1 00:00:00,000 --> 00:00:01,936 [SQUEAKING] 2 00:00:01,936 --> 00:00:04,356 [RUSTLING] 3 00:00:04,356 --> 00:00:05,324 [CLICKING] 4 00:00:10,660 --> 00:00:12,370 MATTHEW VANDER HEIDEN: Hello, everybody. 5 00:00:12,370 --> 00:00:17,380 Last time, I introduced the idea of the TCA cycle, 6 00:00:17,380 --> 00:00:19,580 tricarboxylic acid cycle. 7 00:00:19,580 --> 00:00:22,570 Also known as the citric acid cycle, because citric acid 8 00:00:22,570 --> 00:00:25,690 is a tricarboxylic acid, as you'll see later today. 9 00:00:25,690 --> 00:00:27,700 Also known as the Krebs cycle, named 10 00:00:27,700 --> 00:00:31,810 after Hans Krebs, who discovered it 11 00:00:31,810 --> 00:00:35,540 in the early part of the last century. 12 00:00:35,540 --> 00:00:38,330 The TCA cycle is the series of reactions 13 00:00:38,330 --> 00:00:40,430 that occurs in the mitochondrial matrix 14 00:00:40,430 --> 00:00:43,550 and it allows the complete oxidation of two carbon 15 00:00:43,550 --> 00:00:47,090 units, derived from many things, including pyruvate, derived 16 00:00:47,090 --> 00:00:52,910 from glucose and glycolysis and enables the complete oxidation 17 00:00:52,910 --> 00:00:55,520 of that carbon to CO2. 18 00:00:55,520 --> 00:00:58,070 Now, it's a cycle because those two carbon 19 00:00:58,070 --> 00:01:03,050 units from pyruvate or other sources enter the cycle, 20 00:01:03,050 --> 00:01:05,930 combine with 4-carbon oxaloacetate, 21 00:01:05,930 --> 00:01:08,240 and combine to make 6-carbon citrate. 22 00:01:08,240 --> 00:01:11,540 Hence the citric acid cycle, or TCA 23 00:01:11,540 --> 00:01:15,800 cycle, that is then oxidized back to 4 carbon units, 24 00:01:15,800 --> 00:01:21,560 forming a cycle that allows cells 25 00:01:21,560 --> 00:01:23,270 to release lots of energy. 26 00:01:23,270 --> 00:01:25,370 Complete oxygen enables, ultimately, 27 00:01:25,370 --> 00:01:28,010 complete oxidation of glucose to CO2. 28 00:01:28,010 --> 00:01:30,560 We've talked many times how this releases energy. 29 00:01:30,560 --> 00:01:32,750 And it also generates lots of intermediates 30 00:01:32,750 --> 00:01:36,260 for the cell that can be used to make other stuff. 31 00:01:36,260 --> 00:01:39,290 Last time, I alluded to the fact that citrate can be 32 00:01:39,290 --> 00:01:42,380 used, say, to make fatty acids. 33 00:01:42,380 --> 00:01:44,360 Today, what I want to do is I want to dive 34 00:01:44,360 --> 00:01:49,010 into the details of how this TCA cycle occurs 35 00:01:49,010 --> 00:01:51,980 and the consequences of the way it works, 36 00:01:51,980 --> 00:01:55,640 and how that affects other aspects of metabolism. 37 00:01:55,640 --> 00:01:57,920 You'll see that it actually affects 38 00:01:57,920 --> 00:02:01,440 the ability of different organisms, 39 00:02:01,440 --> 00:02:03,050 whether or not they can make things 40 00:02:03,050 --> 00:02:05,930 from intermediates in this cycle, 41 00:02:05,930 --> 00:02:07,665 because of how the cycle works. 42 00:02:17,750 --> 00:02:18,395 Now, to start. 43 00:02:21,150 --> 00:02:24,980 Of course, if we're going to start from pyruvate. 44 00:02:27,870 --> 00:02:31,210 Pyruvate, of course, has three carbons. 45 00:02:31,210 --> 00:02:35,760 And so if we're going to generate a two-carbon acetate 46 00:02:35,760 --> 00:02:37,890 group, we have to lose CO2. 