1 00:00:01,000 --> 00:00:04,000 Good morning, class. 2 00:00:04,000 --> 00:00:08,000 I just wanted to spend the first couple minutes clearing up three 3 00:00:08,000 --> 00:00:12,000 issues. None is a major conceptual issue, but we like to focus on 4 00:00:12,000 --> 00:00:17,000 details and get them right, get them correct here as well. 5 00:00:17,000 --> 00:00:21,000 Firstly, I misdrew a reaction last time that described why RNA is 6 00:00:21,000 --> 00:00:26,000 alkali labile, i.e., if we have high pH we call 7 00:00:26,000 --> 00:00:30,000 that an alkali pH, or an alkaline pH, actually, 8 00:00:30,000 --> 00:00:35,000 to use the adjective. And we said that hydroxyl groups can 9 00:00:35,000 --> 00:00:39,000 cause the cleavage of the phosphodiester bonds of RNA but not 10 00:00:39,000 --> 00:00:44,000 DNA. And the way I described that happening is that the alkali group 11 00:00:44,000 --> 00:00:49,000 causes the formation of this five-membered ring right here, 12 00:00:49,000 --> 00:00:53,000 two carbons, two oxygens and a phosphate. And that resolves 13 00:00:53,000 --> 00:00:58,000 eventually to this where there's no longer any connection with the 14 00:00:58,000 --> 00:01:03,000 ribonucleoside monophosphate below. And I drew it like this, 15 00:01:03,000 --> 00:01:07,000 without an oxygen, and that's a no-no because, 16 00:01:07,000 --> 00:01:12,000 in fact, in truth, and as many of you picked up, 17 00:01:12,000 --> 00:01:16,000 this reverts to a two prime hydroxyl. So, please note there's a mistake 18 00:01:16,000 --> 00:01:21,000 there. There's also a couple other mistakes. For example, 19 00:01:21,000 --> 00:01:25,000 in the textbook it gives you the impression that when you polymerize 20 00:01:25,000 --> 00:01:30,000 nucleic acids you use a monophosphate to do so. 21 00:01:30,000 --> 00:01:34,000 And, if you listened to my lecture last time, that doesn't make any 22 00:01:34,000 --> 00:01:38,000 sense, because you need to invest the energy of a triphosphate in 23 00:01:38,000 --> 00:01:42,000 order to create enough energy to generate enough energy for the 24 00:01:42,000 --> 00:01:46,000 polymerization. The textbook is incorrect there. 25 00:01:46,000 --> 00:01:51,000 Textbooks are written by people, for better or worse, 26 00:01:51,000 --> 00:01:55,000 and as such, like everything else, they are a mortal and fallible. So, 27 00:01:55,000 --> 00:01:59,000 the truth of the matter is, when you're polymerizing DNA or RNA you 28 00:01:59,000 --> 00:02:03,000 need one of the four ribonucleoside or deoxyribonucleoside triphosphates 29 00:02:03,000 --> 00:02:08,000 in order to donate the energy that makes possible this polymerization. 30 00:02:08,000 --> 00:02:12,000 And please note that is a mistake in the book. Recall, 31 00:02:12,000 --> 00:02:16,000 as I said last time, the fact that ATP is really the 32 00:02:16,000 --> 00:02:20,000 currency of energy in the cell, and that its energy is stored and 33 00:02:20,000 --> 00:02:24,000 coiled up in this pent up spring where the mutual electrostatic 34 00:02:24,000 --> 00:02:28,000 repulsion between the three negatively charged phosphates 35 00:02:28,000 --> 00:02:33,000 carries with it enormous potential energy. 36 00:02:33,000 --> 00:02:36,000 And some of that potential energy can be realized, 37 00:02:36,000 --> 00:02:40,000 during the synthesis of polymerization of nucleic acids by 38 00:02:40,000 --> 00:02:43,000 cleaving this bond here. One can also generate potential 39 00:02:43,000 --> 00:02:47,000 energy by cleaving this bond here. This is the alpha, the beta and the 40 00:02:47,000 --> 00:02:51,000 gamma-phosphate. And cleavage of either can create 41 00:02:51,000 --> 00:02:54,000 substantial energy, which in turn can, as we'll indicate 42 00:02:54,000 --> 00:02:58,000 shortly, be invested in other reactions. The reaction 43 00:02:58,000 --> 00:03:01,000 of polymerization. A second point I'd like to make to 44 00:03:01,000 --> 00:03:05,000 you is the following, and you'd say it's kind of 45 00:03:05,000 --> 00:03:09,000 coincidence. The currency of energy in the cell is ATP, 46 00:03:09,000 --> 00:03:13,000 adenosine triphosphate, we see its structure here, 47 00:03:13,000 --> 00:03:17,000 and this happens to be one of the four precursors of the RNA. 48 00:03:17,000 --> 00:03:21,000 So, the same molecule is used in these two different ostensively 49 00:03:21,000 --> 00:03:25,000 unrelated applications. One, to polymerize to make RNA 50 00:03:25,000 --> 00:03:29,000 where genetic information is stored and conveyed. 51 00:03:29,000 --> 00:03:33,000 Or, alternatively it's used here in this context in order to serve as a 52 00:03:33,000 --> 00:03:37,000 currency for energy. High energy as ATP. ADP with a 53 00:03:37,000 --> 00:03:41,000 little lower energy. AMP monophosphate with even lower 54 00:03:41,000 --> 00:03:46,000 energy. And you might ask yourself, scratch your head and say why is the 55 00:03:46,000 --> 00:03:50,000 same molecule used for these two different things? 56 00:03:50,000 --> 00:03:54,000 In fact, there are yet other applications of these 57 00:03:54,000 --> 00:03:58,000 ribonucleosides which also seem to be unrelated to the storage or the 58 00:03:58,000 --> 00:04:02,000 conveyance of genetic information. And it is believed, 59 00:04:02,000 --> 00:04:06,000 probably correctly, that the reason why the same molecule is used for 60 00:04:06,000 --> 00:04:10,000 these totally different applications is that early in the evolution of 61 00:04:10,000 --> 00:04:14,000 life on this planet there really were a rather small number of 62 00:04:14,000 --> 00:04:18,000 biological molecules that existed. Indeed, as we'll mention again 63 00:04:18,000 --> 00:04:22,000 later, it's probably the case that the first organisms didn't use DNA 64 00:04:22,000 --> 00:04:26,000 as genomes. It's an article of faith with us that one stores 65 00:04:26,000 --> 00:04:30,000 genetic information in DNA molecules. 66 00:04:30,000 --> 00:04:33,000 And I implied that quite explicitly last time. But, 67 00:04:33,000 --> 00:04:37,000 the fact of the matter is, it's probably the case that the 68 00:04:37,000 --> 00:04:41,000 first organism, the first pre-cellular life forms 69 00:04:41,000 --> 00:04:44,000 used RNA as the genetic material, RNA to store things, replicating RNA 70 00:04:44,000 --> 00:04:48,000 via double-stranded RNA molecules as a way of archiving genetic 71 00:04:48,000 --> 00:04:52,000 information. And only later during the evolution of life on this planet, 72 00:04:52,000 --> 00:04:56,000 when that later was we can't tell, but it could have been a hundred or 73 00:04:56,000 --> 00:05:00,000 two hundred years later. Obviously, if we're talking about 74 00:05:00,000 --> 00:05:04,000 the origin of life as between 3. and 3.5 billion years ago, we can't 75 00:05:04,000 --> 00:05:09,000 really localize that in time very well, but only later was DNA 76 00:05:09,000 --> 00:05:13,000 assigned the job of storing, in a stable fashion, genetic 77 00:05:13,000 --> 00:05:18,000 information. And as a consequence, we come to realize as well yet 78 00:05:18,000 --> 00:05:23,000 another discovery, which is that all the catalysts that 79 00:05:23,000 --> 00:05:27,000 we're going to talk about today, the enzymes as we call them, almost 80 00:05:27,000 --> 00:05:32,000 all modern-day enzymes are proteins. And we talked about them briefly 81 00:05:32,000 --> 00:05:36,000 before. But over the last 15 years, 20 years there's been the discovery 82 00:05:36,000 --> 00:05:40,000 that certain RNA molecules also posses the ability to catalyze 83 00:05:40,000 --> 00:05:45,000 certain kinds of reactions. When I was taking biochemistry, 84 00:05:45,000 --> 00:05:49,000 if somebody would have told me that, I would have called the psychiatric 85 00:05:49,000 --> 00:05:53,000 ward because that was such an outlandish idea. 86 00:05:53,000 --> 00:05:58,000 How can an RNA molecule catalyze a biochemical reaction? 