WEBVTT 00:00:01.000 --> 00:00:04.000 Good morning, class. 00:00:04.000 --> 00:00:08.000 I just wanted to spend the first couple minutes clearing up three 00:00:08.000 --> 00:00:12.000 issues. None is a major conceptual issue, but we like to focus on 00:00:12.000 --> 00:00:17.000 details and get them right, get them correct here as well. 00:00:17.000 --> 00:00:21.000 Firstly, I misdrew a reaction last time that described why RNA is 00:00:21.000 --> 00:00:26.000 alkali labile, i.e., if we have high pH we call 00:00:26.000 --> 00:00:30.000 that an alkali pH, or an alkaline pH, actually, 00:00:30.000 --> 00:00:35.000 to use the adjective. And we said that hydroxyl groups can 00:00:35.000 --> 00:00:39.000 cause the cleavage of the phosphodiester bonds of RNA but not 00:00:39.000 --> 00:00:44.000 DNA. And the way I described that happening is that the alkali group 00:00:44.000 --> 00:00:49.000 causes the formation of this five-membered ring right here, 00:00:49.000 --> 00:00:53.000 two carbons, two oxygens and a phosphate. And that resolves 00:00:53.000 --> 00:00:58.000 eventually to this where there's no longer any connection with the 00:00:58.000 --> 00:01:03.000 ribonucleoside monophosphate below. And I drew it like this, 00:01:03.000 --> 00:01:07.000 without an oxygen, and that's a no-no because, 00:01:07.000 --> 00:01:12.000 in fact, in truth, and as many of you picked up, 00:01:12.000 --> 00:01:16.000 this reverts to a two prime hydroxyl. So, please note there's a mistake 00:01:16.000 --> 00:01:21.000 there. There's also a couple other mistakes. For example, 00:01:21.000 --> 00:01:25.000 in the textbook it gives you the impression that when you polymerize 00:01:25.000 --> 00:01:30.000 nucleic acids you use a monophosphate to do so. 00:01:30.000 --> 00:01:34.000 And, if you listened to my lecture last time, that doesn't make any 00:01:34.000 --> 00:01:38.000 sense, because you need to invest the energy of a triphosphate in 00:01:38.000 --> 00:01:42.000 order to create enough energy to generate enough energy for the 00:01:42.000 --> 00:01:46.000 polymerization. The textbook is incorrect there. 00:01:46.000 --> 00:01:51.000 Textbooks are written by people, for better or worse, 00:01:51.000 --> 00:01:55.000 and as such, like everything else, they are a mortal and fallible. So, 00:01:55.000 --> 00:01:59.000 the truth of the matter is, when you're polymerizing DNA or RNA you 00:01:59.000 --> 00:02:03.000 need one of the four ribonucleoside or deoxyribonucleoside triphosphates 00:02:03.000 --> 00:02:08.000 in order to donate the energy that makes possible this polymerization. 00:02:08.000 --> 00:02:12.000 And please note that is a mistake in the book. Recall, 00:02:12.000 --> 00:02:16.000 as I said last time, the fact that ATP is really the 00:02:16.000 --> 00:02:20.000 currency of energy in the cell, and that its energy is stored and 00:02:20.000 --> 00:02:24.000 coiled up in this pent up spring where the mutual electrostatic 00:02:24.000 --> 00:02:28.000 repulsion between the three negatively charged phosphates 00:02:28.000 --> 00:02:33.000 carries with it enormous potential energy. 00:02:33.000 --> 00:02:36.000 And some of that potential energy can be realized, 00:02:36.000 --> 00:02:40.000 during the synthesis of polymerization of nucleic acids by 00:02:40.000 --> 00:02:43.000 cleaving this bond here. One can also generate potential 00:02:43.000 --> 00:02:47.000 energy by cleaving this bond here. This is the alpha, the beta and the 00:02:47.000 --> 00:02:51.000 gamma-phosphate. And cleavage of either can create 00:02:51.000 --> 00:02:54.000 substantial energy, which in turn can, as we'll indicate 00:02:54.000 --> 00:02:58.000 shortly, be invested in other reactions. The reaction 00:02:58.000 --> 00:03:01.000 of polymerization. A second point I'd like to make to 00:03:01.000 --> 00:03:05.000 you is the following, and you'd say it's kind of 00:03:05.000 --> 00:03:09.000 coincidence. The currency of energy in the cell is ATP, 00:03:09.000 --> 00:03:13.000 adenosine triphosphate, we see its structure here, 00:03:13.000 --> 00:03:17.000 and this happens to be one of the four precursors of the RNA. 00:03:17.000 --> 00:03:21.000 So, the same molecule is used in these two different ostensively 00:03:21.000 --> 00:03:25.000 unrelated applications. One, to polymerize to make RNA 00:03:25.000 --> 00:03:29.000 where genetic information is stored and conveyed. 00:03:29.000 --> 00:03:33.000 Or, alternatively it's used here in this context in order to serve as a 00:03:33.000 --> 00:03:37.000 currency for energy. High energy as ATP. ADP with a 00:03:37.000 --> 00:03:41.000 little lower energy. AMP monophosphate with even lower 00:03:41.000 --> 00:03:46.000 energy. And you might ask yourself, scratch your head and say why is the 00:03:46.000 --> 00:03:50.000 same molecule used for these two different things? 00:03:50.000 --> 00:03:54.000 In fact, there are yet other applications of these 00:03:54.000 --> 00:03:58.000 ribonucleosides which also seem to be unrelated to the storage or the 00:03:58.000 --> 00:04:02.000 conveyance of genetic information. And it is believed, 00:04:02.000 --> 00:04:06.000 probably correctly, that the reason why the same molecule is used for 00:04:06.000 --> 00:04:10.000 these totally different applications is that early in the evolution of 00:04:10.000 --> 00:04:14.000 life on this planet there really were a rather small number of 00:04:14.000 --> 00:04:18.000 biological molecules that existed. Indeed, as we'll mention again 00:04:18.000 --> 00:04:22.000 later, it's probably the case that the first organisms didn't use DNA 00:04:22.000 --> 00:04:26.000 as genomes. It's an article of faith with us that one stores 00:04:26.000 --> 00:04:30.000 genetic information in DNA molecules. 00:04:30.000 --> 00:04:33.000 And I implied that quite explicitly last time. But, 00:04:33.000 --> 00:04:37.000 the fact of the matter is, it's probably the case that the 00:04:37.000 --> 00:04:41.000 first organism, the first pre-cellular life forms 00:04:41.000 --> 00:04:44.000 used RNA as the genetic material, RNA to store things, replicating RNA 00:04:44.000 --> 00:04:48.000 via double-stranded RNA molecules as a way of archiving genetic 00:04:48.000 --> 00:04:52.000 information. And only later during the evolution of life on this planet, 00:04:52.000 --> 00:04:56.000 when that later was we can't tell, but it could have been a hundred or 00:04:56.000 --> 00:05:00.000 two hundred years later. Obviously, if we're talking about 00:05:00.000 --> 00:05:04.000 the origin of life as between 3. and 3.5 billion years ago, we can't 00:05:04.000 --> 00:05:09.000 really localize that in time very well, but only later was DNA 00:05:09.000 --> 00:05:13.000 assigned the job of storing, in a stable fashion, genetic 00:05:13.000 --> 00:05:18.000 information. And as a consequence, we come to realize as well yet 00:05:18.000 --> 00:05:23.000 another discovery, which is that all the catalysts that 00:05:23.000 --> 00:05:27.000 we're going to talk about today, the enzymes as we call them, almost 00:05:27.000 --> 00:05:32.000 all modern-day enzymes are proteins. And we talked about them briefly 00:05:32.000 --> 00:05:36.000 before. But over the last 15 years, 20 years there's been the discovery 00:05:36.000 --> 00:05:40.000 that certain RNA molecules also posses the ability to catalyze 00:05:40.000 --> 00:05:45.000 certain kinds of reactions. When I was taking biochemistry, 00:05:45.000 --> 00:05:49.000 if somebody would have told me that, I would have called the psychiatric 00:05:49.000 --> 00:05:53.000 ward because that was such an outlandish idea. 00:05:53.000 --> 00:05:58.000 How can an RNA molecule catalyze a biochemical reaction? 