47 00:02:44,220 --> 00:02:47,280 And as I described a couple lectures ago, 48 00:02:47,280 --> 00:02:49,740 acetate is also the thing that we 49 00:02:49,740 --> 00:02:52,710 can derive from metabolism of ethanol, 50 00:02:52,710 --> 00:02:54,720 showing us two things-- 51 00:02:54,720 --> 00:02:57,750 metabolized glucose to pyruvate or ethanol itself-- 52 00:02:57,750 --> 00:02:59,820 can both be turned into acetate. 53 00:02:59,820 --> 00:03:05,940 But really, the donor is this molecule, acetyl-CoA. 54 00:03:05,940 --> 00:03:09,240 Which is basically also a carboxylic acid, but rather 55 00:03:09,240 --> 00:03:12,180 than being a carboxylic acid, instead makes this 56 00:03:12,180 --> 00:03:15,880 a thioester bond, which activates this carbon 57 00:03:15,880 --> 00:03:35,760 as a leaving group, such that it can combine with oxaloacetate, 58 00:03:35,760 --> 00:03:41,130 which you'll remember from gluconeogenesis, 59 00:03:41,130 --> 00:03:52,760 releasing that S-CoA molecule to generate six-carbon citric. 60 00:04:15,730 --> 00:04:16,690 So, this is citric. 61 00:04:16,690 --> 00:04:20,110 You can see that we've made a bond from this carbon 62 00:04:20,110 --> 00:04:25,700 on the acetate, losing this S-CoA group to this carbon 63 00:04:25,700 --> 00:04:30,290 oxaloacetate to make this six-carbon citrate molecule, 64 00:04:30,290 --> 00:04:32,170 which is a one, two, three-- 65 00:04:32,170 --> 00:04:36,460 three-carboxylic acid, or tricarboxylic acid. 66 00:04:36,460 --> 00:04:39,280 Hence the name citric acid cycle, 67 00:04:39,280 --> 00:04:41,450 tricarboxylic acid cycle. 68 00:04:41,450 --> 00:04:44,590 Now, these six carbons can then-- 69 00:04:44,590 --> 00:04:46,900 or this six-carbon citrate molecule 70 00:04:46,900 --> 00:04:53,740 can then be oxidized, generating two CO2 molecules that 71 00:04:53,740 --> 00:04:56,400 are released, and ultimately reforming 72 00:04:56,400 --> 00:05:01,780 oxaloacetate that can pick up another two-carbon acetyl-CoA 73 00:05:01,780 --> 00:05:05,110 to generate another citrate, and around and around the cycle 74 00:05:05,110 --> 00:05:10,030 goes, allowing in the end the net entry 75 00:05:10,030 --> 00:05:13,840 of two carbons, effectively from acetate, 76 00:05:13,840 --> 00:05:18,640 and release of two carbons as CO2. 77 00:05:18,640 --> 00:05:24,190 Now, as I alluded to, this can come from pyruvate. 78 00:05:24,190 --> 00:05:27,670 It can come from acetate itself, vinegar. 79 00:05:27,670 --> 00:05:29,200 It can come from alcohol. 80 00:05:29,200 --> 00:05:31,000 It turns out that when you break down fat, 81 00:05:31,000 --> 00:05:35,180 you also break it into two carbon units. 82 00:05:35,180 --> 00:05:38,680 And so this cycle becomes very useful for cells, 83 00:05:38,680 --> 00:05:43,600 because it allows the oxidation of many different molecules 84 00:05:43,600 --> 00:05:48,800 to completely turn that carbon into CO2. 85 00:05:48,800 --> 00:05:53,370 Now, if we're going to do this from glucose, 86 00:05:53,370 --> 00:06:05,750 however, you'll remember that pyruvate has three carbons. 87 00:06:05,750 --> 00:06:07,690 And so if we're going to turn pyruvate 88 00:06:07,690 --> 00:06:14,440 into acetate, or acetyl-CoA, we have to lose a carbon of CO2. 89 00:06:14,440 --> 00:06:17,930 We have to lose this carbon as CO2. 