87 00:05:58,000 --> 00:06:02,000 It doesn't have all the side groups that one needs to create the 88 00:06:02,000 --> 00:06:06,000 catalytic sites for reactions. But we now realize, on the basis of 89 00:06:06,000 --> 00:06:10,000 research which actually led to a Nobel Prize being awarded about five 90 00:06:10,000 --> 00:06:14,000 years ago, that RNA molecules are able to catalyze certain kinds of 91 00:06:14,000 --> 00:06:18,000 reactions. And that begins to give us an insight into how life 92 00:06:18,000 --> 00:06:22,000 originated on this planet because RNA molecules may have stored 93 00:06:22,000 --> 00:06:26,000 genetic information, as I said before, RNA molecules, 94 00:06:26,000 --> 00:06:30,000 or their precursors like ATP, may have been their currency for storing 95 00:06:30,000 --> 00:06:34,000 high energy bonds, as is indicated here. 96 00:06:34,000 --> 00:06:38,000 And RNA molecules may well have been the first enzymes to catalyze many 97 00:06:38,000 --> 00:06:43,000 of the reactions in the most primitive life forms that first 98 00:06:43,000 --> 00:06:47,000 existed on this planet. And, therefore, what I'm saying is 99 00:06:47,000 --> 00:06:52,000 that as life developed in the first hundred or two hundred million years, 100 00:06:52,000 --> 00:06:56,000 who knows how long it took, gradually DNA took over the job of 101 00:06:56,000 --> 00:07:01,000 storing information from RNA and gradually proteins took over the job 102 00:07:01,000 --> 00:07:05,000 of mediating catalysis, of acting as enzymes to taking over 103 00:07:05,000 --> 00:07:10,000 the job from RNA molecules. Today there are certain vestigial 104 00:07:10,000 --> 00:07:14,000 biochemical reactions which we believe are relics, 105 00:07:14,000 --> 00:07:18,000 echoes of the beginning of life on earth, which are still mediated by 106 00:07:18,000 --> 00:07:22,000 RNA catalysts. We think that they are throwbacks 107 00:07:22,000 --> 00:07:26,000 to these very early steps, maybe even in pre-cellular life form 108 00:07:26,000 --> 00:07:31,000 where RNA was delegated with the task of acting as a catalyst. 109 00:07:31,000 --> 00:07:35,000 We're going to focus a lot today on the whole issue of biochemical 110 00:07:35,000 --> 00:07:40,000 reactions and the issue of energy. And this gets us into the 111 00:07:40,000 --> 00:07:45,000 realization that there really are two kinds of biochemical reactions. 112 00:07:45,000 --> 00:07:49,000 Some of you may have learned this a long time ago. 113 00:07:49,000 --> 00:07:54,000 Either exergonic reactions that release energy, 114 00:07:54,000 --> 00:07:59,000 that produce energy as they proceed, or conversely endergonic reactions 115 00:07:59,000 --> 00:08:04,000 which require an investment of energy in order to move forward. 116 00:08:04,000 --> 00:08:08,000 So, here, obviously, if this is a high energy state and 117 00:08:08,000 --> 00:08:12,000 we're talking about the free energy of the system, 118 00:08:12,000 --> 00:08:16,000 which is one way to depict in thermodynamic language how much 119 00:08:16,000 --> 00:08:20,000 energy is in a molecule, if we go from a high energy state to 120 00:08:20,000 --> 00:08:24,000 a low energy state then we can draw this like this and we can realize 121 00:08:24,000 --> 00:08:28,000 that in order to conserve energy, the energy that was inherent in this 122 00:08:28,000 --> 00:08:32,000 molecule, the high potential energy is released as this ball or this 123 00:08:32,000 --> 00:08:37,000 molecule rolls down the hill. And, therefore, 124 00:08:37,000 --> 00:08:41,000 the reaction yields energy, it's exergonic. And, conversely, 125 00:08:41,000 --> 00:08:45,000 if we want this reaction to proceed, we need to invest free energy in 126 00:08:45,000 --> 00:08:49,000 order to make it happen. The free energy happens to be, 127 00:08:49,000 --> 00:08:53,000 more often than not, in the form of chemical bonds, 128 00:08:53,000 --> 00:08:57,000 i.e., energy that can be invested, for example, by taking advantage of 129 00:08:57,000 --> 00:09:01,000 the potential energy stored in these phosphodiester, 130 00:09:01,000 --> 00:09:06,000 in these phosphate-phosphate linkages indicated right here. 131 00:09:06,000 --> 00:09:10,000 Here, by the way, is the space-filling model of ATP 132 00:09:10,000 --> 00:09:14,000 just for your information. That's the way it actually would 133 00:09:14,000 --> 00:09:18,000 look in life, and this is the way we actually draw it. 134 00:09:18,000 --> 00:09:22,000 Now, having said that, if we look at the free energy 135 00:09:22,000 --> 00:09:26,000 profile of various biochemical changes then we can depict them, 136 00:09:26,000 --> 00:09:30,000 once again, in this very schematic way here. 137 00:09:30,000 --> 00:09:35,000 And, by the way, free energy is called G, 138 00:09:35,000 --> 00:09:40,000 the Gibbs free energy after Josiah Gibbs who was a thermodynamic wiz in 139 00:09:40,000 --> 00:09:45,000 the 19th century at Yale in New Haven. And here what we see is that 140 00:09:45,000 --> 00:09:50,000 the change in free energy between the reactants and the products is 141 00:09:50,000 --> 00:09:55,000 given by delta G. So, by definition, 142 00:09:55,000 --> 00:10:00,000 we start out the reaction with reactants. 143 00:10:00,000 --> 00:10:03,000 And we end up at the end of the reaction with products. 144 00:10:03,000 --> 00:10:07,000 And, overall, if the reaction is exergonic and will proceed forward, 145 00:10:07,000 --> 00:10:11,000 it releases energy. And the net release of energy is indicated here 146 00:10:11,000 --> 00:10:15,000 by delta G. But, more often than not, 147 00:10:15,000 --> 00:10:18,000 biochemical reactions that are energetically favored, 148 00:10:18,000 --> 00:10:22,000 that are exergonic actually can't happen spontaneously. 149 00:10:22,000 --> 00:10:26,000 They don't happen spontaneously because, for various reasons, 150 00:10:26,000 --> 00:10:30,000 they have to pass through an intermediate state. 151 00:10:30,000 --> 00:10:34,000 Which actually represents a much higher free energy than the initial 152 00:10:34,000 --> 00:10:38,000 reactants posses. And this higher free energy, 153 00:10:38,000 --> 00:10:42,000 that they need to acquire in order to move over the hill and down into 154 00:10:42,000 --> 00:10:46,000 the valley, is called the energy of activation, the activation energy. 155 00:10:46,000 --> 00:10:50,000 And, therefore, if I were to supply these reactants with energy, 156 00:10:50,000 --> 00:10:54,000 for instance, let's say I were to heat up these reactants and 157 00:10:54,000 --> 00:10:58,000 therefore give them a higher degree of thermal energy which they might 158 00:10:58,000 --> 00:11:03,000 be able to use to move up to this high energy state. 159 00:11:03,000 --> 00:11:06,000 I supplied them with free energy by giving them heat. 160 00:11:06,000 --> 00:11:09,000 Then they might be able to move up to here and then roll down the hill. 161 00:11:09,000 --> 00:11:12,000 But in the absence of actually actively intervening and supplying 162 00:11:12,000 --> 00:11:16,000 them that energy, they'll remain right here, 163 00:11:16,000 --> 00:11:19,000 and they may remain right there for a million years, 164 00:11:19,000 --> 00:11:22,000 even though in principle, if they were to reach down here, 165 00:11:22,000 --> 00:11:26,000 they would be much happier in terms of reaching a much lower 166 00:11:26,000 --> 00:11:30,000 energy state. To state the obvious, 167 00:11:30,000 --> 00:11:34,000 all these kinds of reactions wish to reach the lowest energy state 168 00:11:34,000 --> 00:11:38,000 possible. But in real-time it can't happen if there is a high energy of 169 00:11:38,000 --> 00:11:42,000 activation. Now, what do enzymes do? 170 00:11:42,000 --> 00:11:47,000 As always, I'm glad I asked that question. What they do is they 171 00:11:47,000 --> 00:11:51,000 lower the energy of activation. And this is in one sense obvious, 172 00:11:51,000 --> 00:11:55,000 and in one sense it's subtle, because enzymes have no affect on 173 00:11:55,000 --> 00:11:59,000 the free energy state of the reactants, they have no affect on 174 00:11:59,000 --> 00:12:03,000 the free energy of the products. All they do is to lower the hump, 175 00:12:03,000 --> 00:12:07,000 and they may lower it very substantially. 