00:05:58.000 --> 00:06:02.000 It doesn't have all the side groups that one needs to create the 00:06:02.000 --> 00:06:06.000 catalytic sites for reactions. But we now realize, on the basis of 00:06:06.000 --> 00:06:10.000 research which actually led to a Nobel Prize being awarded about five 00:06:10.000 --> 00:06:14.000 years ago, that RNA molecules are able to catalyze certain kinds of 00:06:14.000 --> 00:06:18.000 reactions. And that begins to give us an insight into how life 00:06:18.000 --> 00:06:22.000 originated on this planet because RNA molecules may have stored 00:06:22.000 --> 00:06:26.000 genetic information, as I said before, RNA molecules, 00:06:26.000 --> 00:06:30.000 or their precursors like ATP, may have been their currency for storing 00:06:30.000 --> 00:06:34.000 high energy bonds, as is indicated here. 00:06:34.000 --> 00:06:38.000 And RNA molecules may well have been the first enzymes to catalyze many 00:06:38.000 --> 00:06:43.000 of the reactions in the most primitive life forms that first 00:06:43.000 --> 00:06:47.000 existed on this planet. And, therefore, what I'm saying is 00:06:47.000 --> 00:06:52.000 that as life developed in the first hundred or two hundred million years, 00:06:52.000 --> 00:06:56.000 who knows how long it took, gradually DNA took over the job of 00:06:56.000 --> 00:07:01.000 storing information from RNA and gradually proteins took over the job 00:07:01.000 --> 00:07:05.000 of mediating catalysis, of acting as enzymes to taking over 00:07:05.000 --> 00:07:10.000 the job from RNA molecules. Today there are certain vestigial 00:07:10.000 --> 00:07:14.000 biochemical reactions which we believe are relics, 00:07:14.000 --> 00:07:18.000 echoes of the beginning of life on earth, which are still mediated by 00:07:18.000 --> 00:07:22.000 RNA catalysts. We think that they are throwbacks 00:07:22.000 --> 00:07:26.000 to these very early steps, maybe even in pre-cellular life form 00:07:26.000 --> 00:07:31.000 where RNA was delegated with the task of acting as a catalyst. 00:07:31.000 --> 00:07:35.000 We're going to focus a lot today on the whole issue of biochemical 00:07:35.000 --> 00:07:40.000 reactions and the issue of energy. And this gets us into the 00:07:40.000 --> 00:07:45.000 realization that there really are two kinds of biochemical reactions. 00:07:45.000 --> 00:07:49.000 Some of you may have learned this a long time ago. 00:07:49.000 --> 00:07:54.000 Either exergonic reactions that release energy, 00:07:54.000 --> 00:07:59.000 that produce energy as they proceed, or conversely endergonic reactions 00:07:59.000 --> 00:08:04.000 which require an investment of energy in order to move forward. 00:08:04.000 --> 00:08:08.000 So, here, obviously, if this is a high energy state and 00:08:08.000 --> 00:08:12.000 we're talking about the free energy of the system, 00:08:12.000 --> 00:08:16.000 which is one way to depict in thermodynamic language how much 00:08:16.000 --> 00:08:20.000 energy is in a molecule, if we go from a high energy state to 00:08:20.000 --> 00:08:24.000 a low energy state then we can draw this like this and we can realize 00:08:24.000 --> 00:08:28.000 that in order to conserve energy, the energy that was inherent in this 00:08:28.000 --> 00:08:32.000 molecule, the high potential energy is released as this ball or this 00:08:32.000 --> 00:08:37.000 molecule rolls down the hill. And, therefore, 00:08:37.000 --> 00:08:41.000 the reaction yields energy, it's exergonic. And, conversely, 00:08:41.000 --> 00:08:45.000 if we want this reaction to proceed, we need to invest free energy in 00:08:45.000 --> 00:08:49.000 order to make it happen. The free energy happens to be, 00:08:49.000 --> 00:08:53.000 more often than not, in the form of chemical bonds, 00:08:53.000 --> 00:08:57.000 i.e., energy that can be invested, for example, by taking advantage of 00:08:57.000 --> 00:09:01.000 the potential energy stored in these phosphodiester, 00:09:01.000 --> 00:09:06.000 in these phosphate-phosphate linkages indicated right here. 00:09:06.000 --> 00:09:10.000 Here, by the way, is the space-filling model of ATP 00:09:10.000 --> 00:09:14.000 just for your information. That's the way it actually would 00:09:14.000 --> 00:09:18.000 look in life, and this is the way we actually draw it. 00:09:18.000 --> 00:09:22.000 Now, having said that, if we look at the free energy 00:09:22.000 --> 00:09:26.000 profile of various biochemical changes then we can depict them, 00:09:26.000 --> 00:09:30.000 once again, in this very schematic way here. 00:09:30.000 --> 00:09:35.000 And, by the way, free energy is called G, 00:09:35.000 --> 00:09:40.000 the Gibbs free energy after Josiah Gibbs who was a thermodynamic wiz in 00:09:40.000 --> 00:09:45.000 the 19th century at Yale in New Haven. And here what we see is that 00:09:45.000 --> 00:09:50.000 the change in free energy between the reactants and the products is 00:09:50.000 --> 00:09:55.000 given by delta G. So, by definition, 00:09:55.000 --> 00:10:00.000 we start out the reaction with reactants. 00:10:00.000 --> 00:10:03.000 And we end up at the end of the reaction with products. 00:10:03.000 --> 00:10:07.000 And, overall, if the reaction is exergonic and will proceed forward, 00:10:07.000 --> 00:10:11.000 it releases energy. And the net release of energy is indicated here 00:10:11.000 --> 00:10:15.000 by delta G. But, more often than not, 00:10:15.000 --> 00:10:18.000 biochemical reactions that are energetically favored, 00:10:18.000 --> 00:10:22.000 that are exergonic actually can't happen spontaneously. 00:10:22.000 --> 00:10:26.000 They don't happen spontaneously because, for various reasons, 00:10:26.000 --> 00:10:30.000 they have to pass through an intermediate state. 00:10:30.000 --> 00:10:34.000 Which actually represents a much higher free energy than the initial 00:10:34.000 --> 00:10:38.000 reactants posses. And this higher free energy, 00:10:38.000 --> 00:10:42.000 that they need to acquire in order to move over the hill and down into 00:10:42.000 --> 00:10:46.000 the valley, is called the energy of activation, the activation energy. 00:10:46.000 --> 00:10:50.000 And, therefore, if I were to supply these reactants with energy, 00:10:50.000 --> 00:10:54.000 for instance, let's say I were to heat up these reactants and 00:10:54.000 --> 00:10:58.000 therefore give them a higher degree of thermal energy which they might 00:10:58.000 --> 00:11:03.000 be able to use to move up to this high energy state. 00:11:03.000 --> 00:11:06.000 I supplied them with free energy by giving them heat. 00:11:06.000 --> 00:11:09.000 Then they might be able to move up to here and then roll down the hill. 00:11:09.000 --> 00:11:12.000 But in the absence of actually actively intervening and supplying 00:11:12.000 --> 00:11:16.000 them that energy, they'll remain right here, 00:11:16.000 --> 00:11:19.000 and they may remain right there for a million years, 00:11:19.000 --> 00:11:22.000 even though in principle, if they were to reach down here, 00:11:22.000 --> 00:11:26.000 they would be much happier in terms of reaching a much lower 00:11:26.000 --> 00:11:30.000 energy state. To state the obvious, 00:11:30.000 --> 00:11:34.000 all these kinds of reactions wish to reach the lowest energy state 00:11:34.000 --> 00:11:38.000 possible. But in real-time it can't happen if there is a high energy of 00:11:38.000 --> 00:11:42.000 activation. Now, what do enzymes do? 00:11:42.000 --> 00:11:47.000 As always, I'm glad I asked that question. What they do is they 00:11:47.000 --> 00:11:51.000 lower the energy of activation. And this is in one sense obvious, 00:11:51.000 --> 00:11:55.000 and in one sense it's subtle, because enzymes have no affect on 00:11:55.000 --> 00:11:59.000 the free energy state of the reactants, they have no affect on 00:11:59.000 --> 00:12:03.000 the free energy of the products. All they do is to lower the hump, 00:12:03.000 --> 00:12:07.000 and they may lower it very substantially. 