90 00:06:17,930 --> 00:06:21,700 Now, we saw this before, that we can do this via-- 91 00:06:21,700 --> 00:06:26,800 this is exactly how we generated ethanol 92 00:06:26,800 --> 00:06:32,830 when we did fermentation of pyruvate to ethanol, 93 00:06:32,830 --> 00:06:36,130 but that molecule didn't generate acetate, 94 00:06:36,130 --> 00:06:38,530 It generated acetaldehyde. 95 00:06:38,530 --> 00:06:42,190 The difference, of course, being whether or not this carbon gets 96 00:06:42,190 --> 00:06:44,830 oxidized in the case of acetate to the acid, 97 00:06:44,830 --> 00:06:47,200 or in acetaldehyde in ethanol metabolism, 98 00:06:47,200 --> 00:06:49,570 where it remains an aldehyde. 99 00:06:49,570 --> 00:06:52,330 Now obviously, you can make ethanol and then 100 00:06:52,330 --> 00:06:54,580 turn that ethanol into acetate. 101 00:06:54,580 --> 00:06:57,800 And that's a pathway that I guess certainly would work. 102 00:06:57,800 --> 00:07:01,420 However, that's not the way it happens in most organisms. 103 00:07:01,420 --> 00:07:07,180 Most organisms actually directly produce 104 00:07:07,180 --> 00:07:11,870 acetate, or more correctly, acetyl-CoA from pyruvate. 105 00:07:11,870 --> 00:07:14,200 So let's take a look at that reaction. 106 00:07:18,500 --> 00:07:20,370 So here once again is pyruvate. 107 00:07:24,490 --> 00:07:27,910 And if we turn that pyruvate into 108 00:07:27,910 --> 00:07:34,590 this two-carbon acetyl-CoA, let's 109 00:07:34,590 --> 00:07:37,390 look here what has to happen. 110 00:07:37,390 --> 00:07:42,060 Well, the first thing is that we have 111 00:07:42,060 --> 00:07:48,050 to decarboxylate this molecule. 112 00:07:48,050 --> 00:07:51,610 And so that generates a CO2. 113 00:07:51,610 --> 00:07:55,420 We have to lose that one carbon. 114 00:07:55,420 --> 00:08:00,010 Also, as I pointed out, if we do this 115 00:08:00,010 --> 00:08:02,320 as if we did it in ethanol metabolism, 116 00:08:02,320 --> 00:08:04,700 we'd be left with an aldehyde. 117 00:08:04,700 --> 00:08:10,930 But this is an acid, and so this carbon also has to be oxidized. 118 00:08:10,930 --> 00:08:12,040 We know how to do that. 119 00:08:12,040 --> 00:08:19,240 We can donate those electrons to something like NAD, 120 00:08:19,240 --> 00:08:23,050 so the NAD is reduced to NADH. 121 00:08:23,050 --> 00:08:30,770 And we had to add this S-CoA molecule, which 122 00:08:30,770 --> 00:08:33,450 I'll come to in a minute. 123 00:08:33,450 --> 00:08:39,730 And so each of these steps ends up 124 00:08:39,730 --> 00:08:43,720 making a fairly complicated reaction. 125 00:08:43,720 --> 00:08:46,798 Obviously, several co-factors are going to be involved. 126 00:08:46,798 --> 00:08:49,090 You should be able to guess that just by looking at it. 127 00:08:49,090 --> 00:08:51,400 Obviously, I already drew up there NAD. 128 00:08:51,400 --> 00:08:54,460 If we're going to decarboxylate, remember 129 00:08:54,460 --> 00:09:03,750 this is a alpha carboxylic, an alpha 130 00:09:03,750 --> 00:09:08,170 ketoacid-- the ketone group is alpha to the carboxylic acid. 131 00:09:08,170 --> 00:09:11,190 And so if we're doing alpha decarboxylation, 132 00:09:11,190 --> 00:09:13,500 as you might guess, we need a co-factor. 133 00:09:13,500 --> 00:09:15,210 That co-factor, as I told you before, 134 00:09:15,210 --> 00:09:17,850 is the co-factor factor thiamine pyrophosphate. 