176 00:12:07,000 --> 00:12:11,000 And because they lower it substantially, 177 00:12:11,000 --> 00:12:15,000 it might be that some of the reactants here may, 178 00:12:15,000 --> 00:12:18,000 just through a chance, acquisition of thermal energy, 179 00:12:18,000 --> 00:12:22,000 be able to move over the much lowered hump and move down into this 180 00:12:22,000 --> 00:12:26,000 state right here. Now, the actual difference in the 181 00:12:26,000 --> 00:12:30,000 Gibbs free energy is totally unaffected. 182 00:12:30,000 --> 00:12:35,000 All that happens is that the enzyme, by lowering the energy of activation, 183 00:12:35,000 --> 00:12:40,000 make this possible in real-time. The fact is that ultimately, if one 184 00:12:40,000 --> 00:12:46,000 were to plot many kinds of reactions, many reactions, 185 00:12:46,000 --> 00:12:51,000 as is indicated here, have a very high activation energy, 186 00:12:51,000 --> 00:12:56,000 and therefore we look at it like this. But there could be other 187 00:12:56,000 --> 00:13:02,000 reactions which might have an activation energy that looks like 188 00:13:02,000 --> 00:13:07,000 this, almost nothing at all. And these reactions could happen 189 00:13:07,000 --> 00:13:11,000 spontaneously at room temperature in the absence of any intervention by 190 00:13:11,000 --> 00:13:15,000 an enzyme. For example, let's say we're talking about a 191 00:13:15,000 --> 00:13:19,000 carboxyl group which discharges a proton. We've talked about that 192 00:13:19,000 --> 00:13:23,000 already. Well, that reaction happens spontaneously 193 00:13:23,000 --> 00:13:27,000 at room temperature. It doesn‘t need an enzyme to make 194 00:13:27,000 --> 00:13:31,000 it happen. It can happen because there's essentially not 195 00:13:31,000 --> 00:13:35,000 energy of activation. But the great majority of 196 00:13:35,000 --> 00:13:39,000 biochemical reactions do have such an activation energy, 197 00:13:39,000 --> 00:13:44,000 and therefore do require a lowering like this in order to take place. 198 00:13:44,000 --> 00:13:49,000 Now, let's imagine other versions of the energy profile of a reaction. 199 00:13:49,000 --> 00:13:53,000 And keep in mind that what I'm showing here on the abscissa is just 200 00:13:53,000 --> 00:13:58,000 the course of the reaction. You could imagine I'm not really 201 00:13:58,000 --> 00:14:05,000 plotting time. I'm just talking about a situation 202 00:14:05,000 --> 00:14:13,000 where to the left the reaction hasn't happened and to the right it 203 00:14:13,000 --> 00:14:21,000 has happened. Can you see this over there? Then I won't write over 204 00:14:21,000 --> 00:14:30,000 there. All right. Let's see if this works. 205 00:14:30,000 --> 00:14:36,000 Boy, here we are in the 21st century and we still haven't worked this out. 206 00:14:36,000 --> 00:14:42,000 OK. Everybody can see this right here, right? OK. 207 00:14:42,000 --> 00:14:48,000 So, look. Let's imagine we have a reaction that looks like this, 208 00:14:48,000 --> 00:14:54,000 a reaction profile that looks like this, where these two energies are 209 00:14:54,000 --> 00:15:00,000 actually equivalent. OK? I've tried to draw them on. 210 00:15:00,000 --> 00:15:04,000 Well, they're not exactly, but they're pretty much on exactly 211 00:15:04,000 --> 00:15:08,000 the same level. And let's say we start out with a 212 00:15:08,000 --> 00:15:13,000 large number of molecules right over here. Now, if there were an enzyme 213 00:15:13,000 --> 00:15:17,000 around, the enzyme might lower the activation energy and, 214 00:15:17,000 --> 00:15:21,000 in so doing, make it possible for molecules to tunnel through this 215 00:15:21,000 --> 00:15:26,000 hill and move over here. The fact that when a molecule gets 216 00:15:26,000 --> 00:15:30,000 over here it has the same free energy as over there means that the 217 00:15:30,000 --> 00:15:35,000 catalyst may, in principle, also facilitate a back reaction. 218 00:15:35,000 --> 00:15:38,000 What do I mean by a back reaction? I mean going in exactly the 219 00:15:38,000 --> 00:15:42,000 opposite direction. And so, once molecules over here 220 00:15:42,000 --> 00:15:46,000 are formed, the energy lowering affects of the enzyme may allow them 221 00:15:46,000 --> 00:15:50,000 to move in both directions. And, therefore, what we will have 222 00:15:50,000 --> 00:15:54,000 is ultimately the establishment of an equilibrium. 223 00:15:54,000 --> 00:15:58,000 If these two energy states are equivalent then, 224 00:15:58,000 --> 00:16:02,000 I will tell you, 50% of the molecules end up here and 50% of the 225 00:16:02,000 --> 00:16:06,000 molecules end up here. And here we're beginning now to 226 00:16:06,000 --> 00:16:10,000 wrestle between two different independent concepts, 227 00:16:10,000 --> 00:16:14,000 the rate of the reaction and the equilibrium state of the reaction. 228 00:16:14,000 --> 00:16:19,000 Note that the enzyme has no affect whatsoever on the equilibrium state. 229 00:16:19,000 --> 00:16:23,000 These two are at equal free energies, the equilibrium state. 230 00:16:23,000 --> 00:16:27,000 Whether the energy barrier is this high or whether it's this 231 00:16:27,000 --> 00:16:32,000 high is irrelevant. The fact is if the enzyme makes 232 00:16:32,000 --> 00:16:36,000 possible this motion back and forth, the ultimate equilibrium state will 233 00:16:36,000 --> 00:16:40,000 be 50% of the molecules here and 50% of the molecules there. 234 00:16:40,000 --> 00:16:44,000 And, therefore, the enzyme really only affects the rate at which the 235 00:16:44,000 --> 00:16:48,000 reaction takes place. Will it happen in a microsecond or 236 00:16:48,000 --> 00:16:52,000 will it happen in a day or will it happen in a million years? 237 00:16:52,000 --> 00:16:56,000 The enzyme has no affect whatsoever on the ultimate end product, 238 00:16:56,000 --> 00:17:00,000 which in this case is the equilibrium. 239 00:17:00,000 --> 00:17:06,000 Of course, there is a simple mathematic formalism which relates 240 00:17:06,000 --> 00:17:12,000 the difference in free energies with the equilibrium. 241 00:17:12,000 --> 00:17:18,000 Here we might have a situation where 80% of the molecules end up at 242 00:17:18,000 --> 00:17:24,000 equilibrium over here and 20% end up here. Or, we might end up as a 243 00:17:24,000 --> 00:17:30,000 state where 99. % of the molecules end up here and 0. 244 00:17:30,000 --> 00:17:34,000 % of the molecules end up here. But that ultimate equilibrium state 245 00:17:34,000 --> 00:17:38,000 is no way influenced by the enzyme. They just make it happen in 246 00:17:38,000 --> 00:17:42,000 real-time. And, therefore, to repeat and echo a 247 00:17:42,000 --> 00:17:46,000 point I made last time, if most biochemical reactions are to 248 00:17:46,000 --> 00:17:50,000 occur in real-time, i.e., in the order of seconds or 249 00:17:50,000 --> 00:17:54,000 minutes, an enzyme has to be around to make sure they happen. 250 00:17:54,000 --> 00:17:58,000 In the absence of such an enzyme of its intermediation, 251 00:17:58,000 --> 00:18:02,000 it just won't happen in real-time. Even though, in principle, 252 00:18:02,000 --> 00:18:06,000 it's energetically favored. So, let's just keep that very much 253 00:18:06,000 --> 00:18:11,000 in mind in the course of discussions that happen. And let's just begin 254 00:18:11,000 --> 00:18:15,000 now to look at an important energy-generating reaction in the 255 00:18:15,000 --> 00:18:20,000 cell which is called glycolysis. We already know the prefix glycol. 256 00:18:20,000 --> 00:18:24,000 Glyco refers to sugar. And lysis, L-Y-S-I-S refers to the breakdown of 257 00:18:24,000 --> 00:18:29,000 a certain compound. I am not going to ask you, 258 00:18:29,000 --> 00:18:33,000 nor is anyone else in the room going to ask you to memorize this 259 00:18:33,000 --> 00:18:37,000 sequence of reactions. But I'd like you to look at it and 260 00:18:37,000 --> 00:18:41,000 see what take-home lessons we can distill out of that, 261 00:18:41,000 --> 00:18:45,000 what wisdom we can learn from looking at such a complex series of 262 00:18:45,000 --> 00:18:49,000 reactions. Perhaps, the first thing we can learn is that 263 00:18:49,000 --> 00:18:53,000 when we think about biochemical reactions, we don't think of them as 264 00:18:53,000 --> 00:18:57,000 happening in isolation. Here I'm talking about, 265 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, 266 00:19:01,000 --> 00:19:05,000 and there might be a back reaction to reach equilibrium. 