Isocitrate dehydrogenase (NADP+) kinase

Transcription

Kevin Ahern: I hope you had a good weekend? I hope. I had a nice weekend. I went to the coast and got away, which is why I didn't get the video posted until Sunday. I know there were several of you who were very anxious to see that, so I'm sorry it took a few minutes. There was actually a problem on the OSU site on Friday so I couldn't get the thing done on Friday afternoon, which is what I prefer to do. That's why it waited until yesterday. I have not yet officially announced a time for the BB450 review session for Exam 2. I am planning on that being tomorrow night at 5:00 p.m. I will videotape, as before, and I will announce that for sure when I get a room secured. I haven't done that yet, so I need to do that. But I'll send an email out to the classroom listserv. I'll also post the information for it on the class web page. Material for Exam 2 will go through whatever I cover today. Today is the end of material for Exam 2. Clear as mud? We're just about done talking about metabolic control, and this last thing I'm going to talk about is sort of an add-on in the chapter. It's kind of like your authors of the textbook weren't quite sure where to put this one, so they stuck it here under metabolic control. This refers to the reaction types that enzymes catalyze and it's actually a fairly interesting set of information. The reaction types are shown down here and this categorization of the reactions that enzymes catalyze is an attempt to systematize the classification of enzymes, making them systematic. There are, it turns out, six separate categories of reactions that are catalyzed by enzymes. When we take all of the reactions that are catalyzed by enzymes then we can place them into six groups based on the chemical reaction being catalyzed. They're shown on the screen. I'll give you examples or show you some examples of these, but the six different types are oxidation-reduction, ligation requiring ATP cleavage, isomerization, group transfer, hydrolytic, and addition or removal of functional groups. Let me show you some examples of these types of reactions. Oxidation-reduction reactions I talked about last time, and they involve loss of electrons by one substance and gain of electrons by another substance. In this case, the top case, we see succinate, which we will study next term in learning about the citric acid cycle. We see succinate losing electrons to FAD, meaning succinate is becoming oxidized. FAD is becoming reduced, and the products of those reactions are fumarate plus FADH2. Whenever you see an electron carrier in a reaction, you can pretty much assume it's a redox reaction, because the electron carrier is there to carry electrons. Here's another reaction we'll talk about next term, malate going to oxaloacetate. Malate has the electrons that it loses. It gives electrons to NAD to make NADH, and oxaloacetate is a product. So oxidation-reduction reactions are very easy to spot, largely because of the involvement of the electron carriers. Ligation reactions are also fairly easy to understand. Ligation reactions generally involve the joining together of two different molecules, the joining together of two different molecules. We can see, in this case, pyruvate is being joined to carbon dioxide. We'll talk about this reaction in about a week or so. In this reaction, carbon dioxide is joined with to three-carbon pyruvate to make a four-carbon molecule oxaloacetate. So joining two substances together is what is involved in ligation reactions. Isomerization reactions are also very easy to recognize. We're changing the configuration of a molecule. We're not causing oxidation or reduction or breakage or ligation or any of that. We're simply rearranging it. In this case, citrate, we can see here, is being converted to isocitrate, and basically this hydroxyl group is moving from here over to here, swapping places with a hydrogen. So a rearrangement reaction is an isomerization. You'll also notice that the reaction types that I give here are slightly different than the ones that were in the table, it's jumping around, slightly different than the ones they gave in the table. I would prefer that you use my categorizations than the one that's in that table. Group transfer may not sound very intuitive by its name, but it basically involves the movement of a portion of one molecule to another molecule. Notice that I said a "portion" of one molecule to another molecule. A ligation joins two molecules. A group transfer transfers a portion of one to another. In this reaction, which we'll talk about later today, glucose is gaining a phosphate from ATP. That gives it glucose 6-phosphate and leaves behind ADP. This is a prime example of a