00:12:07.000 --> 00:12:11.000 And because they lower it substantially, 00:12:11.000 --> 00:12:15.000 it might be that some of the reactants here may, 00:12:15.000 --> 00:12:18.000 just through a chance, acquisition of thermal energy, 00:12:18.000 --> 00:12:22.000 be able to move over the much lowered hump and move down into this 00:12:22.000 --> 00:12:26.000 state right here. Now, the actual difference in the 00:12:26.000 --> 00:12:30.000 Gibbs free energy is totally unaffected. 00:12:30.000 --> 00:12:35.000 All that happens is that the enzyme, by lowering the energy of activation, 00:12:35.000 --> 00:12:40.000 make this possible in real-time. The fact is that ultimately, if one 00:12:40.000 --> 00:12:46.000 were to plot many kinds of reactions, many reactions, 00:12:46.000 --> 00:12:51.000 as is indicated here, have a very high activation energy, 00:12:51.000 --> 00:12:56.000 and therefore we look at it like this. But there could be other 00:12:56.000 --> 00:13:02.000 reactions which might have an activation energy that looks like 00:13:02.000 --> 00:13:07.000 this, almost nothing at all. And these reactions could happen 00:13:07.000 --> 00:13:11.000 spontaneously at room temperature in the absence of any intervention by 00:13:11.000 --> 00:13:15.000 an enzyme. For example, let's say we're talking about a 00:13:15.000 --> 00:13:19.000 carboxyl group which discharges a proton. We've talked about that 00:13:19.000 --> 00:13:23.000 already. Well, that reaction happens spontaneously 00:13:23.000 --> 00:13:27.000 at room temperature. It doesn‘t need an enzyme to make 00:13:27.000 --> 00:13:31.000 it happen. It can happen because there's essentially not 00:13:31.000 --> 00:13:35.000 energy of activation. But the great majority of 00:13:35.000 --> 00:13:39.000 biochemical reactions do have such an activation energy, 00:13:39.000 --> 00:13:44.000 and therefore do require a lowering like this in order to take place. 00:13:44.000 --> 00:13:49.000 Now, let's imagine other versions of the energy profile of a reaction. 00:13:49.000 --> 00:13:53.000 And keep in mind that what I'm showing here on the abscissa is just 00:13:53.000 --> 00:13:58.000 the course of the reaction. You could imagine I'm not really 00:13:58.000 --> 00:14:05.000 plotting time. I'm just talking about a situation 00:14:05.000 --> 00:14:13.000 where to the left the reaction hasn't happened and to the right it 00:14:13.000 --> 00:14:21.000 has happened. Can you see this over there? Then I won't write over 00:14:21.000 --> 00:14:30.000 there. All right. Let's see if this works. 00:14:30.000 --> 00:14:36.000 Boy, here we are in the 21st century and we still haven't worked this out. 00:14:36.000 --> 00:14:42.000 OK. Everybody can see this right here, right? OK. 00:14:42.000 --> 00:14:48.000 So, look. Let's imagine we have a reaction that looks like this, 00:14:48.000 --> 00:14:54.000 a reaction profile that looks like this, where these two energies are 00:14:54.000 --> 00:15:00.000 actually equivalent. OK? I've tried to draw them on. 00:15:00.000 --> 00:15:04.000 Well, they're not exactly, but they're pretty much on exactly 00:15:04.000 --> 00:15:08.000 the same level. And let's say we start out with a 00:15:08.000 --> 00:15:13.000 large number of molecules right over here. Now, if there were an enzyme 00:15:13.000 --> 00:15:17.000 around, the enzyme might lower the activation energy and, 00:15:17.000 --> 00:15:21.000 in so doing, make it possible for molecules to tunnel through this 00:15:21.000 --> 00:15:26.000 hill and move over here. The fact that when a molecule gets 00:15:26.000 --> 00:15:30.000 over here it has the same free energy as over there means that the 00:15:30.000 --> 00:15:35.000 catalyst may, in principle, also facilitate a back reaction. 00:15:35.000 --> 00:15:38.000 What do I mean by a back reaction? I mean going in exactly the 00:15:38.000 --> 00:15:42.000 opposite direction. And so, once molecules over here 00:15:42.000 --> 00:15:46.000 are formed, the energy lowering affects of the enzyme may allow them 00:15:46.000 --> 00:15:50.000 to move in both directions. And, therefore, what we will have 00:15:50.000 --> 00:15:54.000 is ultimately the establishment of an equilibrium. 00:15:54.000 --> 00:15:58.000 If these two energy states are equivalent then, 00:15:58.000 --> 00:16:02.000 I will tell you, 50% of the molecules end up here and 50% of the 00:16:02.000 --> 00:16:06.000 molecules end up here. And here we're beginning now to 00:16:06.000 --> 00:16:10.000 wrestle between two different independent concepts, 00:16:10.000 --> 00:16:14.000 the rate of the reaction and the equilibrium state of the reaction. 00:16:14.000 --> 00:16:19.000 Note that the enzyme has no affect whatsoever on the equilibrium state. 00:16:19.000 --> 00:16:23.000 These two are at equal free energies, the equilibrium state. 00:16:23.000 --> 00:16:27.000 Whether the energy barrier is this high or whether it's this 00:16:27.000 --> 00:16:32.000 high is irrelevant. The fact is if the enzyme makes 00:16:32.000 --> 00:16:36.000 possible this motion back and forth, the ultimate equilibrium state will 00:16:36.000 --> 00:16:40.000 be 50% of the molecules here and 50% of the molecules there. 00:16:40.000 --> 00:16:44.000 And, therefore, the enzyme really only affects the rate at which the 00:16:44.000 --> 00:16:48.000 reaction takes place. Will it happen in a microsecond or 00:16:48.000 --> 00:16:52.000 will it happen in a day or will it happen in a million years? 00:16:52.000 --> 00:16:56.000 The enzyme has no affect whatsoever on the ultimate end product, 00:16:56.000 --> 00:17:00.000 which in this case is the equilibrium. 00:17:00.000 --> 00:17:06.000 Of course, there is a simple mathematic formalism which relates 00:17:06.000 --> 00:17:12.000 the difference in free energies with the equilibrium. 00:17:12.000 --> 00:17:18.000 Here we might have a situation where 80% of the molecules end up at 00:17:18.000 --> 00:17:24.000 equilibrium over here and 20% end up here. Or, we might end up as a 00:17:24.000 --> 00:17:30.000 state where 99. % of the molecules end up here and 0. 00:17:30.000 --> 00:17:34.000 % of the molecules end up here. But that ultimate equilibrium state 00:17:34.000 --> 00:17:38.000 is no way influenced by the enzyme. They just make it happen in 00:17:38.000 --> 00:17:42.000 real-time. And, therefore, to repeat and echo a 00:17:42.000 --> 00:17:46.000 point I made last time, if most biochemical reactions are to 00:17:46.000 --> 00:17:50.000 occur in real-time, i.e., in the order of seconds or 00:17:50.000 --> 00:17:54.000 minutes, an enzyme has to be around to make sure they happen. 00:17:54.000 --> 00:17:58.000 In the absence of such an enzyme of its intermediation, 00:17:58.000 --> 00:18:02.000 it just won't happen in real-time. Even though, in principle, 00:18:02.000 --> 00:18:06.000 it's energetically favored. So, let's just keep that very much 00:18:06.000 --> 00:18:11.000 in mind in the course of discussions that happen. And let's just begin 00:18:11.000 --> 00:18:15.000 now to look at an important energy-generating reaction in the 00:18:15.000 --> 00:18:20.000 cell which is called glycolysis. We already know the prefix glycol. 00:18:20.000 --> 00:18:24.000 Glyco refers to sugar. And lysis, L-Y-S-I-S refers to the breakdown of 00:18:24.000 --> 00:18:29.000 a certain compound. I am not going to ask you, 00:18:29.000 --> 00:18:33.000 nor is anyone else in the room going to ask you to memorize this 00:18:33.000 --> 00:18:37.000 sequence of reactions. But I'd like you to look at it and 00:18:37.000 --> 00:18:41.000 see what take-home lessons we can distill out of that, 00:18:41.000 --> 00:18:45.000 what wisdom we can learn from looking at such a complex series of 00:18:45.000 --> 00:18:49.000 reactions. Perhaps, the first thing we can learn is that 00:18:49.000 --> 00:18:53.000 when we think about biochemical reactions, we don't think of them as 00:18:53.000 --> 00:18:57.000 happening in isolation. Here I'm talking about, 00:18:57.000 --> 00:19:01.000 for example, in this case I could be talking A plus B going to C plus D, 00:19:01.000 --> 00:19:05.000 and there might be a back reaction to reach equilibrium. 