267 00:19:05,000 --> 00:19:09,000 And we're just isolating that simple reaction from all others around it. 268 00:19:09,000 --> 00:19:13,000 But in the real world in living cells most reactions are parts of 269 00:19:13,000 --> 00:19:17,000 very long pathways where each of these steps here indicates one of 270 00:19:17,000 --> 00:19:21,000 the others, a step in the pathway. What we're interested in here is 271 00:19:21,000 --> 00:19:25,000 how glucose, which I advertised two lectures ago as being an important 272 00:19:25,000 --> 00:19:30,000 energy source, is actually broken down. 273 00:19:30,000 --> 00:19:34,000 How does the cell harvest the energy, which is inherent in glucose, 274 00:19:34,000 --> 00:19:38,000 in order to generate, among other things, ATP, which we've said 275 00:19:38,000 --> 00:19:42,000 repeatedly is the energy currency? ATP is used by hundreds of 276 00:19:42,000 --> 00:19:47,000 different biochemical reactions in order to make them happen. 277 00:19:47,000 --> 00:19:51,000 These other biochemical reactions are endergonic, 278 00:19:51,000 --> 00:19:55,000 they require the investment of energy, and almost invariably, 279 00:19:55,000 --> 00:19:59,000 but not invariably, but almost invariably the cell will grab hold 280 00:19:59,000 --> 00:20:04,000 of an ATP molecule, break it down usually to AMP or ADP. 281 00:20:04,000 --> 00:20:08,000 And then utilize the energy, which derives from breaking down ATP, 282 00:20:08,000 --> 00:20:13,000 it will invest that energy in an endergonic reaction, 283 00:20:13,000 --> 00:20:18,000 which in the otherwise would not happen. So, here we reach the idea 284 00:20:18,000 --> 00:20:23,000 that perhaps by investing energy in a reaction, the equilibrium is 285 00:20:23,000 --> 00:20:28,000 shifted. Because by investing energy, actually, 286 00:20:28,000 --> 00:20:33,000 the cell is able to lower the free energy state between these two. 287 00:20:33,000 --> 00:20:36,000 And that makes it possible for their equilibrium to be much more favored. 288 00:20:36,000 --> 00:20:40,000 Let's look at this glycolytic pathway. Glycolytic refers, 289 00:20:40,000 --> 00:20:44,000 obviously, to glycolysis. And here we start out with glucose. 290 00:20:44,000 --> 00:20:48,000 We're drawing it out flat rather than the circular structure we 291 00:20:48,000 --> 00:20:52,000 talked about last time. And let's look at what happens here, 292 00:20:52,000 --> 00:20:56,000 again, not because anyone wants you to memorize this, 293 00:20:56,000 --> 00:21:00,000 but because some of the details are in themselves very illustrative. 294 00:21:00,000 --> 00:21:04,000 The goal of this exercise is to create ATP for the cell, 295 00:21:04,000 --> 00:21:08,000 but the first step in the reaction is actually totally 296 00:21:08,000 --> 00:21:12,000 counterproductive. Look at the first thing that 297 00:21:12,000 --> 00:21:16,000 happens. The first thing that happens is that the cell invests an 298 00:21:16,000 --> 00:21:20,000 ATP molecule to make glucose-6-phosphate. 299 00:21:20,000 --> 00:21:24,000 I've advertised the goal of this is to generate ATP from ADP, 300 00:21:24,000 --> 00:21:28,000 adenosine diphosphate. But the first thing here, 301 00:21:28,000 --> 00:21:32,000 this is an endergonic reaction in which the cell invests energy to 302 00:21:32,000 --> 00:21:35,000 create this molecule here. So, this doesn't make sense. 303 00:21:35,000 --> 00:21:38,000 But ostensively it must make sense, at one level or another, because you 304 00:21:38,000 --> 00:21:42,000 and I, we're all here, and everybody in this room, 305 00:21:42,000 --> 00:21:45,000 at least this moment is metabolically active. 306 00:21:45,000 --> 00:21:48,000 All right. So, we've got this molecule here, 307 00:21:48,000 --> 00:21:51,000 glucose-6-phosphate. And this can isomerize. 308 00:21:51,000 --> 00:21:55,000 You see, here's glucose-6-phosphate, fructose-6-phosphate. 309 00:21:55,000 --> 00:21:58,000 And, the fact of the matter is, there's no oxidation reduction 310 00:21:58,000 --> 00:22:02,000 reaction here. It's just an isomerization. 311 00:22:02,000 --> 00:22:06,000 And this molecule and this molecule are virtually in the same free 312 00:22:06,000 --> 00:22:10,000 energy state. It happens to be the case that their profile will look 313 00:22:10,000 --> 00:22:14,000 very much like the one I drew you before. Their energy profile will 314 00:22:14,000 --> 00:22:18,000 look like this. And one needs an enzyme to lower it, 315 00:22:18,000 --> 00:22:22,000 but there's no energy that needs to be invested in converting one to the 316 00:22:22,000 --> 00:22:26,000 other because they're very similar molecules and therefore incomparable 317 00:22:26,000 --> 00:22:31,000 free energy states. Now look at the next step. 318 00:22:31,000 --> 00:22:35,000 The next step is again another ostensively totally 319 00:22:35,000 --> 00:22:39,000 counterproductive way of generating energy. Because, 320 00:22:39,000 --> 00:22:44,000 once again, ATP, the gamma-phosphate, its energy is invested in creating a 321 00:22:44,000 --> 00:22:48,000 dephosphorylated hexose, fructose 1, 6-diphosphate where the 322 00:22:48,000 --> 00:22:52,000 numbers refer obviously to the identities of the carbon. 323 00:22:52,000 --> 00:22:57,000 And now we have a dephosphorylated fructose molecule. 324 00:22:57,000 --> 00:23:01,000 And so here you can actually see what the three-dimensional, 325 00:23:01,000 --> 00:23:05,000 what we would imagine closer to what the three-dimensional structures of 326 00:23:05,000 --> 00:23:09,000 these molecules look like. And we shouldn't focus this time on 327 00:23:09,000 --> 00:23:13,000 whether it's this or this. For all practical purposes, 328 00:23:13,000 --> 00:23:17,000 let's just focus on this pathway here. And here, 329 00:23:17,000 --> 00:23:21,000 for the first time, what now happens is that this hexose 330 00:23:21,000 --> 00:23:25,000 is broken down into two trioses, i.e., into two three carbon sugars. 331 00:23:25,000 --> 00:23:30,000 And this is a slightly exergonic reaction. 332 00:23:30,000 --> 00:23:33,000 It yields, it happens without the investment of energy. 333 00:23:33,000 --> 00:23:37,000 And there's an enzyme, once again, that's required in order 334 00:23:37,000 --> 00:23:40,000 to catalyze it. But let's be really clear now. 335 00:23:40,000 --> 00:23:44,000 Now we have to follow the fate of two molecules. 336 00:23:44,000 --> 00:23:47,000 The first triose and the second triose. They have different names, 337 00:23:47,000 --> 00:23:51,000 but we're not going to focus on the names. One thing you notice about 338 00:23:51,000 --> 00:23:54,000 these trioses is that they're readily interconvertible. 339 00:23:54,000 --> 00:23:58,000 Once again, we can image that we have a situation that 340 00:23:58,000 --> 00:24:02,000 looks like this. These are flipping back and forth. 341 00:24:02,000 --> 00:24:06,000 And therefore, for all practical purposes from our point of view, 342 00:24:06,000 --> 00:24:10,000 these two are equivalent because they can be exchanged virtually 343 00:24:10,000 --> 00:24:14,000 instantaneously one with the other. Now, so far we've actually expended 344 00:24:14,000 --> 00:24:19,000 energy. We haven't harvested energy. But, keep in mind, 345 00:24:19,000 --> 00:24:23,000 the old economic dictum; you have to invest money to make money. 346 00:24:23,000 --> 00:24:27,000 And that's what's going on here. The first thing that happens is we 347 00:24:27,000 --> 00:24:32,000 have an oxidation reaction. What's an oxidation reaction? 348 00:24:32,000 --> 00:24:36,000 We want to strip some electrons, a pair of electrons off of this 349 00:24:36,000 --> 00:24:41,000 particular triose, the 3 carbon sugar. 