group transfer type reaction, the phosphate being the group that's being transferred. Hydrolytic reactions, as their name implies, you've already seen a bunch of these, are reactions that involve cleavage of a molecule using water. The proteases, for example, that we talked about, used water to break peptide bonds and they are an example of hydrolytic reactions. You see a prime one on the screen right there. The last one is probably the hardest to understand, and, to be honest with you, I don't place much emphasis on it, but I'll just give you a notion of what it is. The last group is called lyases, and lyases are enzymes that basically catalyze the splitting of a molecule. That's a little bit of a simplification of what they do, but you can see in this reaction from glycolysis that fructose 1,6-bisphosphate is being split in half, and so lyases cause the breakage of bonds to split molecules into two. That's one way of thinking of them, and, for our purposes, that's all we really need to pay attention to. I'm not going to expect you to memorize the names of these enzymes, but I do think that you should know the categories. The six categories. I'm not going to say, "Give me an example "of an enzyme that is a lyase," for example. I think that's just busy work. But understanding the six categories is useful. Now, the six categories actually, as I said, are an attempt to systematize the naming of enzymes. This gave rise to what was called the "EC number. " The EC number, "EC" stands for "Enzyme Commission," and they were the group that came up with these categories and they are involved in classifying enzymes into each of the six different categories. So when we see, for example, an enzyme that is an oxidoreductase, it would all fit into a category like this and it would have a specific EC number. So the EC number, I don't remember the numbers off the top of my head, but I think this is Category 1, all oxidoreductases would have, in their numbering scheme that corresponds to them, the very first digit corresponding to the category that they're in. So, in this case, an oxidoreductase is Category 1. All of the enzymes in the EC commission that are oxidoreductase would have as their first number, number 1. They would have a 1, point something, point something, point something, point something. Each point would designate a little bit more specificity about the type of reaction that the enzyme catalyzes. But, six general groups, and that first digit of the EC number is what is critical for identifying these six groups. Yes, sir? Student: Do the EC numbers correspond to the order you gave those to us in? Kevin Ahern: I don't know if they correspond to the order, to be honest with you. I'm not worried about you memorizing those numbers, but know that there are six. That's basically what I want to say. I'll just show you this last thing as a trivia item. It's not something I expect you to memorize or anything, but it shows the sort of central importance of the molecule ADP in a variety of places, as it appears in biochemistry. ADP is, of course, a part of ATP. ADP is shown in blue, red and yellow. That same ADP is found in NADH. It's found in NADPH, which is not shown on here. It's found in FAD, and it's also found in coenzyme A. I don't know why it's not red over here but it's also found in coenzyme A. You don't need to know that. I'm just showing you that for trivia purposes, but these molecules play very, very important roles inside of cells, and ADP is a significant component of every one of them. So much for metabolic controls. We're going to turn our attention now, for the first time, to the pathways of metabolism. "Metabolism" I'm going to define for you as a collection of reactions that are found in cells. Metabolism we can think of as the chemical reactions or the biochemical reactions going on inside of cells. As I showed you on the road map the other day, these molecules or these reactions are actually linked into highway-like pathways, that one molecule leads to another, leads to another, leads to another. As such, that means that all of metabolism in a given cell is linked. There's no escaping that. They're all linked. Now, I want to emphasize to you that a given pathway, we're going to be talking about glycolysis, a given pathway is a man-made invention. It's a man-made invention. "But it's there, it's on the table." Well, that's right. But where I call it glycolysis really depends upon where I define the starting point and the ending point. If we look at that big road map, it's kind of like saying, "Where is the road to Portland?" The road to Portland could start in Ashland, if we're thinking about southern Oregon. It could start in Salem. It could start in Seattle. So where