00:19:05.000 --> 00:19:09.000 And we're just isolating that simple reaction from all others around it. 00:19:09.000 --> 00:19:13.000 But in the real world in living cells most reactions are parts of 00:19:13.000 --> 00:19:17.000 very long pathways where each of these steps here indicates one of 00:19:17.000 --> 00:19:21.000 the others, a step in the pathway. What we're interested in here is 00:19:21.000 --> 00:19:25.000 how glucose, which I advertised two lectures ago as being an important 00:19:25.000 --> 00:19:30.000 energy source, is actually broken down. 00:19:30.000 --> 00:19:34.000 How does the cell harvest the energy, which is inherent in glucose, 00:19:34.000 --> 00:19:38.000 in order to generate, among other things, ATP, which we've said 00:19:38.000 --> 00:19:42.000 repeatedly is the energy currency? ATP is used by hundreds of 00:19:42.000 --> 00:19:47.000 different biochemical reactions in order to make them happen. 00:19:47.000 --> 00:19:51.000 These other biochemical reactions are endergonic, 00:19:51.000 --> 00:19:55.000 they require the investment of energy, and almost invariably, 00:19:55.000 --> 00:19:59.000 but not invariably, but almost invariably the cell will grab hold 00:19:59.000 --> 00:20:04.000 of an ATP molecule, break it down usually to AMP or ADP. 00:20:04.000 --> 00:20:08.000 And then utilize the energy, which derives from breaking down ATP, 00:20:08.000 --> 00:20:13.000 it will invest that energy in an endergonic reaction, 00:20:13.000 --> 00:20:18.000 which in the otherwise would not happen. So, here we reach the idea 00:20:18.000 --> 00:20:23.000 that perhaps by investing energy in a reaction, the equilibrium is 00:20:23.000 --> 00:20:28.000 shifted. Because by investing energy, actually, 00:20:28.000 --> 00:20:33.000 the cell is able to lower the free energy state between these two. 00:20:33.000 --> 00:20:36.000 And that makes it possible for their equilibrium to be much more favored. 00:20:36.000 --> 00:20:40.000 Let's look at this glycolytic pathway. Glycolytic refers, 00:20:40.000 --> 00:20:44.000 obviously, to glycolysis. And here we start out with glucose. 00:20:44.000 --> 00:20:48.000 We're drawing it out flat rather than the circular structure we 00:20:48.000 --> 00:20:52.000 talked about last time. And let's look at what happens here, 00:20:52.000 --> 00:20:56.000 again, not because anyone wants you to memorize this, 00:20:56.000 --> 00:21:00.000 but because some of the details are in themselves very illustrative. 00:21:00.000 --> 00:21:04.000 The goal of this exercise is to create ATP for the cell, 00:21:04.000 --> 00:21:08.000 but the first step in the reaction is actually totally 00:21:08.000 --> 00:21:12.000 counterproductive. Look at the first thing that 00:21:12.000 --> 00:21:16.000 happens. The first thing that happens is that the cell invests an 00:21:16.000 --> 00:21:20.000 ATP molecule to make glucose-6-phosphate. 00:21:20.000 --> 00:21:24.000 I've advertised the goal of this is to generate ATP from ADP, 00:21:24.000 --> 00:21:28.000 adenosine diphosphate. But the first thing here, 00:21:28.000 --> 00:21:32.000 this is an endergonic reaction in which the cell invests energy to 00:21:32.000 --> 00:21:35.000 create this molecule here. So, this doesn't make sense. 00:21:35.000 --> 00:21:38.000 But ostensively it must make sense, at one level or another, because you 00:21:38.000 --> 00:21:42.000 and I, we're all here, and everybody in this room, 00:21:42.000 --> 00:21:45.000 at least this moment is metabolically active. 00:21:45.000 --> 00:21:48.000 All right. So, we've got this molecule here, 00:21:48.000 --> 00:21:51.000 glucose-6-phosphate. And this can isomerize. 00:21:51.000 --> 00:21:55.000 You see, here's glucose-6-phosphate, fructose-6-phosphate. 00:21:55.000 --> 00:21:58.000 And, the fact of the matter is, there's no oxidation reduction 00:21:58.000 --> 00:22:02.000 reaction here. It's just an isomerization. 00:22:02.000 --> 00:22:06.000 And this molecule and this molecule are virtually in the same free 00:22:06.000 --> 00:22:10.000 energy state. It happens to be the case that their profile will look 00:22:10.000 --> 00:22:14.000 very much like the one I drew you before. Their energy profile will 00:22:14.000 --> 00:22:18.000 look like this. And one needs an enzyme to lower it, 00:22:18.000 --> 00:22:22.000 but there's no energy that needs to be invested in converting one to the 00:22:22.000 --> 00:22:26.000 other because they're very similar molecules and therefore incomparable 00:22:26.000 --> 00:22:31.000 free energy states. Now look at the next step. 00:22:31.000 --> 00:22:35.000 The next step is again another ostensively totally 00:22:35.000 --> 00:22:39.000 counterproductive way of generating energy. Because, 00:22:39.000 --> 00:22:44.000 once again, ATP, the gamma-phosphate, its energy is invested in creating a 00:22:44.000 --> 00:22:48.000 dephosphorylated hexose, fructose 1, 6-diphosphate where the 00:22:48.000 --> 00:22:52.000 numbers refer obviously to the identities of the carbon. 00:22:52.000 --> 00:22:57.000 And now we have a dephosphorylated fructose molecule. 00:22:57.000 --> 00:23:01.000 And so here you can actually see what the three-dimensional, 00:23:01.000 --> 00:23:05.000 what we would imagine closer to what the three-dimensional structures of 00:23:05.000 --> 00:23:09.000 these molecules look like. And we shouldn't focus this time on 00:23:09.000 --> 00:23:13.000 whether it's this or this. For all practical purposes, 00:23:13.000 --> 00:23:17.000 let's just focus on this pathway here. And here, 00:23:17.000 --> 00:23:21.000 for the first time, what now happens is that this hexose 00:23:21.000 --> 00:23:25.000 is broken down into two trioses, i.e., into two three carbon sugars. 00:23:25.000 --> 00:23:30.000 And this is a slightly exergonic reaction. 00:23:30.000 --> 00:23:33.000 It yields, it happens without the investment of energy. 00:23:33.000 --> 00:23:37.000 And there's an enzyme, once again, that's required in order 00:23:37.000 --> 00:23:40.000 to catalyze it. But let's be really clear now. 00:23:40.000 --> 00:23:44.000 Now we have to follow the fate of two molecules. 00:23:44.000 --> 00:23:47.000 The first triose and the second triose. They have different names, 00:23:47.000 --> 00:23:51.000 but we're not going to focus on the names. One thing you notice about 00:23:51.000 --> 00:23:54.000 these trioses is that they're readily interconvertible. 00:23:54.000 --> 00:23:58.000 Once again, we can image that we have a situation that 00:23:58.000 --> 00:24:02.000 looks like this. These are flipping back and forth. 00:24:02.000 --> 00:24:06.000 And therefore, for all practical purposes from our point of view, 00:24:06.000 --> 00:24:10.000 these two are equivalent because they can be exchanged virtually 00:24:10.000 --> 00:24:14.000 instantaneously one with the other. Now, so far we've actually expended 00:24:14.000 --> 00:24:19.000 energy. We haven't harvested energy. But, keep in mind, 00:24:19.000 --> 00:24:23.000 the old economic dictum; you have to invest money to make money. 00:24:23.000 --> 00:24:27.000 And that's what's going on here. The first thing that happens is we 00:24:27.000 --> 00:24:32.000 have an oxidation reaction. What's an oxidation reaction? 00:24:32.000 --> 00:24:36.000 We want to strip some electrons, a pair of electrons off of this 00:24:36.000 --> 00:24:41.000 particular triose, the 3 carbon sugar. 