135 00:09:17,850 --> 00:09:21,940 And finally, there's this S-CoA group. 136 00:09:21,940 --> 00:09:25,320 We need to describe what that is. 137 00:09:25,320 --> 00:09:29,190 However, before delving into those, and there's 138 00:09:29,190 --> 00:09:32,160 actually a couple other factors that are needed as well, 139 00:09:32,160 --> 00:09:36,150 I want to mention one other issue 140 00:09:36,150 --> 00:09:40,980 about this reaction, in that this reaction happens 141 00:09:40,980 --> 00:09:44,820 in the mitochondria, because that's also 142 00:09:44,820 --> 00:09:46,770 where the TCA cycle happens. 143 00:09:46,770 --> 00:09:50,820 So, recall that if we divide the cell into two compartments 144 00:09:50,820 --> 00:09:53,880 like an eukaryotic cell, here we have the cytosol 145 00:09:53,880 --> 00:09:56,640 and the mitochondria in a eukaryotic cell. 146 00:09:56,640 --> 00:09:59,640 As we've already talked about, having different compartments 147 00:09:59,640 --> 00:10:02,393 helps facilitate different metabolic reactions, 148 00:10:02,393 --> 00:10:04,560 because you can have different conditions in the two 149 00:10:04,560 --> 00:10:05,860 compartments. 150 00:10:05,860 --> 00:10:15,240 And so as we said, glycolysis occurs in the cytosol. 151 00:10:15,240 --> 00:10:17,090 Glucose to pyruvate. 152 00:10:17,090 --> 00:10:26,720 And last time I mentioned, the TCA cycle 153 00:10:26,720 --> 00:10:31,340 occurs in the mitochondria. 154 00:10:31,340 --> 00:10:34,700 And so that means if we're going to fully oxidize 155 00:10:34,700 --> 00:10:38,900 the pyruvate carbon to CO2 using the TCA 156 00:10:38,900 --> 00:10:41,510 cycle in the mitochondria, that pyruvate 157 00:10:41,510 --> 00:10:44,840 has to get from the cytosol to the mitochondria, or at least 158 00:10:44,840 --> 00:10:47,408 carbons from the pyruvate have to get there. 159 00:10:47,408 --> 00:10:48,950 It turns out, you'll see in a minute, 160 00:10:48,950 --> 00:10:50,870 acetyl-CoA is a very large group. 161 00:10:50,870 --> 00:10:53,750 And so pyruvate itself is transported 162 00:10:53,750 --> 00:10:55,640 into the mitochondria. 163 00:10:55,640 --> 00:10:57,260 And that's where the reaction occurs 164 00:10:57,260 --> 00:11:00,620 to turn it into acetyl-CoA that can then enter the TCA cycle 165 00:11:00,620 --> 00:11:02,540 and oxidize. 166 00:11:02,540 --> 00:11:07,370 However, what this means is that a transporter is actually 167 00:11:07,370 --> 00:11:10,610 needed to get this across the mitochondrial membranes 168 00:11:10,610 --> 00:11:13,220 and into the matrix of the mitochondria where the TCA 169 00:11:13,220 --> 00:11:15,162 cycle happens. 170 00:11:15,162 --> 00:11:17,120 And I like to mention this because it turns out 171 00:11:17,120 --> 00:11:18,290 the pyruvate carrier-- 172 00:11:18,290 --> 00:11:19,790 that is, the transporter-- the way 173 00:11:19,790 --> 00:11:22,820 that it actually gets that pyruvate from the cytosol 174 00:11:22,820 --> 00:11:25,220 into the mitochondria actually was 175 00:11:25,220 --> 00:11:27,050 an unknown thing about metabolism 176 00:11:27,050 --> 00:11:30,380 until 2012, so not that long ago. 177 00:11:30,380 --> 00:11:35,150 Sometimes one can be left with the feeling, 178 00:11:35,150 --> 