we define a given roadway is really a man-made invention, and so glycolysis is like that. For our purposes, glycolysis will be a pathway that starts with glucose and ends at pyruvate, starts at glucose and ends at pyruvate. You'll see, as we get going further into that, that that is a bit of an arbitrary thing. I also want you to keep in mind that when we talk about metabolism and when we talk about these pathways, that the pathways are interconnected. They're interconnected. I could take the road to Portland by going, for example, over to I-5, riding it up to Salem and then cutting over to, say, 99, and then going back up. There's a lot of ways I could take that pathway to Portland, and the metabolic pathways are the same way. Now, the significance for us, of the interconnectedness of pathways is the interconnectedness of the molecules in those pathways. We can't say that “This molecule is in glycolysis,” because, A, glycolysis is a man-made invention, and B, that molecule may be part of what we define as glycolysis, but now if I think of the pathways going out instead of up and down, that molecule might be tied to other things, as well. So there's a very, very strong interconnectedness with that. You'll see that especially next term when I start talking about the citric acid cycle and fatty acid oxidation. Well, let's take an overview of the pathway of glycolysis. The pathway of glycolysis starts with glucose. Glucose is the most common and most abundant sugar on the face of the Earth. It has six carbons. The pathway of glycolysis causes it to be broken into two pieces that are identical, known as pyruvate. So one six-carbon piece gives rise to two three-carbon pieces. Then, at pyruvate, we see that pyruvate has three different "fates," as we describe them, meaning pyruvate can be converted into three different things, depending upon what the cell needs, what the cell has, and the type of cell in which the reaction is occurring... what the cell needs, what the cell has, and the type of reaction in which this reaction is occurring. Let's just very briefly go through these. I'll probably remind you of them later when I get to pyruvate, talking about the pathway. Pyruvate, most commonly, when we're talking about aerobic metabolism, goes to acetyl-CoA. It's not even shown on here, but acetyl-CoA is an intermediate. Acetyl-CoA plus oxygen ultimately leads to carbon dioxide plus water. So the first and most common direction is to go where oxygen is available and it goes to acetyl-CoA. We'll see how that acetyl-CoA gets used next term. But this pathway, this very first and most important pathway, requires oxygen. Well, what if cells don't have oxygen? If cells don't have oxygen—and yes, that does occur in our bodies, when we're exercising heavily our muscles can't get oxygen fast enough—they have to have a backup way of generating energy, as we shall see. In our cells, when they're lacking oxygen, pyruvate gets converted to lactate. Notice, again, lactate is not the same as lactose. Lactose is a sugar. Lactate is this guy over here. Well, what if it's not my cells? What if it is a bacterial cell or a yeast cell and it runs out of oxygen? That, of course, that pathway goes from pyruvate and leads us to ethanol. It's the foundation of brewing, micro brewing and all these great things that happen with that. For any of you who've ever made your own beer and so forth, you know that you mix all the stuff up and then you cap it off so that there's no oxygen available, and that lack of oxygen is what leads to the production of ethanol, and we'll see later why that's the case. So, three different fates for pyruvate. No oxygen leads to either ethanol, in the case of bacteria and yeast, or lactate, in the case of us. In any organism that is aerobic when there's oxygen available it goes to acetyl-CoA. Questions on that? I'm hearing none. Let's dive into glycolysis. This figure is a fairly good overview of the process of glycolysis. It has all the players on it. You can see glucose, glucose 6 - phosphate, blah, blah, blah. The first question I get is, "Well, what of this do we have to know?" Well, I'm going to spell that out for you fairly explicitly right here. First of all, you need to know the names of the ten intermediates. Second of all, you need to know the names of anything that you could easily learn. What does that mean? Well, I've told you previously that you need to know the structure of glucose, and glucose is right there. Duh, right? If you know the structure of glucose, then learning the structure of glucose 6-phosphate means you simply need to know where to put the phosphate onto there. Duh-uh. You also had to know the structure of fructose, and fructose, if you put a phosphate on