00:24:41.000 --> 00:24:46.000 And by stripping off a pair of electrons we donate the electrons 00:24:46.000 --> 00:24:50.000 from NAD+ to NADH. And here these structures are given 00:24:50.000 --> 00:24:55.000 in your book. But NADH, it turns out, is the electrons are 00:24:55.000 --> 00:25:00.000 pulled away from the triose and they're used to reduce NAD+ to NADH. 00:25:00.000 --> 00:25:04.000 Keep in mind that in an oxidation reaction, one molecule that's being 00:25:04.000 --> 00:25:08.000 oxidized is deprived, is denied a pair of electrons. 00:25:08.000 --> 00:25:12.000 The other molecule that's being reduced, in this case NAD, 00:25:12.000 --> 00:25:16.000 acquires a pair of electrons. And you can focus, if you want, 00:25:16.000 --> 00:25:20.000 about the charge of these molecules, one or the other. But, keep in mind, 00:25:20.000 --> 00:25:24.000 that in these oxidation reduction reactions, whether it's plus charged 00:25:24.000 --> 00:25:28.000 or minus charged is irrelevant. The real name of the game is the 00:25:28.000 --> 00:25:31.000 electrons. Forget about the protons, 00:25:31.000 --> 00:25:35.000 whether it has a plus charge or it's neutral. The real name of the game 00:25:35.000 --> 00:25:38.000 here is that two electrons are being used to reduce this molecule to this. 00:25:38.000 --> 00:25:42.000 By the way, third mistake I forgot to tell you before, 00:25:42.000 --> 00:25:45.000 there's a double-bond in one of the pyrimidines in the book that doesn't 00:25:45.000 --> 00:25:49.000 make any sense. Whoever finds it gets a prize, 00:25:49.000 --> 00:25:53.000 but no one's figured out what the prize is yet. So, 00:25:53.000 --> 00:25:56.000 this double bond gets reduced. You see the difference between this 00:25:56.000 --> 00:26:00.000 and this over here. And this NADH, it turns out, 00:26:00.000 --> 00:26:04.000 is a high energy molecule. The street value of NADH is three 00:26:04.000 --> 00:26:10.000 ATPs, i.e., in the mitochondria NADH can be used to generate three ATPs, 00:26:10.000 --> 00:26:15.000 and that's worth something. So, NADH on its own is a high energy 00:26:15.000 --> 00:26:20.000 molecule. It can't be used for that many things, but it can be pulled 00:26:20.000 --> 00:26:26.000 into the mitochondria where it's converted to three ATPs. 00:26:26.000 --> 00:26:31.000 So, we say, well, we're starting to make some money out of this 00:26:31.000 --> 00:26:37.000 investment because we've made, in fact, these NADHs. 00:26:37.000 --> 00:26:41.000 See right here. Why do we say two NADHs? 00:26:41.000 --> 00:26:46.000 Because there are two trioses we're working with, and each one of the 00:26:46.000 --> 00:26:50.000 trioses gives you an NADH. So, everything that's going on 00:26:50.000 --> 00:26:55.000 after this, starting from the top here, is now double because we're 00:26:55.000 --> 00:26:59.000 looking at the parallel behaviors of two identical three carbon sugars. 00:26:59.000 --> 00:27:04.000 So, here we've so far generated, in principle, six ATPs. 00:27:04.000 --> 00:27:08.000 How much did we invest already up to this point? Two. 00:27:08.000 --> 00:27:13.000 We invested two but we harvested six. Already we're starting to make 00:27:13.000 --> 00:27:18.000 a little money because I told you the street value of an NADH is three 00:27:18.000 --> 00:27:23.000 ATPs on the black market. OK, so what happens next? 00:27:23.000 --> 00:27:27.000 Next is another good thing. Each of the trioses, one can 00:27:27.000 --> 00:27:32.000 actually cause each of the trioses to generate an ATP molecule 00:27:32.000 --> 00:27:36.000 from an ADP. What happens here? 00:27:36.000 --> 00:27:40.000 It turns out that this phosphate over here is actually in a pretty 00:27:40.000 --> 00:27:43.000 high energy state, in no small part because of electron 00:27:43.000 --> 00:27:47.000 negative-negative repulsion. And by stripping this phosphate off 00:27:47.000 --> 00:27:51.000 this high energy phosphate stripped off of this molecule here, 00:27:51.000 --> 00:27:54.000 whose name we will ignore, allows us to phosphorylate an ATP. 00:27:54.000 --> 00:27:58.000 And since there are two trioses being converted, we're 00:27:58.000 --> 00:28:02.000 going to get two ATPs. So, in effect, 00:28:02.000 --> 00:28:06.000 now we're actually ahead. We started out investing two, 00:28:06.000 --> 00:28:10.000 we got six back from the NADHs, and we're getting two back here. 00:28:10.000 --> 00:28:14.000 So, we've made two ATPs. This is a good thing. Keep in mind, 00:28:14.000 --> 00:28:19.000 ADP is lower energy, ATP is a high energy. Once again, 00:28:19.000 --> 00:28:23.000 we have an isomerization where these two molecules are at comparable 00:28:23.000 --> 00:28:27.000 states here and here, where the phosphate just jumps over 00:28:27.000 --> 00:28:32.000 to this state. And this hydrolyzes spontaneously 00:28:32.000 --> 00:28:37.000 and we get this molecule right over here, phosphoenolpyruvate at the end. 00:28:37.000 --> 00:28:42.000 And, once again, we harvest two ATPs, 00:28:42.000 --> 00:28:47.000 one ATP from each of the trioses. And we end up, at the end of this 00:28:47.000 --> 00:28:52.000 reaction, with pyruvate. And you'll say this is terrific 00:28:52.000 --> 00:28:57.000 because we invested two ATPs, we harvested four, plus we got six 00:28:57.000 --> 00:29:03.000 from the NADHs, right? Two NADHs, each NADH gives us three 00:29:03.000 --> 00:29:11.000 each, so let's do the arithmetic. Let's do the balance sheet. We 00:29:11.000 --> 00:29:18.000 invested to begin with, with the one glucose, we invested 00:29:18.000 --> 00:29:26.000 two ATPs. That was early on. Then the return was first two NADHs, 00:29:26.000 --> 00:29:33.000 which I've told you equals six ATPs. Because an NADH is worth three ATPs. 00:29:33.000 --> 00:29:39.000 This is so far good. And now subsequently we've made four ATPs so 00:29:39.000 --> 00:29:46.000 that the net yield looks pretty useful. Six plus four is ten minus 00:29:46.000 --> 00:29:52.000 two, a profit of eight ATPs from one glucose molecule. 00:29:52.000 --> 00:29:59.000 This is terrific you may say, but there's a rub. 00:29:59.000 --> 00:30:04.000 There's a catch. If glycolysis is occurring in the 00:30:04.000 --> 00:30:10.000 absence of oxygen, if that happens, then we have a 00:30:10.000 --> 00:30:15.000 problem here, because the only way that these NADHs can generate ATP is 00:30:15.000 --> 00:30:21.000 if there is oxygen around to take these electron pairs and use them to 00:30:21.000 --> 00:30:27.000 reduce an oxygen molecule. That is, by the way, part of the 00:30:27.000 --> 00:30:32.000 reason we breathe. Keep in mind that when you generate 00:30:32.000 --> 00:30:36.000 an NADH from an NAD molecule, you need to regenerate the NAD. 00:30:36.000 --> 00:30:40.000 You can't just accumulate more and more NADHs. You need to regenerate 00:30:40.000 --> 00:30:44.000 the NAD. And, therefore, this NADH, 00:30:44.000 --> 00:30:48.000 with their electron pairs, the electron pairs have some to be 00:30:48.000 --> 00:30:52.000 disposed of. You have to regenerate NAD. You can't just make more and 00:30:52.000 --> 00:30:56.000 more and more of this. So, how do cells get rid of it? 00:30:56.000 --> 00:31:00.000 Well, how they get rid of it is simple. 00:31:00.000 --> 00:31:05.000 You take the electron pairs and you slap them onto oxygen, 00:31:05.000 --> 00:31:10.000 and that's really called combustion. And you get a lot of energy out of 00:31:10.000 --> 00:31:16.000 that. But what happens if all of this is occurring anaerobically? 00:31:16.000 --> 00:31:21.000 Anaerobically means the reaction is occurring in the absence of oxygen. 