350 00:24:41,000 --> 00:24:46,000 And by stripping off a pair of electrons we donate the electrons 351 00:24:46,000 --> 00:24:50,000 from NAD+ to NADH. And here these structures are given 352 00:24:50,000 --> 00:24:55,000 in your book. But NADH, it turns out, is the electrons are 353 00:24:55,000 --> 00:25:00,000 pulled away from the triose and they're used to reduce NAD+ to NADH. 354 00:25:00,000 --> 00:25:04,000 Keep in mind that in an oxidation reaction, one molecule that's being 355 00:25:04,000 --> 00:25:08,000 oxidized is deprived, is denied a pair of electrons. 356 00:25:08,000 --> 00:25:12,000 The other molecule that's being reduced, in this case NAD, 357 00:25:12,000 --> 00:25:16,000 acquires a pair of electrons. And you can focus, if you want, 358 00:25:16,000 --> 00:25:20,000 about the charge of these molecules, one or the other. But, keep in mind, 359 00:25:20,000 --> 00:25:24,000 that in these oxidation reduction reactions, whether it's plus charged 360 00:25:24,000 --> 00:25:28,000 or minus charged is irrelevant. The real name of the game is the 361 00:25:28,000 --> 00:25:31,000 electrons. Forget about the protons, 362 00:25:31,000 --> 00:25:35,000 whether it has a plus charge or it's neutral. The real name of the game 363 00:25:35,000 --> 00:25:38,000 here is that two electrons are being used to reduce this molecule to this. 364 00:25:38,000 --> 00:25:42,000 By the way, third mistake I forgot to tell you before, 365 00:25:42,000 --> 00:25:45,000 there's a double-bond in one of the pyrimidines in the book that doesn't 366 00:25:45,000 --> 00:25:49,000 make any sense. Whoever finds it gets a prize, 367 00:25:49,000 --> 00:25:53,000 but no one's figured out what the prize is yet. So, 368 00:25:53,000 --> 00:25:56,000 this double bond gets reduced. You see the difference between this 369 00:25:56,000 --> 00:26:00,000 and this over here. And this NADH, it turns out, 370 00:26:00,000 --> 00:26:04,000 is a high energy molecule. The street value of NADH is three 371 00:26:04,000 --> 00:26:10,000 ATPs, i.e., in the mitochondria NADH can be used to generate three ATPs, 372 00:26:10,000 --> 00:26:15,000 and that's worth something. So, NADH on its own is a high energy 373 00:26:15,000 --> 00:26:20,000 molecule. It can't be used for that many things, but it can be pulled 374 00:26:20,000 --> 00:26:26,000 into the mitochondria where it's converted to three ATPs. 375 00:26:26,000 --> 00:26:31,000 So, we say, well, we're starting to make some money out of this 376 00:26:31,000 --> 00:26:37,000 investment because we've made, in fact, these NADHs. 377 00:26:37,000 --> 00:26:41,000 See right here. Why do we say two NADHs? 378 00:26:41,000 --> 00:26:46,000 Because there are two trioses we're working with, and each one of the 379 00:26:46,000 --> 00:26:50,000 trioses gives you an NADH. So, everything that's going on 380 00:26:50,000 --> 00:26:55,000 after this, starting from the top here, is now double because we're 381 00:26:55,000 --> 00:26:59,000 looking at the parallel behaviors of two identical three carbon sugars. 382 00:26:59,000 --> 00:27:04,000 So, here we've so far generated, in principle, six ATPs. 383 00:27:04,000 --> 00:27:08,000 How much did we invest already up to this point? Two. 384 00:27:08,000 --> 00:27:13,000 We invested two but we harvested six. Already we're starting to make 385 00:27:13,000 --> 00:27:18,000 a little money because I told you the street value of an NADH is three 386 00:27:18,000 --> 00:27:23,000 ATPs on the black market. OK, so what happens next? 387 00:27:23,000 --> 00:27:27,000 Next is another good thing. Each of the trioses, one can 388 00:27:27,000 --> 00:27:32,000 actually cause each of the trioses to generate an ATP molecule 389 00:27:32,000 --> 00:27:36,000 from an ADP. What happens here? 390 00:27:36,000 --> 00:27:40,000 It turns out that this phosphate over here is actually in a pretty 391 00:27:40,000 --> 00:27:43,000 high energy state, in no small part because of electron 392 00:27:43,000 --> 00:27:47,000 negative-negative repulsion. And by stripping this phosphate off 393 00:27:47,000 --> 00:27:51,000 this high energy phosphate stripped off of this molecule here, 394 00:27:51,000 --> 00:27:54,000 whose name we will ignore, allows us to phosphorylate an ATP. 395 00:27:54,000 --> 00:27:58,000 And since there are two trioses being converted, we're 396 00:27:58,000 --> 00:28:02,000 going to get two ATPs. So, in effect, 397 00:28:02,000 --> 00:28:06,000 now we're actually ahead. We started out investing two, 398 00:28:06,000 --> 00:28:10,000 we got six back from the NADHs, and we're getting two back here. 399 00:28:10,000 --> 00:28:14,000 So, we've made two ATPs. This is a good thing. Keep in mind, 400 00:28:14,000 --> 00:28:19,000 ADP is lower energy, ATP is a high energy. Once again, 401 00:28:19,000 --> 00:28:23,000 we have an isomerization where these two molecules are at comparable 402 00:28:23,000 --> 00:28:27,000 states here and here, where the phosphate just jumps over 403 00:28:27,000 --> 00:28:32,000 to this state. And this hydrolyzes spontaneously 404 00:28:32,000 --> 00:28:37,000 and we get this molecule right over here, phosphoenolpyruvate at the end. 405 00:28:37,000 --> 00:28:42,000 And, once again, we harvest two ATPs, 406 00:28:42,000 --> 00:28:47,000 one ATP from each of the trioses. And we end up, at the end of this 407 00:28:47,000 --> 00:28:52,000 reaction, with pyruvate. And you'll say this is terrific 408 00:28:52,000 --> 00:28:57,000 because we invested two ATPs, we harvested four, plus we got six 409 00:28:57,000 --> 00:29:03,000 from the NADHs, right? Two NADHs, each NADH gives us three 410 00:29:03,000 --> 00:29:11,000 each, so let's do the arithmetic. Let's do the balance sheet. We 411 00:29:11,000 --> 00:29:18,000 invested to begin with, with the one glucose, we invested 412 00:29:18,000 --> 00:29:26,000 two ATPs. That was early on. Then the return was first two NADHs, 413 00:29:26,000 --> 00:29:33,000 which I've told you equals six ATPs. Because an NADH is worth three ATPs. 414 00:29:33,000 --> 00:29:39,000 This is so far good. And now subsequently we've made four ATPs so 415 00:29:39,000 --> 00:29:46,000 that the net yield looks pretty useful. Six plus four is ten minus 416 00:29:46,000 --> 00:29:52,000 two, a profit of eight ATPs from one glucose molecule. 417 00:29:52,000 --> 00:29:59,000 This is terrific you may say, but there's a rub. 418 00:29:59,000 --> 00:30:04,000 There's a catch. If glycolysis is occurring in the 419 00:30:04,000 --> 00:30:10,000 absence of oxygen, if that happens, then we have a 420 00:30:10,000 --> 00:30:15,000 problem here, because the only way that these NADHs can generate ATP is 421 00:30:15,000 --> 00:30:21,000 if there is oxygen around to take these electron pairs and use them to 422 00:30:21,000 --> 00:30:27,000 reduce an oxygen molecule. That is, by the way, part of the 423 00:30:27,000 --> 00:30:32,000 reason we breathe. Keep in mind that when you generate 424 00:30:32,000 --> 00:30:36,000 an NADH from an NAD molecule, you need to regenerate the NAD. 425 00:30:36,000 --> 00:30:40,000 You can't just accumulate more and more NADHs. You need to regenerate 426 00:30:40,000 --> 00:30:44,000 the NAD. And, therefore, this NADH, 427 00:30:44,000 --> 00:30:48,000 with their electron pairs, the electron pairs have some to be 428 00:30:48,000 --> 00:30:52,000 disposed of. You have to regenerate NAD. You can't just make more and 429 00:30:52,000 --> 00:30:56,000 more and more of this. So, how do cells get rid of it? 430 00:30:56,000 --> 00:31:00,000 Well, how they get rid of it is simple. 431 00:31:00,000 --> 00:31:05,000 You take the electron pairs and you slap them onto oxygen, 432 00:31:05,000 --> 00:31:10,000 and that's really called combustion. And you get a lot of energy out of 433 00:31:10,000 --> 00:31:16,000 that. But what happens if all of this is occurring anaerobically? 434 00:31:16,000 --> 00:31:21,000 Anaerobically means the reaction is occurring in the absence of oxygen. 