it, you have fructose 6-phosphate right there, so that's an easy one. Fructose 1,6-bisphosphate, fructose, you put a phosphate on positions 1 and 6, and you've got that guy there. So those four, given the fact that you already are supposed to know glucose and fructose, should be no-brainers for you. I will mention other ones, depending upon how far I get through this pathway today in the lecture, that you'll be responsible for, and I'll save those to see how far along that I get. This overview of the pathway, we can also see in blue, over here, the names of the enzymes. The enzyme names you are responsible for, yes. These are very important enzymes in the body, and the enzyme names actually tell you something about what's going on in the reaction. Here's hexokinase, "hexo - " meaning "six," "kinase" meaning "puts a phosphate onto." This guy puts a phosphate onto six-carbon sugars and, guess what, glucose is a six-carbon sugar, so hexokinase puts a phosphate onto glucose. So the ten names of the intermediates, the ten names of the enzymes, and the molecules that it's easy to learn the structure of, and I'll point out two more for you. Let's look at this process. We see that the process is divided on your screen into two main phases, and those two main phases are actually different than what the last version of your book did. If you have the 6th edition of the book, you will see it splits it into three phases. I was never very fond of the three phases, so I'm glad they went to this two-phase model. The two phases are known as "energy investment" and "energy realization" or "energy generation," you can call it whatever you want. It means that in the first phase of glycolysis we have to put energy into the molecules, and in the second phase of glycolysis we get more energy out than we put in. Now I want to emphasize, that glycolysis is a source of ATP in the cell. It is not usually a very large one. Only when cells are hurting does glycolysis become a very large one. When are cells hurting? One of the times might be if they're low on oxygen. Then that ATP that they can get from glycolysis turns out to be very, very important. I'll also give you some numbers here that we will talk about again probably more next term than this term, but this gives you an idea of the importance or the relative importance of glycolysis in the scheme of things. If I start with glucose and I go down to pyruvate, what you will discover is I produce a net gain of two ATPs... two ATPs. That's not a lot, but I also generate some other things along the way that are very useful. I produce, first of all, some NADH. I produce two of those, and I also produce two pyruvates. Now, if I take these NADHs and I take those pyruvates, and I oxidize them all the way down—this happens in the citric acid cycle and in the electron transport system if I oxidize them all the way down and I count the number of ATPs that I get, depending on who's doing the counting, you get about 38 ATPs. You get 38 ATPs, and you think, "Wow, this is just basically getting the process started," and that's correct. But you only get those 38 ATPs under one condition and that's if there's plenty of oxygen. If there's not plenty of oxygen, you're stuck with two ATPs. So it's important to be able to get as much energy out of glucose as possible if you want to be efficient. If you don't want to be efficient, that's fine. It turns out that's good. Did you know that? Not being efficient is good? Any speculation on when you might not want to be very efficient? Student: When you're running a marathon? Kevin Ahern: Well, when you're running a marathon, you'd kind of like to be efficient, I think. Yeah. Student: Maybe when you're asleep? Kevin Ahern: Maybe when you're asleep? Well, not really. I can think of a better condition. It's a condition I found myself in earlier this term. Student: Well, I was going to say when you're stressed, you don't want to be overloaded, but... Kevin Ahern: No, It's not when you're stressed. Student: When you're on a diet? Kevin Ahern: When you're on a diet! Absolutely! You want to be the least efficient when you're on a diet because the more it takes to burn to make ATP, the more stuff you're going to burn up. I'll tell you later about how people have this notion about going aerobic is the best thing that you can do and I'm going to try to convince you that going anaerobic is the best thing that you could do if you're trying to lose weight. We'll talk about that in times to come. Let's look at the energy investment phase. The energy investment phase we can roughly think of as about the first five or six reactions of glycolysis... It sort of depends on how we count them. In this phase, we have to use energy from ATP to get the