00:11:38,690 listening to these metabolism classes or reading a textbook, 179 00:11:38,690 --> 00:11:42,350 that everything about metabolism has been known forever, 180 00:11:42,350 --> 00:11:43,940 but it's actually not true. 181 00:11:43,940 --> 00:11:47,510 Here's a very key, central part of the pathway that 182 00:11:47,510 --> 00:11:49,760 was actually just discovered, at least 183 00:11:49,760 --> 00:11:52,220 at the time of this lecture, less than 10 years ago. 184 00:11:55,040 --> 00:11:59,340 It also illustrates that not all metabolism is completely 185 00:11:59,340 --> 00:12:02,510 understood. 186 00:12:02,510 --> 00:12:06,980 Now, let's get back to this reaction, how you turn pyruvate 187 00:12:06,980 --> 00:12:09,590 into acetyl-CoA. 188 00:12:09,590 --> 00:12:11,900 And I want to go through now and point out 189 00:12:11,900 --> 00:12:15,000 that several co-factors are needed. 190 00:12:15,000 --> 00:12:19,460 And so I already mentioned one of them, S-CoA, 191 00:12:19,460 --> 00:12:26,490 which is shorthand for coenzyme A. So, 192 00:12:26,490 --> 00:12:29,870 I need to tell you what that is. 193 00:12:29,870 --> 00:12:34,850 You hopefully are already familiar with TPP plus, 194 00:12:34,850 --> 00:12:36,170 thiamine pyrophosphate. 195 00:12:36,170 --> 00:12:39,290 We talked about that when we talked 196 00:12:39,290 --> 00:12:42,890 about how you do alpha decarboxylation to generate 197 00:12:42,890 --> 00:12:45,440 ethanol from pyruvate, also used here 198 00:12:45,440 --> 00:12:48,620 for the alpha decarboxylation reaction. 199 00:12:48,620 --> 00:12:51,200 Redox reaction happens, and so we 200 00:12:51,200 --> 00:12:57,760 needed NAD plus to get converted into NADH. 201 00:12:57,760 --> 00:13:01,120 We talked a lot about how that serves as an electron carrier, 202 00:13:01,120 --> 00:13:04,120 but it turns out that there's two additional electron 203 00:13:04,120 --> 00:13:07,390 carriers that are involved in this reaction. 204 00:13:07,390 --> 00:13:11,950 One of them is called FAD and the other one 205 00:13:11,950 --> 00:13:14,155 is called lipoic acid. 206 00:13:18,120 --> 00:13:19,700 Now you might say, why do we need 207 00:13:19,700 --> 00:13:22,070 all these electron carriers? 208 00:13:22,070 --> 00:13:24,260 Well, these are just different molecules 209 00:13:24,260 --> 00:13:30,600 that can carry two electrons, similar to NADH. 210 00:13:30,600 --> 00:13:34,430 And effectively, what these can do by having multiple electron 211 00:13:34,430 --> 00:13:35,480 carriers-- 212 00:13:35,480 --> 00:13:39,680 one can build chains of oxidation and reduction 213 00:13:39,680 --> 00:13:40,940 reactions. 214 00:13:40,940 --> 00:13:44,120 And it turns out these chains of oxidation reduction reactions 215 00:13:44,120 --> 00:13:48,200 really become central to energy transfers in biology, 216 00:13:48,200 --> 00:13:50,690 because building these chains allows 217 00:13:50,690 --> 00:13:54,020 more easy stepwise release of energy 218 00:13:54,020 --> 00:13:57,660 as one moves across these oxidation reduction reactions. 219 00:13:57,660 --> 00:14:00,170 Which remember, as I alluded to earlier, really 220 00:14:00,170 --> 00:14:06,890 are at the core of bioenergetics and a lot of what allows energy 221 00:14:06,890 --> 00:14:09,980 release from these pathways. 