435 00:31:21,000 --> 00:31:27,000 Well, if you have a yeast that's growing 14 feet underground, 436 00:31:27,000 --> 00:31:31,000 this is happening anaerobically. If you have a yeast that's 437 00:31:31,000 --> 00:31:35,000 fermenting in a big keg to make wine or beer, it's also probably 438 00:31:35,000 --> 00:31:39,000 happening anaerobically. If you start running in a 100 yard 439 00:31:39,000 --> 00:31:43,000 sprint, or let's say you had to run a mile, then initially there's 440 00:31:43,000 --> 00:31:47,000 enough oxygen, there's a lot of oxygen around to 441 00:31:47,000 --> 00:31:51,000 allow you to get rid of these NADHs and dump the electrons that they 442 00:31:51,000 --> 00:31:55,000 have acquired onto the oxygen molecule. And that's fine. 443 00:31:55,000 --> 00:31:59,000 That's worth a lot because, in effect, what you're doing is 444 00:31:59,000 --> 00:32:03,000 you're taking oxygen and hydrogen and you're combusting them together. 445 00:32:03,000 --> 00:32:07,000 And that's great. But as you start running down the 446 00:32:07,000 --> 00:32:12,000 street, soon the oxygen supply to your muscles is going to run out, 447 00:32:12,000 --> 00:32:16,000 and soon a lot of the energy production in your muscles happens 448 00:32:16,000 --> 00:32:21,000 anaerobically. Why? Because you can't get oxygen 449 00:32:21,000 --> 00:32:26,000 quickly enough to your muscles, and therefore, for a period of time, 450 00:32:26,000 --> 00:32:30,000 you start feeling that burning sensation in your muscles because 451 00:32:30,000 --> 00:32:35,000 oxidation of NADH isn't happening. And these NADHs instead are 452 00:32:35,000 --> 00:32:40,000 regenerated by another way. How are they regenerated? The 453 00:32:40,000 --> 00:32:45,000 electron pairs of the NADHs, must be, are dumped back onto this 454 00:32:45,000 --> 00:32:50,000 molecule right here, pyruvate. They're not used to make 455 00:32:50,000 --> 00:32:55,000 ATP because they can't be used to make ATP because there's no oxygen 456 00:32:55,000 --> 00:33:01,000 around to accept the electron pairs that these NADHs have acquired. 457 00:33:01,000 --> 00:33:05,000 And so, what happens with these valuable NADHs? 458 00:33:05,000 --> 00:33:09,000 Under anaerobic conditions this doesn't happen. 459 00:33:09,000 --> 00:33:14,000 These NADHs are used instead, their electrons are donated to our 460 00:33:14,000 --> 00:33:18,000 friend pyruvate here, these three carbon sugar. 461 00:33:18,000 --> 00:33:23,000 And what happens, when they are donated back to the pyruvate, 462 00:33:23,000 --> 00:33:27,000 in order to regenerate NAD you need more NAD to pick up to use later in 463 00:33:27,000 --> 00:33:32,000 the reaction, to use over again in another reaction. 464 00:33:32,000 --> 00:33:36,000 When you donate the electrons from NADH back onto pyruvate, 465 00:33:36,000 --> 00:33:41,000 what happens? You get lactic acid. Lactic acid is what makes your 466 00:33:41,000 --> 00:33:45,000 muscles burn when you're running very quickly and you can't get 467 00:33:45,000 --> 00:33:50,000 enough oxygen into them to begin to burn up the NADH. 468 00:33:50,000 --> 00:33:55,000 So, instead of using NADH to generate ATP, it's diverted to make 469 00:33:55,000 --> 00:34:00,000 lactic acid. That's in one sense good because you regenerate NAD. 470 00:34:00,000 --> 00:34:04,000 Why do you need to regenerate NAD? Because you need a lot of NAD 471 00:34:04,000 --> 00:34:09,000 around for the earlier steps in the reaction. Keep in mind, 472 00:34:09,000 --> 00:34:13,000 early in the reaction you need NAD here. If you don't regenerate it 473 00:34:13,000 --> 00:34:18,000 then glycolysis grinds to a halt. So, even though you make NADH and 474 00:34:18,000 --> 00:34:23,000 it's a good thing in principle, in practice it has to be recycled. 475 00:34:23,000 --> 00:34:27,000 And if it's not recycled to make more new NAD to allow this step to 476 00:34:27,000 --> 00:34:32,000 happen then the whole glycolytic reaction will shut down 477 00:34:32,000 --> 00:34:37,000 and you're in a mess. However, sadly, 478 00:34:37,000 --> 00:34:41,000 in the absence of oxygen, the only way to recycle this is to 479 00:34:41,000 --> 00:34:46,000 dump these electrons not onto oxygen which is energy rich, 480 00:34:46,000 --> 00:34:50,000 it's dump them back onto pyruvic acid creating lactic acid. 481 00:34:50,000 --> 00:34:55,000 So, you reduce this bond right here. So, you get CH, 482 00:34:55,000 --> 00:35:00,000 COH. This bond right here is reduced and you get lactic acid. 483 00:35:00,000 --> 00:35:04,000 So, instead of a carbonyl bond here you have CH and COH right here, 484 00:35:04,000 --> 00:35:09,000 that's a reduction reaction. And now you're able to regenerate the 485 00:35:09,000 --> 00:35:13,000 NAD. And now you say that's a great thing. But, keep in mind, 486 00:35:13,000 --> 00:35:18,000 that now the entire glycolytic reaction, how much is our net profit 487 00:35:18,000 --> 00:35:23,000 now? Before I was gloating about the fact that we made eight ATPs, 488 00:35:23,000 --> 00:35:27,000 we netted eight ATPs out of this. What are we back down to now? 489 00:35:27,000 --> 00:35:32,000 What's the whole net yield now? Well, the TAs can't answer. 490 00:35:32,000 --> 00:35:36,000 It's two, because we invested two and we got out four. 491 00:35:36,000 --> 00:35:40,000 And it's only two. Now, why is this so interesting? 492 00:35:40,000 --> 00:35:45,000 Well, until about six hundred million years ago there wasn't that 493 00:35:45,000 --> 00:35:49,000 much oxygen in the atmosphere. And in the absence of oxygen this 494 00:35:49,000 --> 00:35:54,000 is almost the only reaction that could be used in order to generate 495 00:35:54,000 --> 00:35:58,000 energy. And about six hundred million years ago more and more 496 00:35:58,000 --> 00:36:03,000 oxygen from photosynthesis became dumped into the atmosphere. 497 00:36:03,000 --> 00:36:08,000 And soon oxygen became available to organisms like our ancestors. 498 00:36:08,000 --> 00:36:13,000 And then they could actually begin to recycle this NADH in a much more 499 00:36:13,000 --> 00:36:18,000 productive way. And as a consequence what happened, 500 00:36:18,000 --> 00:36:23,000 instead of having glycolysis yielding two, we could go up to this 501 00:36:23,000 --> 00:36:28,000 theoretical eight because the NADHs could now deposit their electrons on 502 00:36:28,000 --> 00:36:33,000 oxygen, which is much more profitable. 503 00:36:33,000 --> 00:36:39,000 In fact, I've just told you now that in the absence of oxygen you can 504 00:36:39,000 --> 00:36:45,000 only make two ATPs. I will tell you, without providing 505 00:36:45,000 --> 00:36:51,000 it to you, that in the presence of oxygen you can make 34 ATPs. 506 00:36:51,000 --> 00:36:57,000 And 34 is, we can agree, much better than two in the 507 00:36:57,000 --> 00:37:01,000 presence of oxygen. Higher life forms could not evolve 508 00:37:01,000 --> 00:37:05,000 until this much more effective way of generating energy became 509 00:37:05,000 --> 00:37:09,000 available. And, therefore, if our ancestors who 510 00:37:09,000 --> 00:37:13,000 lived longer than six hundred million years ago were very sluggish 511 00:37:13,000 --> 00:37:17,000 and they weren't very smart, the reason why they were sluggish 512 00:37:17,000 --> 00:37:21,000 and they weren't very smart is because they couldn't generate the 513 00:37:21,000 --> 00:37:25,000 energy that was required to efficiently drive metabolism. 514 00:37:25,000 --> 00:37:29,000 The metabolism, anaerobic metabolism, i. 515 00:37:29,000 --> 00:37:33,000 ., occurring in the absence of energy, is extremely inefficient. 