00:31:21.000 --> 00:31:27.000 Well, if you have a yeast that's growing 14 feet underground, 00:31:27.000 --> 00:31:31.000 this is happening anaerobically. If you have a yeast that's 00:31:31.000 --> 00:31:35.000 fermenting in a big keg to make wine or beer, it's also probably 00:31:35.000 --> 00:31:39.000 happening anaerobically. If you start running in a 100 yard 00:31:39.000 --> 00:31:43.000 sprint, or let's say you had to run a mile, then initially there's 00:31:43.000 --> 00:31:47.000 enough oxygen, there's a lot of oxygen around to 00:31:47.000 --> 00:31:51.000 allow you to get rid of these NADHs and dump the electrons that they 00:31:51.000 --> 00:31:55.000 have acquired onto the oxygen molecule. And that's fine. 00:31:55.000 --> 00:31:59.000 That's worth a lot because, in effect, what you're doing is 00:31:59.000 --> 00:32:03.000 you're taking oxygen and hydrogen and you're combusting them together. 00:32:03.000 --> 00:32:07.000 And that's great. But as you start running down the 00:32:07.000 --> 00:32:12.000 street, soon the oxygen supply to your muscles is going to run out, 00:32:12.000 --> 00:32:16.000 and soon a lot of the energy production in your muscles happens 00:32:16.000 --> 00:32:21.000 anaerobically. Why? Because you can't get oxygen 00:32:21.000 --> 00:32:26.000 quickly enough to your muscles, and therefore, for a period of time, 00:32:26.000 --> 00:32:30.000 you start feeling that burning sensation in your muscles because 00:32:30.000 --> 00:32:35.000 oxidation of NADH isn't happening. And these NADHs instead are 00:32:35.000 --> 00:32:40.000 regenerated by another way. How are they regenerated? The 00:32:40.000 --> 00:32:45.000 electron pairs of the NADHs, must be, are dumped back onto this 00:32:45.000 --> 00:32:50.000 molecule right here, pyruvate. They're not used to make 00:32:50.000 --> 00:32:55.000 ATP because they can't be used to make ATP because there's no oxygen 00:32:55.000 --> 00:33:01.000 around to accept the electron pairs that these NADHs have acquired. 00:33:01.000 --> 00:33:05.000 And so, what happens with these valuable NADHs? 00:33:05.000 --> 00:33:09.000 Under anaerobic conditions this doesn't happen. 00:33:09.000 --> 00:33:14.000 These NADHs are used instead, their electrons are donated to our 00:33:14.000 --> 00:33:18.000 friend pyruvate here, these three carbon sugar. 00:33:18.000 --> 00:33:23.000 And what happens, when they are donated back to the pyruvate, 00:33:23.000 --> 00:33:27.000 in order to regenerate NAD you need more NAD to pick up to use later in 00:33:27.000 --> 00:33:32.000 the reaction, to use over again in another reaction. 00:33:32.000 --> 00:33:36.000 When you donate the electrons from NADH back onto pyruvate, 00:33:36.000 --> 00:33:41.000 what happens? You get lactic acid. Lactic acid is what makes your 00:33:41.000 --> 00:33:45.000 muscles burn when you're running very quickly and you can't get 00:33:45.000 --> 00:33:50.000 enough oxygen into them to begin to burn up the NADH. 00:33:50.000 --> 00:33:55.000 So, instead of using NADH to generate ATP, it's diverted to make 00:33:55.000 --> 00:34:00.000 lactic acid. That's in one sense good because you regenerate NAD. 00:34:00.000 --> 00:34:04.000 Why do you need to regenerate NAD? Because you need a lot of NAD 00:34:04.000 --> 00:34:09.000 around for the earlier steps in the reaction. Keep in mind, 00:34:09.000 --> 00:34:13.000 early in the reaction you need NAD here. If you don't regenerate it 00:34:13.000 --> 00:34:18.000 then glycolysis grinds to a halt. So, even though you make NADH and 00:34:18.000 --> 00:34:23.000 it's a good thing in principle, in practice it has to be recycled. 00:34:23.000 --> 00:34:27.000 And if it's not recycled to make more new NAD to allow this step to 00:34:27.000 --> 00:34:32.000 happen then the whole glycolytic reaction will shut down 00:34:32.000 --> 00:34:37.000 and you're in a mess. However, sadly, 00:34:37.000 --> 00:34:41.000 in the absence of oxygen, the only way to recycle this is to 00:34:41.000 --> 00:34:46.000 dump these electrons not onto oxygen which is energy rich, 00:34:46.000 --> 00:34:50.000 it's dump them back onto pyruvic acid creating lactic acid. 00:34:50.000 --> 00:34:55.000 So, you reduce this bond right here. So, you get CH, 00:34:55.000 --> 00:35:00.000 COH. This bond right here is reduced and you get lactic acid. 00:35:00.000 --> 00:35:04.000 So, instead of a carbonyl bond here you have CH and COH right here, 00:35:04.000 --> 00:35:09.000 that's a reduction reaction. And now you're able to regenerate the 00:35:09.000 --> 00:35:13.000 NAD. And now you say that's a great thing. But, keep in mind, 00:35:13.000 --> 00:35:18.000 that now the entire glycolytic reaction, how much is our net profit 00:35:18.000 --> 00:35:23.000 now? Before I was gloating about the fact that we made eight ATPs, 00:35:23.000 --> 00:35:27.000 we netted eight ATPs out of this. What are we back down to now? 00:35:27.000 --> 00:35:32.000 What's the whole net yield now? Well, the TAs can't answer. 00:35:32.000 --> 00:35:36.000 It's two, because we invested two and we got out four. 00:35:36.000 --> 00:35:40.000 And it's only two. Now, why is this so interesting? 00:35:40.000 --> 00:35:45.000 Well, until about six hundred million years ago there wasn't that 00:35:45.000 --> 00:35:49.000 much oxygen in the atmosphere. And in the absence of oxygen this 00:35:49.000 --> 00:35:54.000 is almost the only reaction that could be used in order to generate 00:35:54.000 --> 00:35:58.000 energy. And about six hundred million years ago more and more 00:35:58.000 --> 00:36:03.000 oxygen from photosynthesis became dumped into the atmosphere. 00:36:03.000 --> 00:36:08.000 And soon oxygen became available to organisms like our ancestors. 00:36:08.000 --> 00:36:13.000 And then they could actually begin to recycle this NADH in a much more 00:36:13.000 --> 00:36:18.000 productive way. And as a consequence what happened, 00:36:18.000 --> 00:36:23.000 instead of having glycolysis yielding two, we could go up to this 00:36:23.000 --> 00:36:28.000 theoretical eight because the NADHs could now deposit their electrons on 00:36:28.000 --> 00:36:33.000 oxygen, which is much more profitable. 00:36:33.000 --> 00:36:39.000 In fact, I've just told you now that in the absence of oxygen you can 00:36:39.000 --> 00:36:45.000 only make two ATPs. I will tell you, without providing 00:36:45.000 --> 00:36:51.000 it to you, that in the presence of oxygen you can make 34 ATPs. 00:36:51.000 --> 00:36:57.000 And 34 is, we can agree, much better than two in the 00:36:57.000 --> 00:37:01.000 presence of oxygen. Higher life forms could not evolve 00:37:01.000 --> 00:37:05.000 until this much more effective way of generating energy became 00:37:05.000 --> 00:37:09.000 available. And, therefore, if our ancestors who 00:37:09.000 --> 00:37:13.000 lived longer than six hundred million years ago were very sluggish 00:37:13.000 --> 00:37:17.000 and they weren't very smart, the reason why they were sluggish 00:37:17.000 --> 00:37:21.000 and they weren't very smart is because they couldn't generate the 00:37:21.000 --> 00:37:25.000 energy that was required to efficiently drive metabolism. 00:37:25.000 --> 00:37:29.000 The metabolism, anaerobic metabolism, i. 00:37:29.000 --> 00:37:33.000 ., occurring in the absence of energy, is extremely inefficient. 