222 00:14:09,980 --> 00:14:13,490 What I mean by this will be more explicit 223 00:14:13,490 --> 00:14:18,350 as we go through what some of these co-factors look like. 224 00:14:18,350 --> 00:14:25,550 So, I'm not going to draw TPP plus or NAD again, but let's 225 00:14:25,550 --> 00:14:30,090 define what some of these other cofactors look like. 226 00:14:30,090 --> 00:14:39,410 So, let's start with coenzyme A. So, coenzyme A, it turns out, 227 00:14:39,410 --> 00:14:41,210 is useful. 228 00:14:41,210 --> 00:14:46,360 It's actually involved in lots of acylation reactions. 229 00:14:46,360 --> 00:14:48,070 What's an acylation reaction? 230 00:14:48,070 --> 00:14:52,750 Well, that's basically if you're making a carbon-carbon bond 231 00:14:52,750 --> 00:14:56,200 by adding a molecule of greater than one carbon, so two carbons 232 00:14:56,200 --> 00:14:58,030 or greater, to something else. 233 00:14:58,030 --> 00:15:00,070 That's an acylation reaction, as we 234 00:15:00,070 --> 00:15:04,810 did with adding the two-carbon acetate to oxaloacetate 235 00:15:04,810 --> 00:15:06,460 to make citrate. 236 00:15:06,460 --> 00:15:08,680 And you'll actually see coenzyme A will come up 237 00:15:08,680 --> 00:15:11,110 in this in many, many lectures throughout the rest 238 00:15:11,110 --> 00:15:12,400 of the course. 239 00:15:12,400 --> 00:15:16,750 Why this becomes useful is because it activates 240 00:15:16,750 --> 00:15:25,530 this basically carbon to the left of, in this case, 241 00:15:25,530 --> 00:15:26,450 the ketone. 242 00:15:26,450 --> 00:15:29,330 Because, by having that thioester there, 243 00:15:29,330 --> 00:15:33,620 ends up activating that carbon and allows it to carry out 244 00:15:33,620 --> 00:15:37,070 these acylation reactions. 245 00:15:37,070 --> 00:15:41,000 Coenzyme A itself is derived from a vitamin 246 00:15:41,000 --> 00:15:44,285 called pantothenic acid. 247 00:15:48,670 --> 00:15:52,720 And as a vitamin, this is something 248 00:15:52,720 --> 00:15:55,370 that you have to get from the diet. 249 00:15:55,370 --> 00:15:58,690 Now, when we abbreviate coenzyme A, 250 00:15:58,690 --> 00:16:02,440 which is often abbreviated S-CoA, 251 00:16:02,440 --> 00:16:04,960 you get the sense that it's this little tiny molecule 252 00:16:04,960 --> 00:16:06,700 that's just stuck on sulfur-- 253 00:16:06,700 --> 00:16:09,610 stuck on to the end to make this thioester bond. 254 00:16:09,610 --> 00:16:12,730 But it turns out coenzyme A is actually a giant molecule, one 255 00:16:12,730 --> 00:16:19,390 of the reasons why you actually synthesize acetyl-CoA 256 00:16:19,390 --> 00:16:20,800 in the mitochondria, because it's 257 00:16:20,800 --> 00:16:24,170 hard to transport this giant molecule. 258 00:16:24,170 --> 00:16:28,990 And so this is what pantothenic acid looks like. 259 00:16:47,920 --> 00:16:53,400 So, if I put an acid group here and a hydroxyl group there, 260 00:16:53,400 --> 00:16:55,530 that would be pantothenic acid, the thing 261 00:16:55,530 --> 00:17:00,075 that's in your cereal, on the side of your cereal box. 262 00:17:05,470 --> 00:17:07,690 Then there's these additional pieces. 