. But it's superimposable on its mirror image. How could the enzyme ever figure this out? How did that happen? So look at this. This is really cool. So here's the molecule. So do you see here? This is the same as this. And so let's change these into letters to simplify. So here we have x, y, z, and z. And so this enzyme, I've just told you, can discriminate between this bond and this bond. So let's just do a hypothetical example. Say a complementary amino acid has this prime symbol. So z has a complementarity to z prime, y to y prime, x to x prime. And now what we're all going to do in our head is rotate this molecule such that this z is here. And what happens to the arrangement of this x and the y in relation to x prime and y prime? So now we have this face pointing it. What if we switch to this face when we rotate that? Do you see what happens? What is x bound to? Y prime. Isn't it like, it's like that they have those ads on TV about, you know, look at the dot and it's like actually not moving. It's almost like an illusion here. So this face is discriminable by the enzyme because the enzyme can position whatever complementary amino acids it wants at each of these positions. And so when we flip this around, so you know, I don't know exactly what amino acid is putting there, but it could discriminate these because you'll have z bound to z prime, x will bind to y prime, y will bind to x prime. So this enzyme can hold this thing, exposing just one of these protons to a general base because what we're doing an elimination reaction here. And so we can hold the substrate precisely and put a general, because we're holding it precisely, we can come in with an amino acid, a general base and deprotonate just one of those hydrogen atoms. So if you look in the next picture, right, so here we have a general base. This substrate is held precisely and the enzyme can discriminate the two prokaryl faces of the substrate. And so this base pulls off this proton here and not this proton because there's just not a base there. The substrate is held specifically. Okay. Is this important in evolution? I don't know. It's beautiful. It's elegant. Does it matter? It's an observation. It's a beautiful illustration of how enzymes interact with their substrates. Initially, people were debating, they say, there's no way, man. This econotase must not be part of the TCA cycle because how could it create chirality out of nowhere? So it led for decades. People said, this must not be in the TCA cycle. And then they said, ah, someone just got a paper out and said, oh, yeah. If I turn that just right, it can discriminate the prokaryl faces. Okay. So we're actually going to spend a whole lecture on econotase, the last lecture in the class. This enzyme not only does it have this beautiful holding of the prokaryl faces of the substrate, it also is involved in regulation of iron. And we'll see that at the end of the class. So it's multi-purpose enzyme. So this is obviously heavily regulated. What have I told you before about what you can predict about where pathways are regulated? Highly exergonic steps, right? I.e., the ones that are early in this cycle that are dehydrogenases, these are regulating them. This is the gatekeeper to the furnace, right? So, you know, and also, once we convert, once we bring pyruvate into the cell, this is a transporter and it could, it could be an equilibrium. It could go back out of the cell. So we're also trapping the pyruvate into the mitochondria, I should say, go into the mitochondria. So that's simpler. If you just keep pulling in pyruvate, that will potentially just go back out eventually from the mitochondria. But we convert it here. And so this is perhaps the most regulated step logically. But then the highly exergonic steps, citrate synthase. We saw how that was highly exergonic isocitrate dehydrogenase and this one as well. And so let's think about this. A lot of this makes sense. So if we already have enough ATP or we already have enough product, that means the whole pathway is backing up. We have enough NADH. It's, you know, we don't need to make more. But fatty acids. Why do fatty acids inhibit this? Can you imagine? Why does what, so have you ever guys, have you guys ever heard of beta oxidation? It is a lecture in the course later on. So as it turns out, fatty acids are catabolized to acetyl-CoA. And so fatty acids might be short circuiting this, right? So these need to be regulated. So if we have acetyl-CoA coming in from fatty acids, we don't need it coming in from pyruvate. And so we have regulation all over the place. We're sensing the redox status. We're keeping an even balance of the substrates here. And remember, there's talk. So we mentioned PFK1, citrate inhibits. You're like, where's this happening on the planet Mars? Where's it happening? It is happening in glycolysis. Where? Cytoplasm. Where is, where is the Krebs cycle happening? So what must there be? A shuttle. There must be something that can transport citrate out. And once transported out, what is the inhibition of PFK doing to help in the regulation of the overall process? That's right. So it's balancing these pathways out. So you can, this alsteric modulator doesn't bind at the active site. It bonds at a regulatory site. And so you have the binding affinity of that alsteric regular tune just such that the rate of glycolysis is in sync with the rate of the Krebs cycle. So it's important for these to talk. Why not have citrate inhibit hexokinase? Milk drinkers. I gave you a little hint there. Milky. What is the milky reason? Is that a bit disturbing? What is it? Why not? So, yeah, hey, come on. You want to, you want to really control things the first step, man. Why not inhibit that? What is milk? What is, or what kind of disaccharide occurs in milk? Lactate. What, what is that? It's a disaccharide, or lactose, not like, so lact, what is lactose? That's in milk. It's a disaccharide of glucose and galactose. Now, how important is hexokinase to galactose? It doesn't use it at all. So if we had citrate regulating hexokinase, galact, every time you drink some milk, it would short circuit that regulation, right? Because galactose is coming in at a later stage, right? So if you go back to those slides, you'll see there's a logic. Everything is precisely positioned because people start drinking milk in the enzymes. I'm like, whoa, wait a minute. I'm out of sync, man, with all that lactose. Okay, isn't that cool? I think a lot of things are cool that you probably don't. Okay, so we've oxidized carbon dioxide, and we have all these metabolites in between.  