process started. So here's a catabolic process, and we'll see that in several ways. glycolysis is an unusual catabolic process, here's a catabolic process that is sort of breaking the rules and requiring us to put energy in before we can get energy out. Most catabolic processes don't do that. There's two different places, as you can see on the screen here, where ATP is used. In this first phase, no ATP is generated. The ATP is generated only in the second phase, as we shall see. Well, let's take a look at the reactions. Here's the first reaction. Glucose combines with ATP to produce glucose 6-phosphate and ADP. I showed you this reaction earlier. It's catalyzed by the enzyme known as hexokinase. By the way, I'm also going to tell you, in a few cases, the Delta G zero primes for reactions, and I think you should know the generalities, not the absolute numbers. This guy has a fairly negative Delta G zero prime. If I tell you about the Delta G zero prime for a reaction being fairly negative, fairly positive, you should know those. This is one of them. This reaction has a Delta G zero prime that's fairly negative, meaning that, if we start this reaction with all concentrations of everything equal, that it will go strongly in the forward direction. What if we try to do this reaction with phosphate instead of ATP? The enzyme will actually do it. If we measure the Delta G zero prime for the reaction using phosphate instead of ATP, what we discover is that the Delta G zero prime for the reaction is very positive. Well, this is a prime example of where coupling the hydrolysis of ATP to an energetically unfavorable reaction converts it into a favorable reaction... a prime example of that. The same reaction with phosphate instead of ATP has a Delta G zero prime that's very positive, meaning not very favorable in the forward direction. It's going to be much more favorable in the reverse direction. However, when I link it to ATP and the hydrolysis of ATP yields energy that drives this reaction forwards. This reaction is an interesting reaction. It's one of three reactions in glycolysis that is one of the regulated reactions, meaning the enzyme itself is regulated. This one turns out to have an unusual regulation and I'm going to spend very little time talking about that. I won't talk about regulation until at least Wednesday. But this is one of three. Glycolysis is also unusual in having not one regulatory step, but three steps that are controlled. Student: [unintelligible] Kevin Ahern: What's that? Student: [unintelligible] Kevin Ahern: In the pathway. The second step of—oh, by the way, the induced fit, I've showed you guys that before, but you may recall when I talked about glycolysis before and I said that, remember how the substrate changes the enzyme upon binding it? And the example I actually gave you was this enzyme. Hexokinase starts out, it's got two molecules that it's got to bind and it's got to crunch together to transfer that phosphate from one to the other. We can think of these as rather like the jaws, the teeth of the jaws, right here, where up above I could have, let's say, ATP, and down below I could have glucose. The binding of both of these causes the jaws to close so that the phosphate of the ATP is brought into close proximity of glucose, and the phosphate is able to jump. When that phosphate jumps, the jaws open and the molecules come back out. This is a really good example of an enzyme that has an induced fit. It changes its shape as it binds to its substrate. You can see that here, a little bit. They're only showing you the glucose. They're not showing you the ATP. But you can see that the addition of glucose, in this case, is converting the unbound enzyme to the bound enzyme, shown in red. You can literally see those jaws closing down. Reaction two of glycolysis is a reaction catalyzed by phosphoglucose isomerase. It simply involves the conversion of the glucose 6-phosphate to fructose 6-phosphate. We see it goes through a linear intermediate in order to do this, and all that's happening here is an aldehyde group, right here, is being converted to a ketone group. So the position of the double-bonded oxygen is moving in the molecule. That is an isomerization. Student: Could you say the name of the enzyme one more time, please? Kevin Ahern: Phosphoglucose isomerase or glucose phosphate isomerase. You see it listed both ways. Reaction number three is one of the most important reactions of glycolysis. This reaction, first of all, is catalyzed by the enzyme known as PFK, phosphofructokinase, and, yes, you're more than welcome to call it "PFK." Later in the term you'll see that we refer to this as PFK1 and you can call it either PFK1 or PFK at this point, because we haven't