516 00:37:33,000 --> 00:37:39,000 It just doesn't happen very well. Now, what actually happens if we 517 00:37:39,000 --> 00:37:45,000 have oxygen around? Well, what happens is something 518 00:37:45,000 --> 00:37:51,000 like this. We take the pyruvate, which is the product of glycolysis 519 00:37:51,000 --> 00:37:57,000 and which is this much more primitive pathway, 520 00:37:57,000 --> 00:38:02,000 and we dump it into the mitochondria. And now we generate through this 521 00:38:02,000 --> 00:38:08,000 cycle here, which I'm not asking you memorize, please, 522 00:38:08,000 --> 00:38:13,000 don't do that. We generate the reactions which go from here and get 523 00:38:13,000 --> 00:38:19,000 us up to this 34 ATP yield per glucose. And the essence of the 524 00:38:19,000 --> 00:38:24,000 citric acid cycle, which happens in the mitochondria, 525 00:38:24,000 --> 00:38:30,000 keep in mind that the mitochondria look like this. 526 00:38:30,000 --> 00:38:34,000 Keep in mind that the mitochondrion are the decedents of bacteria which 527 00:38:34,000 --> 00:38:39,000 parasitized the cytoplasm of cells probably 1.5 billion years ago. 528 00:38:39,000 --> 00:38:43,000 But if we now look at what happens in the mitochondrion, 529 00:38:43,000 --> 00:38:48,000 the pyruvate that we generated in the cytosol, in the soluble part of 530 00:38:48,000 --> 00:38:53,000 the cytoplasm is now pumped into the mitochondria, and there's a whole 531 00:38:53,000 --> 00:38:57,000 series of reactions that go on here, which takes this three-carbon 532 00:38:57,000 --> 00:39:02,000 sugar. The first thing that happens is that 533 00:39:02,000 --> 00:39:06,000 carbon is boiled off. Carbon dioxide, that's released. 534 00:39:06,000 --> 00:39:10,000 Now we're down to a two carbon sugar. And then this two carbon 535 00:39:10,000 --> 00:39:14,000 sugar is added to a four carbon sugar and progressively oxidized. 536 00:39:14,000 --> 00:39:19,000 And as it's oxidized what's spun off? Well, what's spun off is, 537 00:39:19,000 --> 00:39:23,000 for example, there's NADH which is spun off, there's ATP. 538 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. 539 00:39:27,000 --> 00:39:32,000 Here is a cousin of NADH. It's called FADH which, 540 00:39:32,000 --> 00:39:36,000 once again, generates a high energy molecule. Once again, 541 00:39:36,000 --> 00:39:41,000 the carbon molecules are oxidized, electrons are stripped away and used 542 00:39:41,000 --> 00:39:45,000 to create these high energy molecules, FADH and NADH. 543 00:39:45,000 --> 00:39:49,000 By the way, FADH, a cousin of NADH, is only worth two ATPs on the open 544 00:39:49,000 --> 00:39:54,000 market. Whereas, NADH, as I've told you repeatedly, 545 00:39:54,000 --> 00:39:58,000 is worth three. And by the time we add up all of the NADHs that have 546 00:39:58,000 --> 00:40:03,000 been generated by this cycling and the carbon dioxides that are 547 00:40:03,000 --> 00:40:07,000 releases, at the end of this cycle here we start with two carbons, 548 00:40:07,000 --> 00:40:12,000 add it to four and we get a six carbon molecule. 549 00:40:12,000 --> 00:40:16,000 We spew off some carbon dioxides here and go back to four carbon 550 00:40:16,000 --> 00:40:20,000 sugar. Add another two, go up to six carbons. Go around 551 00:40:20,000 --> 00:40:24,000 again, spin around the wheel. And each time we do that we 552 00:40:24,000 --> 00:40:28,000 generate a lot of NADHs, we generate a lot of FADHs, 553 00:40:28,000 --> 00:40:33,000 and we generate a lot of ATP. In all cases, these are highly 554 00:40:33,000 --> 00:40:39,000 profitable reactions simply because the NADHs and the FADHs can be used 555 00:40:39,000 --> 00:40:45,000 in the mitochondrion to generate ATP. So, let's look at the energy 556 00:40:45,000 --> 00:40:51,000 profile of the entire thing. Put it all together. This is where 557 00:40:51,000 --> 00:40:57,000 we started out at the beginning, and this is the end of glycolysis, 558 00:40:57,000 --> 00:41:02,000 OK? So, now we're adding up the energy 559 00:41:02,000 --> 00:41:06,000 profiles of the whole sequence of reactions that constituted 560 00:41:06,000 --> 00:41:10,000 glycolysis, which begins up here and ends right here because pyruvate, 561 00:41:10,000 --> 00:41:14,000 as you will recall, is the product of glycolysis, 562 00:41:14,000 --> 00:41:18,000 the first step. The Krebs Cycle happens, 563 00:41:18,000 --> 00:41:22,000 or sometimes it's called the Citric Acid Cycle. So, 564 00:41:22,000 --> 00:41:26,000 let's just get these words straight. Citric Acid Cycle because it 565 00:41:26,000 --> 00:41:30,000 happens to be one of the cycles, or it's sometimes called the Krebs 566 00:41:30,000 --> 00:41:35,000 Cycle after the person who really discovered it, Krebs. 567 00:41:35,000 --> 00:41:39,000 The Krebs Cycle begins here. You see how the shading changes 568 00:41:39,000 --> 00:41:43,000 from pyruvate. And here we go all the way down 569 00:41:43,000 --> 00:41:47,000 there. And let's now look at what happens in terms of energy exchange. 570 00:41:47,000 --> 00:41:51,000 Recall that early on we needed to invest ATPs to kick up the energy 571 00:41:51,000 --> 00:41:55,000 state up to here. We invested ATPs at this stage 572 00:41:55,000 --> 00:42:00,000 right here, and then we began to get some back. 573 00:42:00,000 --> 00:42:04,000 We got these two NADHs, one NADH coming from each of the 574 00:42:04,000 --> 00:42:08,000 three carbon sugars. We got some more ATPs here and we 575 00:42:08,000 --> 00:42:12,000 got some more ATPs here, but these NADHs could not be used 576 00:42:12,000 --> 00:42:17,000 productively for generating ATP in the absence of oxygen, 577 00:42:17,000 --> 00:42:21,000 but in the presence of oxygen now we can begin to use these very 578 00:42:21,000 --> 00:42:25,000 profitably. Each of these makes three ATPs and each of these, 579 00:42:25,000 --> 00:42:30,000 obviously, makes ATPs. And then let's look at what happens 580 00:42:30,000 --> 00:42:34,000 in the mitochondrion. Keep in mind here's the borderline 581 00:42:34,000 --> 00:42:38,000 between the cytosol, the cytoplasm and the mitochondrion. 582 00:42:38,000 --> 00:42:42,000 Here is where the oxygen is actually used and here we generate 583 00:42:42,000 --> 00:42:46,000 all these NADHs here, here and here, FADHs. And I keep 584 00:42:46,000 --> 00:42:50,000 saying, and it's still true, just in spite of the fact I keep 585 00:42:50,000 --> 00:42:54,000 saying it, that these NADHs can be converted to ATPs, 586 00:42:54,000 --> 00:42:58,000 and the ATPs can then be diffused, transmitted throughout the entire 587 00:42:58,000 --> 00:43:02,000 cell where they're then used invested in endergonic reactions. 588 00:43:02,000 --> 00:43:06,000 Here we see all these NADHs. And look at the overall change in 589 00:43:06,000 --> 00:43:11,000 free energy. The initial steps in glycolysis didn't really take 590 00:43:11,000 --> 00:43:15,000 advantage. Glucose has inherent in it almost 680 kilocalories per mole 591 00:43:15,000 --> 00:43:20,000 of energy. It's pretty high up here. But by the time we get from here 592 00:43:20,000 --> 00:43:25,000 down to here, there's an enormous release of energy, 593 00:43:25,000 --> 00:43:30,000 it's harvested in the form of these molecules which are then reinvested. 594 00:43:30,000 --> 00:43:34,000 In the absence of oxygen, this entire procedure can only go 595 00:43:34,000 --> 00:43:38,000 from here down to here. And a lot of this drop from six to 596 00:43:38,000 --> 00:43:42,000 seven is futile because we have to reinvest this NADH. 597 00:43:42,000 --> 00:43:47,000 These cannot be used, actually, to generate more ATPs, 598 00:43:47,000 --> 00:43:51,000 as I've said repeatedly. So, this means in the end that we can 599 00:43:51,000 --> 00:43:55,000 generate an enormous amount of energy in the form of these 600 00:43:55,000 --> 00:44:01,000 coupled reactions. Having said that, 601 00:44:01,000 --> 00:44:08,000 let's actually look at what happens inside of the mitochondria. 602 00:44:08,000 --> 00:44:15,000 Inside of the mitochondria there are actually different physical 603 00:44:15,000 --> 00:44:22,000 compartments. See the blue space there, the intermembrane space, 604 00:44:22,000 --> 00:44:30,000 the blue spaces there? The matrix is on the inside. 605 00:44:30,000 --> 00:44:35,000 The intermembrane space is between the two, the inner and the outer 606 00:44:35,000 --> 00:44:40,000 membrane, and outside is the cytoplasm. The outer membrane, 607 00:44:40,000 --> 00:44:45,000 the inner membrane, in between it. So, look what happens, actually, in 608 00:44:45,000 --> 00:44:50,000 the mitochondrion. Those NADHs are used to pump 609 00:44:50,000 --> 00:44:55,000 protons from the inner space of the mitochondrion into the intermembrane 610 00:44:55,000 --> 00:45:00,000 space. I'm not showing you that happening. 