You have to know the structures. Look at all the exams. I don't see them, but I'm still nervous. That's okay. It's all good. But the other reason is because evolution needs lots of diversity. If we're going to start to build products up, we need to take, okay, you know, maybe one TCA intermediate is better than another to make another molecule. And so it's useful to have all these steps, both in glycolysis and the Krebs cycle, because we could say, oh, that's useful, it's sort of close to where I want to go. So you grab one of those out. But in terms of the Krebs cycle, we remember it's carbon neutral. You take one out, you're like, oh, that's bad. We start decreasing all of the metabolites until the Krebs cycle just grinds to a halt. We need to support both the energy generation and anabolic processes. So we have a way to fill these out. So two carbons in, two carbons out, take any of these away, and the whole cycle grounds to a halt. And so we have what's called anoplerotic reactions, and these are filling up reactions. So in human cells, we take three carbon sugars, and we add a carbon to make a four carbon sugar. And that's in the TCA cycle. And once we make one, we've filled them all up. So we take one molecule out anywhere in the cycle. You just have to put one molecule anywhere back into the cycle, and you'll fill it up. Your energy production will not be affected. And so this is a general schematic. This is very useful. We'll come back to this a lot. So maybe a little star on this slide. We'll come back throughout the semester to this. So do you see that the anabolic processes are the blue arrows? So for example, from citrate, we can make fatty acids and sterols, aflacidoglutarate, we can make glutamate, and other amino acids and nucleotides, succinyl-CoA, we can make porphyrins, we've seen those in heme, acetylacetate, can make all kinds of stuff, amino acids, nucleotides, and more amino acids. So these, all of these blue processes, I mean, we need to be able to make amino acids. We need to be able to do all of these things. But anytime we do that, we take a molecule out, slowing energy production. So the anoplarotic reactions in this diagram are in red arrows. And so all of these, you're taking a three-carbon molecule and turning it into a four-carbon molecule. So pyruvate, remember the structure of pyruvate has that enol form, remember? And so that can be converted into acetylacetate, or it can be converted into malate, PEP, similar in structure to pyruvate, three carbons, it can be converted. So, you know, there's, I've mentioned lots here. In general, these are, if you're adding a carbon, these are carboxylase reactions. So in the mitochondria, a great source of carbon is carbon dioxide floating around everywhere. And so we can take any of these metabolites and stick on a carbon dioxide or a bicarbonate to make these other molecules. But to do this, we need a carboxylase reaction. We haven't really seen these so far. And so there's a, because we're doing a new type of chemistry, you might guess there's going to be a new cofactor going on here. And so let's look at just one of these. We don't have time to look at them all. And so this is pretty amazing. So you have bicarbonate and you're going to phosphorylate. So take the phosphate off of ADP, stick it on this bicarbonate until you get carboxyphosphate. You might imagine that's not too stable, man. That's some sick, sick-looking molecule. But it's covered up by the enzyme. And so that allows you to, this can come apart to carbon dioxide. So you start with bicarbonate and get to carbon dioxide. That carbon dioxide can then react with biotin. Do you guys ever touch biotin in the lab? So beyond its ability to bind to streptavidin, which is how we co-opt its usefulness, it's also important in all these carboxylation reactions. So we form a new covalent bond, a carbon dioxide. That's pretty amazing. You would think carbon dioxide is very stable. How could it make a bond? Well it does. This enzyme catalyzes that. And then what we're going to do is confusing. So we have two active sites in this enzyme. You bind the carbon dioxide in one active site and then you rotate. You know, it's on this tether that's actually attached to the enzyme. You rotate to the other active site and you drop it. See, that seems silly. Why not just bind it there in the first place? Well, secretly waiting there is this other molecule, pyruvate. See, pyruvate was already hanging out. It was bound there. And it's not just sort of tossing it like, ah, active site. It's putting it right there. That's where you need to go. It's putting it, pointing these two molecules at each other so that they can, this is not easy chemistry. You're adding a carbon-carbon bond. And so it's very precisely controlling the entropy of this process, helping to accelerate the rate. And so now we have the carbon dioxide binds and makes oxaloacetate. So