00:37:33.000 --> 00:37:39.000 It just doesn't happen very well. Now, what actually happens if we 00:37:39.000 --> 00:37:45.000 have oxygen around? Well, what happens is something 00:37:45.000 --> 00:37:51.000 like this. We take the pyruvate, which is the product of glycolysis 00:37:51.000 --> 00:37:57.000 and which is this much more primitive pathway, 00:37:57.000 --> 00:38:02.000 and we dump it into the mitochondria. And now we generate through this 00:38:02.000 --> 00:38:08.000 cycle here, which I'm not asking you memorize, please, 00:38:08.000 --> 00:38:13.000 don't do that. We generate the reactions which go from here and get 00:38:13.000 --> 00:38:19.000 us up to this 34 ATP yield per glucose. And the essence of the 00:38:19.000 --> 00:38:24.000 citric acid cycle, which happens in the mitochondria, 00:38:24.000 --> 00:38:30.000 keep in mind that the mitochondria look like this. 00:38:30.000 --> 00:38:34.000 Keep in mind that the mitochondrion are the decedents of bacteria which 00:38:34.000 --> 00:38:39.000 parasitized the cytoplasm of cells probably 1.5 billion years ago. 00:38:39.000 --> 00:38:43.000 But if we now look at what happens in the mitochondrion, 00:38:43.000 --> 00:38:48.000 the pyruvate that we generated in the cytosol, in the soluble part of 00:38:48.000 --> 00:38:53.000 the cytoplasm is now pumped into the mitochondria, and there's a whole 00:38:53.000 --> 00:38:57.000 series of reactions that go on here, which takes this three-carbon 00:38:57.000 --> 00:39:02.000 sugar. The first thing that happens is that 00:39:02.000 --> 00:39:06.000 carbon is boiled off. Carbon dioxide, that's released. 00:39:06.000 --> 00:39:10.000 Now we're down to a two carbon sugar. And then this two carbon 00:39:10.000 --> 00:39:14.000 sugar is added to a four carbon sugar and progressively oxidized. 00:39:14.000 --> 00:39:19.000 And as it's oxidized what's spun off? Well, what's spun off is, 00:39:19.000 --> 00:39:23.000 for example, there's NADH which is spun off, there's ATP. 00:39:23.000 --> 00:39:27.000 See, there's an NADH which is spun off. Here's an NADH that's spun off. 00:39:27.000 --> 00:39:32.000 Here is a cousin of NADH. It's called FADH which, 00:39:32.000 --> 00:39:36.000 once again, generates a high energy molecule. Once again, 00:39:36.000 --> 00:39:41.000 the carbon molecules are oxidized, electrons are stripped away and used 00:39:41.000 --> 00:39:45.000 to create these high energy molecules, FADH and NADH. 00:39:45.000 --> 00:39:49.000 By the way, FADH, a cousin of NADH, is only worth two ATPs on the open 00:39:49.000 --> 00:39:54.000 market. Whereas, NADH, as I've told you repeatedly, 00:39:54.000 --> 00:39:58.000 is worth three. And by the time we add up all of the NADHs that have 00:39:58.000 --> 00:40:03.000 been generated by this cycling and the carbon dioxides that are 00:40:03.000 --> 00:40:07.000 releases, at the end of this cycle here we start with two carbons, 00:40:07.000 --> 00:40:12.000 add it to four and we get a six carbon molecule. 00:40:12.000 --> 00:40:16.000 We spew off some carbon dioxides here and go back to four carbon 00:40:16.000 --> 00:40:20.000 sugar. Add another two, go up to six carbons. Go around 00:40:20.000 --> 00:40:24.000 again, spin around the wheel. And each time we do that we 00:40:24.000 --> 00:40:28.000 generate a lot of NADHs, we generate a lot of FADHs, 00:40:28.000 --> 00:40:33.000 and we generate a lot of ATP. In all cases, these are highly 00:40:33.000 --> 00:40:39.000 profitable reactions simply because the NADHs and the FADHs can be used 00:40:39.000 --> 00:40:45.000 in the mitochondrion to generate ATP. So, let's look at the energy 00:40:45.000 --> 00:40:51.000 profile of the entire thing. Put it all together. This is where 00:40:51.000 --> 00:40:57.000 we started out at the beginning, and this is the end of glycolysis, 00:40:57.000 --> 00:41:02.000 OK? So, now we're adding up the energy 00:41:02.000 --> 00:41:06.000 profiles of the whole sequence of reactions that constituted 00:41:06.000 --> 00:41:10.000 glycolysis, which begins up here and ends right here because pyruvate, 00:41:10.000 --> 00:41:14.000 as you will recall, is the product of glycolysis, 00:41:14.000 --> 00:41:18.000 the first step. The Krebs Cycle happens, 00:41:18.000 --> 00:41:22.000 or sometimes it's called the Citric Acid Cycle. So, 00:41:22.000 --> 00:41:26.000 let's just get these words straight. Citric Acid Cycle because it 00:41:26.000 --> 00:41:30.000 happens to be one of the cycles, or it's sometimes called the Krebs 00:41:30.000 --> 00:41:35.000 Cycle after the person who really discovered it, Krebs. 00:41:35.000 --> 00:41:39.000 The Krebs Cycle begins here. You see how the shading changes 00:41:39.000 --> 00:41:43.000 from pyruvate. And here we go all the way down 00:41:43.000 --> 00:41:47.000 there. And let's now look at what happens in terms of energy exchange. 00:41:47.000 --> 00:41:51.000 Recall that early on we needed to invest ATPs to kick up the energy 00:41:51.000 --> 00:41:55.000 state up to here. We invested ATPs at this stage 00:41:55.000 --> 00:42:00.000 right here, and then we began to get some back. 00:42:00.000 --> 00:42:04.000 We got these two NADHs, one NADH coming from each of the 00:42:04.000 --> 00:42:08.000 three carbon sugars. We got some more ATPs here and we 00:42:08.000 --> 00:42:12.000 got some more ATPs here, but these NADHs could not be used 00:42:12.000 --> 00:42:17.000 productively for generating ATP in the absence of oxygen, 00:42:17.000 --> 00:42:21.000 but in the presence of oxygen now we can begin to use these very 00:42:21.000 --> 00:42:25.000 profitably. Each of these makes three ATPs and each of these, 00:42:25.000 --> 00:42:30.000 obviously, makes ATPs. And then let's look at what happens 00:42:30.000 --> 00:42:34.000 in the mitochondrion. Keep in mind here's the borderline 00:42:34.000 --> 00:42:38.000 between the cytosol, the cytoplasm and the mitochondrion. 00:42:38.000 --> 00:42:42.000 Here is where the oxygen is actually used and here we generate 00:42:42.000 --> 00:42:46.000 all these NADHs here, here and here, FADHs. And I keep 00:42:46.000 --> 00:42:50.000 saying, and it's still true, just in spite of the fact I keep 00:42:50.000 --> 00:42:54.000 saying it, that these NADHs can be converted to ATPs, 00:42:54.000 --> 00:42:58.000 and the ATPs can then be diffused, transmitted throughout the entire 00:42:58.000 --> 00:43:02.000 cell where they're then used invested in endergonic reactions. 00:43:02.000 --> 00:43:06.000 Here we see all these NADHs. And look at the overall change in 00:43:06.000 --> 00:43:11.000 free energy. The initial steps in glycolysis didn't really take 00:43:11.000 --> 00:43:15.000 advantage. Glucose has inherent in it almost 680 kilocalories per mole 00:43:15.000 --> 00:43:20.000 of energy. It's pretty high up here. But by the time we get from here 00:43:20.000 --> 00:43:25.000 down to here, there's an enormous release of energy, 00:43:25.000 --> 00:43:30.000 it's harvested in the form of these molecules which are then reinvested. 00:43:30.000 --> 00:43:34.000 In the absence of oxygen, this entire procedure can only go 00:43:34.000 --> 00:43:38.000 from here down to here. And a lot of this drop from six to 00:43:38.000 --> 00:43:42.000 seven is futile because we have to reinvest this NADH. 00:43:42.000 --> 00:43:47.000 These cannot be used, actually, to generate more ATPs, 00:43:47.000 --> 00:43:51.000 as I've said repeatedly. So, this means in the end that we can 00:43:51.000 --> 00:43:55.000 generate an enormous amount of energy in the form of these 00:43:55.000 --> 00:44:01.000 coupled reactions. Having said that, 00:44:01.000 --> 00:44:08.000 let's actually look at what happens inside of the mitochondria. 00:44:08.000 --> 00:44:15.000 Inside of the mitochondria there are actually different physical 00:44:15.000 --> 00:44:22.000 compartments. See the blue space there, the intermembrane space, 00:44:22.000 --> 00:44:30.000 the blue spaces there? The matrix is on the inside. 00:44:30.000 --> 00:44:35.000 The intermembrane space is between the two, the inner and the outer 00:44:35.000 --> 00:44:40.000 membrane, and outside is the cytoplasm. The outer membrane, 00:44:40.000 --> 00:44:45.000 the inner membrane, in between it. So, look what happens, actually, in 00:44:45.000 --> 00:44:50.000 the mitochondrion. Those NADHs are used to pump 00:44:50.000 --> 00:44:55.000 protons from the inner space of the mitochondrion into the intermembrane 00:44:55.000 --> 00:45:00.000 space. I'm not showing you that happening. 