611 00:45:00,000 --> 00:45:05,000 But you'll have to take it on my word. So, protons pictured here are 612 00:45:05,000 --> 00:45:10,000 extracted from NADH and FADH, and they're used to pump protons out 613 00:45:10,000 --> 00:45:15,000 here. And, therefore, protons are moved from here to here. 614 00:45:15,000 --> 00:45:20,000 Obviously, when you pump protons out the pH gets lower on the outside 615 00:45:20,000 --> 00:45:25,000 than it does on the inside, and because there's a gradient, 616 00:45:25,000 --> 00:45:30,000 there's a higher concentration of protons here than on the inside. 617 00:45:30,000 --> 00:45:34,000 The protons begin to accumulate outside here in the intermembrane 618 00:45:34,000 --> 00:45:39,000 space. Are they in the cytoplasm? No. They're in the space between 619 00:45:39,000 --> 00:45:44,000 the inner and the outer membrane. You start to accumulate in this 620 00:45:44,000 --> 00:45:49,000 blue space lots of protons. And this pumping of protons into 621 00:45:49,000 --> 00:45:54,000 the space between the two membranes requires energy, 622 00:45:54,000 --> 00:45:59,000 and the energy comes from our friends NADH and FADH 623 00:45:59,000 --> 00:46:04,000 as it turns out. They are responsible for causing 624 00:46:04,000 --> 00:46:08,000 this accumulation of protons in the space between the inner and the 625 00:46:08,000 --> 00:46:12,000 outer membrane. So, now we get lots of protons out 626 00:46:12,000 --> 00:46:16,000 there. And what happens now, the protons like to flow back in 627 00:46:16,000 --> 00:46:20,000 because there is a higher concentration here as they are 628 00:46:20,000 --> 00:46:24,000 inside the space that's called the mitochondrial matrix, 629 00:46:24,000 --> 00:46:29,000 on the inside of the mitochondrion. So, what happens? 630 00:46:29,000 --> 00:46:32,000 Here, yet another Nobel Prize winning discovery is the discovery 631 00:46:32,000 --> 00:46:36,000 of a very interesting molecule, or complex of proteins I should say, 632 00:46:36,000 --> 00:46:40,000 that looks in three-dimensions roughly like this. 633 00:46:40,000 --> 00:46:44,000 And what this complex does is as the protons flow through the inner 634 00:46:44,000 --> 00:46:48,000 channel here, they're moving down an energy gradient. 635 00:46:48,000 --> 00:46:52,000 They're going from a state of high concentration to a state of low 636 00:46:52,000 --> 00:46:56,000 concentration. What that does, 637 00:46:56,000 --> 00:47:00,000 that diffusional pressure actually yields energy. 638 00:47:00,000 --> 00:47:05,000 And this complex right here harvests that energy in order to convert ADP 639 00:47:05,000 --> 00:47:10,000 into ATP. So, when I talk about NADH as being 640 00:47:10,000 --> 00:47:15,000 worth, each of them being worth three ATPs, what I'm really talking 641 00:47:15,000 --> 00:47:20,000 about is the fact that NADHs can be used to pump protons in the 642 00:47:20,000 --> 00:47:25,000 mitochondria outside here, and these protons can then be used, 643 00:47:25,000 --> 00:47:31,000 can then be pumped, can then flow in this way through this proton pump, 644 00:47:31,000 --> 00:47:36,000 which then uses ADP in the inner cavity of the mitochondria 645 00:47:36,000 --> 00:47:40,000 to create ATP. And here we get finally the 646 00:47:40,000 --> 00:47:44,000 conversion of ADP into ATP. We can realize, finally, this much 647 00:47:44,000 --> 00:47:48,000 promised benefit. And then these ATP molecules are 648 00:47:48,000 --> 00:47:52,000 exported from the mitochondria throughout the entire cell and used 649 00:47:52,000 --> 00:47:56,000 to drive many reactions. We've already encountered one 650 00:47:56,000 --> 00:48:00,000 important set of reactions, and those reactions are the 651 00:48:00,000 --> 00:48:04,000 polymerization of nucleic acids. Now, one final point I want to make 652 00:48:04,000 --> 00:48:08,000 is the following. We've just talked about metabolic, 653 00:48:08,000 --> 00:48:12,000 we've talked about the pathway of energy production in the cell. 654 00:48:12,000 --> 00:48:16,000 And you might have had the illusion, for a brief instant, 655 00:48:16,000 --> 00:48:20,000 that those are all, that's the sum of all the biochemical reactions in 656 00:48:20,000 --> 00:48:24,000 the cell. But, in fact, if we plot out all the 657 00:48:24,000 --> 00:48:28,000 biochemical reactions in the cell, they're much more complicated. Here 658 00:48:28,000 --> 00:48:31,000 is the glycolytic pathway. You see it right down here where 659 00:48:31,000 --> 00:48:35,000 nothing is named? Here is the Krebs Cycle right here. 660 00:48:35,000 --> 00:48:39,000 And we're not even talking about energy here. And as molecules move 661 00:48:39,000 --> 00:48:43,000 down this pathway from here to here to here to here, 662 00:48:43,000 --> 00:48:46,000 some of these molecules are diverted for other applications. 663 00:48:46,000 --> 00:48:50,000 Not for energy production but for other applications. 664 00:48:50,000 --> 00:48:54,000 And what happens out here, they are converted through a series 665 00:48:54,000 --> 00:48:58,000 of complex biochemical steps into other essential biological 666 00:48:58,000 --> 00:49:02,000 molecules. What do I mean by that? 667 00:49:02,000 --> 00:49:06,000 If you give E. coli, a bacterium, you give it a simple carbon source 668 00:49:06,000 --> 00:49:10,000 like glucose and you give it phosphate and you give it a simple 669 00:49:10,000 --> 00:49:14,000 nitrogen source like ammonium acetate or something, 670 00:49:14,000 --> 00:49:19,000 E. coli can, from those simple atoms generate all the amino acids, 671 00:49:19,000 --> 00:49:23,000 can generate the purines and the pyrimidines, can generate all kinds 672 00:49:23,000 --> 00:49:27,000 of different complex biological molecules just from those 673 00:49:27,000 --> 00:49:33,000 simple building blocks. And so, the process of biosynthesis 674 00:49:33,000 --> 00:49:40,000 involves not only the creation of macromolecules, 675 00:49:40,000 --> 00:49:47,000 these steps of what are called intermediary metabolism are used to 676 00:49:47,000 --> 00:49:54,000 synthesize all the other biochemical entities that one needs to make a 677 00:49:54,000 --> 00:50:01,000 cell. They're used to synthesize purines and pyrimidines. 678 00:50:01,000 --> 00:50:05,000 They're used to synthesize lipids, they're used to synthesize amino 679 00:50:05,000 --> 00:50:09,000 acids, and they're used to synthesize literally hundreds of 680 00:50:09,000 --> 00:50:13,000 other compounds. And when we see this chart like 681 00:50:13,000 --> 00:50:18,000 this, and nobody on the face of the planet has ever memorized this chart, 682 00:50:18,000 --> 00:50:22,000 each one of these steps, going from one molecule to the next, 683 00:50:22,000 --> 00:50:26,000 represents another biochemical reaction. And the vast majority of 684 00:50:26,000 --> 00:50:31,000 these biochemical reactions going from A to B to C to D. 685 00:50:31,000 --> 00:50:35,000 Each one of these steps requires the intervention of an enzyme, 686 00:50:35,000 --> 00:50:39,000 a catalyst that is specialized for that particular step. 687 00:50:39,000 --> 00:50:44,000 So, this begins to give you an appreciation of how many distinct 688 00:50:44,000 --> 00:50:48,000 biochemical steps one needs in a cell. The numbers probably to make 689 00:50:48,000 --> 00:50:53,000 a simple cell, you probably need about a thousand 690 00:50:53,000 --> 00:50:57,000 distinct biochemical reactions, each of one of which requires the 691 00:50:57,000 --> 00:51:02,000 involvement of an enzyme. And many of these steps, 692 00:51:02,000 --> 00:51:06,000 importantly, many of these biochemical steps are endergonic 693 00:51:06,000 --> 00:51:11,000 reactions. Where do they get the energy for driving these reactions 694 00:51:11,000 --> 00:51:15,000 forward if they're endergonic? ATP. So, the ATP from the energy 695 00:51:15,000 --> 00:51:20,000 generating furnace down here is the then spread throughout the cell to 696 00:51:20,000 --> 00:51:25,000 power all of these energy consuming reactions. Have a great weekend.