isn't that cool? And obviously, if we're going to stick it on the end, it has to be the enol form. They're not going to be sticking on the end. And so making the enol form, how stable? Like, if I just toss pyruvate in the solution, would it be a keto or enol form? Which is more, they're tautomers, right? So the keto is much more stable. So not only is it secretly positioned, the pyruvate molecule, it's pre-arranged it so that it's ready to go for the chemistry. It's in the very unstable enol form, ready to react with carbon dioxide. Any questions on that one? Okay. So we've covered lipoic acid. That's important in these oxidation reactions. It also farms a thioester bond to our substrate. It's in these dehydrogenase enzymes. Biotin, it helps to convert pyruvate from its keto to its enol form, controls the entropy by putting the carbon dioxide exactly where it needs to be. And we've seen this this thio group is useful in many, so many of these cofactors have a thio group attached to different amino acids. So this is one we haven't seen yet. It's called acyl carrier protein. It has this similar pantothenic arm, right? But it has a sulfhydryl group, and that's going to make thioester linkages. This is very common in bacteria, so erythromycin. Any of you that had were infected, this saved your life. This makes the molecules that make macrolid polyketide molecules. And so bacteria are using that. So one last topic. So I've showed you anaphyluronic reaction, conversion of a three-carbon molecule to a four, filling back up those things, supporting anaphyluric processes. But what about you say, well, why three plus one? Two plus two also equals four. But as it's turned out, through evolution, we're like, there's plants. We don't need to do that. And so plants have the ability to take two carbon molecules, combine them together, right? We've lost that ability. And so you might have seen this, this movie about someone who spent 30 days, and all he ate was McDonald's. Did you notice at the end of it, he looked really pasty, and you just felt bad for the guy? This will be bad, because if you're only eating fat, and he was eating diet drinks, there was actually no sugar going into this man. And he was not able to support anabolic processes, because everything was going into just making energy. And so we need, in plants, they do have this ability to convert a two carbon, to combine two carbon molecules, to make a four carbon molecules. And that's called the glyoxylate cycle. And so acetyl-CoA, so fatty acids, beta-oxidation makes acetyl-CoA, does the same thing in us. We've got the ability to make acetyl-CoA, but we have no enzyme that's able to combine this into a four carbon molecule. So the glyoxylate cycle, two of the steps, three of the steps here, are the same. Only two new things to learn. So we have here citrate, acetyl-CoA, I know where, I was a student here, I know where you're at. It's painful. So acetyl-CoA, enol, aldo condensation, we saw that citrate, a conditase, I labored that enzyme forever, made isocitrate. Now the new steps. Okay, pay attention, I put it in the box. Isocitrate can be converted into glyoxylate, right? So one, two, three, four, five, six carbons is split into a four carbon molecule and a two carbon molecule. And then we can combine this two carbon glyoxylate with an acetyl-CoA to make l-malate. And then we're back into something that we can convert into oxaloacetate. So we've taken two, two carbon molecules and coming out of this pathway is one, four carbon molecules. Besides that, this pathway is carbon neutral. Okay, and so fatty acids, look at this, there's a cellular organization.  What are the oils you use to cook? It's like sesame oil, you know, sunflower oil, plant oils. These are in the seeds. The seeds have fats, acetyl-CoA comes flowing. It's like a wind of acetyl-CoA coming out of the lipid body, going into the glyoxyzone. There's two membranes to cross. Once in here, you could come in as fatty acids as well. The acetyl-CoA can be converted to succinate. But then we're in a glyoxyzone. Look at, it's not coincidence that these things are arranged in the way they are. Fatty acids are coming from here, going into here, being converted into succinate. And then the succinate is transported across this membrane and through the inner mitochondrial membranes, a small molecule less than 5,000 Dalton's diffuses through the outer membrane and goes into the mitochondria.  There's not sugars. But we have evolved, say, hey, there's sugars all over the place. We don't need to do this process anymore. So we've lost this ability. So here's what we learned today. So you might say, why is the font so weird looking? Well, this is where we are. You might say, oh, when is the drop deadline for this class? But look at this. So you have, look at its position. Why did I spend so much time going over every step? It's the central core. It's the skeleton that holds up all metabolism. All the arrows are coming in and out of this central hub. So here's glycolysis. Here's the Krebs cycle. So now the rest of the class is going to be just, you know, how do we take parts out of these processes that we've just learned? But the basic flow is from sugars in your diet coming in, making energy and making anabolic processes possible. All right. Okay. Two carbons in, two carbons out, eight electrons out. And next time we'll be converting NADH or reduce cofactors into ATP, which is more useful for other processes. Test, test, test, test, test, test, test, test, test. Exams for those that came in late, exams are on the third floor between McMillan and Geochem in the stairwell.  Everybody vote? Was this the easiest clicker ever in the history of life? Yeah. It was E, right? D, no? C, physician? A, A.