encountered PFK1 or 2 yet. But this is PFK. Why is this reaction so important? First of all, this reaction, this enzyme, PFK, is the most important regulatory enzyme in the glycolysis pathway. It's the most important regulatory enzyme in the glycolysis pathway, and, as we will see, there are several things that can allosterically affect this enzyme, several things that allosterically affect this enzyme. You'll notice also that we're adding a second ATP in this reaction. So we're converting fructose 6-phosphate to fructose 1,6-bisphosphate, and, by the way, you may use these abbreviations. There's only one abbreviation I'll ask you not to use, and I'll show you that one later. But all the abbreviations that they use here are certainly acceptable in this class, with the exception of the one I'll give you later. ATP is required, and, just as we saw before, this reaction, with the hydrolysis of ATP, the Delta G zero prime of this reaction is very negative. The hydrolysis of ATP helps to drive this reaction forward. If we try to do this reaction with phosphate instead of ATP, we discover it doesn't go forward very well, at all; in fact, it goes very strongly backwards. So the hydrolysis of ATP, again, is making this reaction much more favorable. So at this point, we've built a molecule that has two phosphates into it and, now, in the next step, we're ready to break it apart. We can look at this next step as being a lyase-type reaction because what we see is that this guy is going to get split right in the middle, and this enzyme is the only enzyme in the entire pathway whose name doesn't immediately tell you what it does. It's called “aldolase.” All the other enzyme names in the pathway tell you what the enzyme does. So what's happening here? In this reaction, fructose 1,6-bisphosphate is being split in half. One half becomes dihydroxyacetone phosphate, or DHAP. The other half becomes glyceraldehyde 3-phosphate, or what your book calls “GAP,” and I don't like that. Let's call it “G3P.” G3P, okay? So instead of calling it “GAP,” you're going to call it “G3P.” This reaction is interesting. This reaction is interesting because the Delta G zero prime for this reaction is very positive. It's very positive. Now, if I told you the Delta G zero prime for a reaction is very positive, would that tell you if the reaction goes forwards or backwards? No. It wouldn't tell you anything, because the Delta G would tell you. Right? Delta G zero prime will influence that, but Delta G will not tell you, right? Same as with the other reactions. Delta G zero prime, even though it's very negative, it doesn't determine the direction. Only if I tell you that all the concentrations of things I start with are equal, then it tells me the direction of a reaction, right? Well, how do I have a reaction that has a Delta G zero prime that's very positive, yet the reaction still gets to go forward? What would it take to do that? Student: You need a covalent intermediate? Kevin Ahern: A covalent intermediate. No. Student: A high concentration of the reactant? Kevin Ahern: A high concentration of the reactant is one thing. And what else would help this reaction to go forward? Student: [unintelligible] Kevin Ahern: Well, enzymes don't change Delta G. Student: The Delta Gs in the first two parts of the reaction? Kevin Ahern: Well, that's a good point. So the Delta Gs in the first two parts of the reaction do actually help, and they help by increasing the concentration of reactants, which is related to what Connie said. Student: A decrease in concentration of the products? Kevin Ahern: A decrease in concentration of the products. Remember, these reactions, we see that one's connected to the next, connected to the next, connected to the next. So if I have something sucking away the products of this reaction, those two can work together to overcome this energy barrier, because that's going to change the value of that log term. Increasing concentration of reactant and decreasing concentration of product. Now, as we will see, the cell has a really cool trick for accomplishing both of these things. Student: [unintelligible] Kevin Ahern: I'm sorry? Student: [unintelligible] Kevin Ahern: Oh, yep. That's not the cool trick it uses, either. Blast you! So we'll come back. We'll talk more about the aldolase reaction in a bit. We turn our attention now to the last of the energy investment phase. The last of this energy investment phase involves the conversion of dihydroxyacetone phosphate into glyceraldehyde 3-phosphate. Notice this is a reversible reaction, so it can go either way. But in the direction of glycolysis it moves to the right. In the direction of synthesizing glucose it moves to the left. We'll talk about the synthesis