00:45:00.000 --> 00:45:05.000 But you'll have to take it on my word. So, protons pictured here are 00:45:05.000 --> 00:45:10.000 extracted from NADH and FADH, and they're used to pump protons out 00:45:10.000 --> 00:45:15.000 here. And, therefore, protons are moved from here to here. 00:45:15.000 --> 00:45:20.000 Obviously, when you pump protons out the pH gets lower on the outside 00:45:20.000 --> 00:45:25.000 than it does on the inside, and because there's a gradient, 00:45:25.000 --> 00:45:30.000 there's a higher concentration of protons here than on the inside. 00:45:30.000 --> 00:45:34.000 The protons begin to accumulate outside here in the intermembrane 00:45:34.000 --> 00:45:39.000 space. Are they in the cytoplasm? No. They're in the space between 00:45:39.000 --> 00:45:44.000 the inner and the outer membrane. You start to accumulate in this 00:45:44.000 --> 00:45:49.000 blue space lots of protons. And this pumping of protons into 00:45:49.000 --> 00:45:54.000 the space between the two membranes requires energy, 00:45:54.000 --> 00:45:59.000 and the energy comes from our friends NADH and FADH 00:45:59.000 --> 00:46:04.000 as it turns out. They are responsible for causing 00:46:04.000 --> 00:46:08.000 this accumulation of protons in the space between the inner and the 00:46:08.000 --> 00:46:12.000 outer membrane. So, now we get lots of protons out 00:46:12.000 --> 00:46:16.000 there. And what happens now, the protons like to flow back in 00:46:16.000 --> 00:46:20.000 because there is a higher concentration here as they are 00:46:20.000 --> 00:46:24.000 inside the space that's called the mitochondrial matrix, 00:46:24.000 --> 00:46:29.000 on the inside of the mitochondrion. So, what happens? 00:46:29.000 --> 00:46:32.000 Here, yet another Nobel Prize winning discovery is the discovery 00:46:32.000 --> 00:46:36.000 of a very interesting molecule, or complex of proteins I should say, 00:46:36.000 --> 00:46:40.000 that looks in three-dimensions roughly like this. 00:46:40.000 --> 00:46:44.000 And what this complex does is as the protons flow through the inner 00:46:44.000 --> 00:46:48.000 channel here, they're moving down an energy gradient. 00:46:48.000 --> 00:46:52.000 They're going from a state of high concentration to a state of low 00:46:52.000 --> 00:46:56.000 concentration. What that does, 00:46:56.000 --> 00:47:00.000 that diffusional pressure actually yields energy. 00:47:00.000 --> 00:47:05.000 And this complex right here harvests that energy in order to convert ADP 00:47:05.000 --> 00:47:10.000 into ATP. So, when I talk about NADH as being 00:47:10.000 --> 00:47:15.000 worth, each of them being worth three ATPs, what I'm really talking 00:47:15.000 --> 00:47:20.000 about is the fact that NADHs can be used to pump protons in the 00:47:20.000 --> 00:47:25.000 mitochondria outside here, and these protons can then be used, 00:47:25.000 --> 00:47:31.000 can then be pumped, can then flow in this way through this proton pump, 00:47:31.000 --> 00:47:36.000 which then uses ADP in the inner cavity of the mitochondria 00:47:36.000 --> 00:47:40.000 to create ATP. And here we get finally the 00:47:40.000 --> 00:47:44.000 conversion of ADP into ATP. We can realize, finally, this much 00:47:44.000 --> 00:47:48.000 promised benefit. And then these ATP molecules are 00:47:48.000 --> 00:47:52.000 exported from the mitochondria throughout the entire cell and used 00:47:52.000 --> 00:47:56.000 to drive many reactions. We've already encountered one 00:47:56.000 --> 00:48:00.000 important set of reactions, and those reactions are the 00:48:00.000 --> 00:48:04.000 polymerization of nucleic acids. Now, one final point I want to make 00:48:04.000 --> 00:48:08.000 is the following. We've just talked about metabolic, 00:48:08.000 --> 00:48:12.000 we've talked about the pathway of energy production in the cell. 00:48:12.000 --> 00:48:16.000 And you might have had the illusion, for a brief instant, 00:48:16.000 --> 00:48:20.000 that those are all, that's the sum of all the biochemical reactions in 00:48:20.000 --> 00:48:24.000 the cell. But, in fact, if we plot out all the 00:48:24.000 --> 00:48:28.000 biochemical reactions in the cell, they're much more complicated. Here 00:48:28.000 --> 00:48:31.000 is the glycolytic pathway. You see it right down here where 00:48:31.000 --> 00:48:35.000 nothing is named? Here is the Krebs Cycle right here. 00:48:35.000 --> 00:48:39.000 And we're not even talking about energy here. And as molecules move 00:48:39.000 --> 00:48:43.000 down this pathway from here to here to here to here, 00:48:43.000 --> 00:48:46.000 some of these molecules are diverted for other applications. 00:48:46.000 --> 00:48:50.000 Not for energy production but for other applications. 00:48:50.000 --> 00:48:54.000 And what happens out here, they are converted through a series 00:48:54.000 --> 00:48:58.000 of complex biochemical steps into other essential biological 00:48:58.000 --> 00:49:02.000 molecules. What do I mean by that? 00:49:02.000 --> 00:49:06.000 If you give E. coli, a bacterium, you give it a simple carbon source 00:49:06.000 --> 00:49:10.000 like glucose and you give it phosphate and you give it a simple 00:49:10.000 --> 00:49:14.000 nitrogen source like ammonium acetate or something, 00:49:14.000 --> 00:49:19.000 E. coli can, from those simple atoms generate all the amino acids, 00:49:19.000 --> 00:49:23.000 can generate the purines and the pyrimidines, can generate all kinds 00:49:23.000 --> 00:49:27.000 of different complex biological molecules just from those 00:49:27.000 --> 00:49:33.000 simple building blocks. And so, the process of biosynthesis 00:49:33.000 --> 00:49:40.000 involves not only the creation of macromolecules, 00:49:40.000 --> 00:49:47.000 these steps of what are called intermediary metabolism are used to 00:49:47.000 --> 00:49:54.000 synthesize all the other biochemical entities that one needs to make a 00:49:54.000 --> 00:50:01.000 cell. They're used to synthesize purines and pyrimidines. 00:50:01.000 --> 00:50:05.000 They're used to synthesize lipids, they're used to synthesize amino 00:50:05.000 --> 00:50:09.000 acids, and they're used to synthesize literally hundreds of 00:50:09.000 --> 00:50:13.000 other compounds. And when we see this chart like 00:50:13.000 --> 00:50:18.000 this, and nobody on the face of the planet has ever memorized this chart, 00:50:18.000 --> 00:50:22.000 each one of these steps, going from one molecule to the next, 00:50:22.000 --> 00:50:26.000 represents another biochemical reaction. And the vast majority of 00:50:26.000 --> 00:50:31.000 these biochemical reactions going from A to B to C to D. 00:50:31.000 --> 00:50:35.000 Each one of these steps requires the intervention of an enzyme, 00:50:35.000 --> 00:50:39.000 a catalyst that is specialized for that particular step. 00:50:39.000 --> 00:50:44.000 So, this begins to give you an appreciation of how many distinct 00:50:44.000 --> 00:50:48.000 biochemical steps one needs in a cell. The numbers probably to make 00:50:48.000 --> 00:50:53.000 a simple cell, you probably need about a thousand 00:50:53.000 --> 00:50:57.000 distinct biochemical reactions, each of one of which requires the 00:50:57.000 --> 00:51:02.000 involvement of an enzyme. And many of these steps, 00:51:02.000 --> 00:51:06.000 importantly, many of these biochemical steps are endergonic 00:51:06.000 --> 00:51:11.000 reactions. Where do they get the energy for driving these reactions 00:51:11.000 --> 00:51:15.000 forward if they're endergonic? ATP. So, the ATP from the energy 00:51:15.000 --> 00:51:20.000 generating furnace down here is the then spread throughout the cell to 00:51:20.000 --> 00:51:25.000 power all of these energy consuming reactions. Have a great weekend.