of glucose next week. Student: [unintelligible] Kevin Ahern: The dihydroxyacetone phosphate was one of the two products of the previous reaction. Student: That's the DHAP. Kevin Ahern: That's the DHAP. So in this reaction, we're converting DHAP into G3P. Now we have two G3Ps. This simplifies things for keeping track of stuff, because now every reaction is just duplicated. We have two copies of everything that's reacting, from this point forward, and they're two copies of the same thing. This reaction is catalyzed by the enzyme known as triose phosphate isomerase, and it is a prime example of something we talked about earlier in the term. Triose phosphate isomerase is a perfect enzyme. It's a perfect enzyme, meaning that it has a very high kcat over Km value. It's limited primarily only by the rate with which the substrate diffuses to the enzyme. The reason that this enzyme is perfect, it appears, is because there's actually an unstable intermediate that is produced in the mechanism of the enzyme. The faster the reaction goes, the less time that unstable intermediate is around to cause problems. By making the enzyme perfect, we make the reaction go so fast the unstable intermediate doesn't have a chance to fall apart. Perfect enzymes commonly have that strategy. Well, the upshot of this is, at this point, we now have two molecules of glyceraldehyde 3-phosphate and we are ready to move to the oxidation phase of glycolysis... the oxidation phase. By the way, there's the unstable intermediate right there. You don't need to know that, but just to tell you I wasn't lying, you know it's there. In the energy generation phase, what we see is that there are two places that ATPs are produced. But remember that, since every molecule is duplicated, the ATPs themselves are also duplicated. So we're going to produce, in this second phase, a total of four ATPs. That's how we get our net gain of two. Let's start the process off with a bang, and starting that process off with a bang is this mouthful of an enzyme, glyceraldehyde 3-phosphate dehydrogenase. By the way, whenever you see the word "dehydrogenase" involved in an enzyme name, it's always a redox enzyme. That, of course, is reaffirmed by the fact there's an NAD plus that is present here. This reaction is actually two reactions that are occurring on this molecule. Remember that we're starting with two of these G3Ps. Everything we've got two of, from now on, so we've got two G3Ps, we have two NADs, we produce two of these, and we produce two of these, and we produce two of these. What's happening in this molecule? The first thing that happens in this molecule is an oxidation. An oxidation is occurring. The NAD is giving us a clue to that, and the structure of this aldehyde is the clue. Notice we have an aldehyde to start with. Over here, we have an ester. That means this guy had to have gotten oxidized to an acid first. So the oxidation happens and then, after the oxidation happens, phosphate is put onto the acid, making the ester. So oxidation to produce an acid, then addition of the phosphate to the acid to produce the ester. The oxidation converts NAD into NADH, as you can see here. This reaction is interesting. If we compare it, for example, to the hexokinase reaction or the PFK reaction, those reactions were putting a phosphate onto something, right? And I told you that putting a phosphate onto something required hydrolysis of ATP. That's why they used ATP. We're putting a phosphate onto something and we're not using ATP. What does that tell you about this reaction? This reaction is energetically favorable, by the way. What does it tell you? It says we have to have an energy source, right? Previously we had an energy source from ATP. Now we have to have some other energy source. What do you suppose the energy source is? Student: The 1,3-BPG has a higher [unintelligible]? Kevin Ahern: Well, this molecule has a higher energy, sure, but we have to make this molecule. Student: [unintelligible] Kevin Ahern: What's that? Student: Is it the NAD-plus? Kevin Ahern: No, it's not the NAD-plus. It's the oxidation reaction. Remember that oxidation produces energy. So this oxidation that's occurring in this process is giving sufficient energy to add that phosphate. In the previous reactions we saw the energy came from ATP. Here the energy is coming from oxidation. So putting together an oxidation, in this case, with a phosphate, gives us a high energy intermediate, and 1,3-bisphosphoglycerate, this guy's loaded with energy. 1,3-bisphosphoglycerate, or 1,3-BPG, is one of those molecules on that table I showed you last time that had a higher energy than ATP did. It's got a higher energy than ATP does. This guy is full of energy. That negative charge and that negative charge really repel each other. They really don't want to be together. This is not the next reaction. This is the mechanism of that reaction. There's the oxidation. There's the phosphate coming on, so you can see the sort of two steps to this process. In the next reaction, we generate our first ATPs. This enzyme is known as phosphoglycerate kinase. If you want to call it PG kinase, that's fine, too. In this reaction, a phosphate from 1,3-BPG is being transferred onto ADP to make ATP, and that leaves us behind with 3-phosphoglycerate. Now, in order for this reaction to go forwards efficiently and, yes, it does—in order for this reaction to go forwards efficiently, this guy has got to have a lot of energy,and it does. 1,3-BPG has more energy than ATP does, so therefore it can transfer the phosphate onto ADP and make ATP very efficiently. This type of a reaction is a type of a reaction that we haven't seen previously. We're actually making ATP using this step. It turns out there are three ways of making ATP. One is what you see on the screen. It's what known as "substrate-level phosphorylation"... substrate-level phosphorylation. In this mechanism, a high energy molecule is transferring a phosphate to ADP to make ATP. That's only one of three ways that cells make ATP. The second way that cells make ATP is what's called "oxidative phosphorylation." We'll talk about that next term when we talk about the electron transport system. Oxidative phosphorylation in animals is, by far, the most abundant form of making ATP, the most abundant way of making ATP. The third type, or the third mechanism for making ATP is what's called "photophosphorylation" and that's what plants use in photosynthesis. Now this is a relatively minor way of making ATP. Substrate-level phosphorylation doesn't contribute very much to the overall ATP pool. Questions? We're getting close. We're getting close. The last three reactions you see are all bundled together. We're going to make it through. Actually, we might not make it through. Maybe we won't. Yay! I'll just take up the time with something else that you'll be responsible for, so... But I'll spend a little bit of time on this first reaction. This first reaction is kind of cool. I don't know why your book bundles all three of them, as if they're not important, because this one turns out to be a really important reaction. Look at what's happening in this reaction: 3-phosphoglycerate is being converted to 2-phosphoglycerate. It looks like it's a simple isomerization reaction, and overall, it is, but you'll notice the enzyme's class is not described as in isomerase. It's described as a mutase. What the heck is a mutase? Well, the mutase tells us something about the mechanism that this uses. So if I had an isomerase, what would an isomerase do? Well, it would grab that phosphate on position number 3, and it would move it to position number 2, and we would have the same end product. Well, how does a mutase work? A mutase works by making an interesting intermediate. It makes an intermediate by putting an additional phosphate on. So at one place I have two phosphates on, and then it removes this phosphate and leaves the other one behind. Well, maybe you can see where this is headed. The intermediate in this process of a mutase is known as "2,3-BPG." 2,3-BPG is an intermediate. It's a byproduct and it's a stable byproduct of this reaction. 2,3-BPG, you remember, I said was a byproduct of metabolism. It binds to hemoglobin and favors the release of oxygen. The more glycolysis I have going on, the more likely some of that 2,3-BPG is going to escape from the enzyme, not make this thing, and now go out and affect hemoglobin. The mechanism of this enzyme is actually telling our body, "Here's where a lot of glycolysis is going on." It's a really cool thing. 2,3-BPG can be released from the enzyme, without making this, in some cases, and at a low efficiency, let's say 5% efficiency, 2,3-BPG is released and this is not made. The more I have glycolysis going on, the more 2,3-BPG is made, and, of course, this is a flag that this is a cell that's needing energy very quickly, very rapidly. So 2,3-BPG is produced in that way. Questions about that? You look like you've been struck dumb. "Oh, my God! I have seen the light!" Right? "I have seen it, I know." And just so that you won't feel like I'm rushing you through it, why don't we just call it right there for the exam? How's that? Oh, one question over here, yeah? Student: [unintelligible] Kevin Ahern: Yep, this equilibrium is fairly, Delta G zero prime is fairly close to zero. See you guys on Wednesday. Exam material stops right here. Captioning provided by Disability Access Services at Oregon State University [END]