263 00:17:07,690 --> 00:17:17,380 This is the active end of the molecule, 264 00:17:17,380 --> 00:17:19,839 that's that sulfur from the S-CoA, 265 00:17:19,839 --> 00:17:25,180 and then this side of the molecule is esterified to two 266 00:17:25,180 --> 00:17:28,810 phosphates, which are esterified to-- 267 00:17:31,477 --> 00:17:33,060 I'm not going to draw it out, but this 268 00:17:33,060 --> 00:17:39,200 would have an adenine base and a phosphate there. 269 00:17:39,200 --> 00:17:47,540 So basically, this is ADP, with a phosphate 270 00:17:47,540 --> 00:17:51,740 added to the 3 prime position of the ADP molecule, 271 00:17:51,740 --> 00:17:56,000 added to pantothenic acid, added to this short chain 272 00:17:56,000 --> 00:17:57,560 with the sulfur on the end. 273 00:17:57,560 --> 00:18:01,550 And this whole molecule together is coenzyme A. 274 00:18:01,550 --> 00:18:03,530 And so when we say acetyl-CoA, it's 275 00:18:03,530 --> 00:18:12,630 this giant molecule, S-thioester to the carbonyl, 276 00:18:12,630 --> 00:18:16,950 the acid on acetate, to CH3. 277 00:18:16,950 --> 00:18:20,680 And so that would be, basically, acetyl-CoA. 278 00:18:20,680 --> 00:18:24,030 And so one convenient thing about this 279 00:18:24,030 --> 00:18:26,610 is it's much easier than drawing that big molecule, 280 00:18:26,610 --> 00:18:30,930 but it is a little bit misleading in terms 281 00:18:30,930 --> 00:18:32,670 of its size. 282 00:18:32,670 --> 00:18:35,970 So, that's coenzyme A. 283 00:18:35,970 --> 00:18:42,240 Next one I want to talk about is FAD 284 00:18:42,240 --> 00:18:51,120 which stands for flavin adenine dinucleotide. 285 00:18:54,600 --> 00:18:58,110 So, flavin adenine dinucleotide is an electron carrier, 286 00:18:58,110 --> 00:18:59,835 just like NAD plus. 287 00:19:05,330 --> 00:19:11,480 And it's derived from the vitamin riboflavin, 288 00:19:11,480 --> 00:19:14,480 also referred to as vitamin B2. 289 00:19:17,240 --> 00:19:21,410 Another thing from the side of your cereal box, and basically 290 00:19:21,410 --> 00:19:23,440 looks like this. 291 00:19:23,440 --> 00:19:31,670 So, like NAD, it's a dinucleotide. 292 00:19:40,140 --> 00:19:45,270 And so here's ADP, just like we do for CoA, or just 293 00:19:45,270 --> 00:19:47,430 like one end of NAD. 294 00:19:47,430 --> 00:19:51,330 One difference is that the other nucleotide down here 295 00:19:51,330 --> 00:19:54,010 actually isn't technically a sugar. 296 00:19:54,010 --> 00:19:57,240 It's ribitol instead of ribose. 297 00:19:57,240 --> 00:19:58,240 What does that mean? 298 00:19:58,240 --> 00:19:59,940 It doesn't have an aldehyde. 299 00:19:59,940 --> 00:20:04,710 Instead, it is just a five-carbon chain 300 00:20:04,710 --> 00:20:08,010 where all of the carbons are alcohols. 301 00:20:14,450 --> 00:20:16,250 And so since there's no aldehyde, 302 00:20:16,250 --> 00:20:18,200 it doesn't form a ring. 303 00:20:45,150 --> 00:20:49,680 And the base on this end, as a nicotinamide, 304 00:20:49,680 --> 00:21:07,180 is this flavin group, which looks like this. 305 00:21:07,180 --> 00:21:15,740 And so this is FAD, which is in the oxidized form. 306 00:21:15,740 --> 00:21:19,250 Turns out, in the oxidized form, FAD 307 00:21:19,250 --> 00:21:25,520 is yellow, hence riboflavin-- "flavin", yellow. 308 00:21:25,520 --> 00:21:27,410 And the reason it's yellow-- you see 309 00:21:27,410 --> 00:21:30,770 there's a conjugated double bond here across this part. 310 00:21:30,770 --> 00:21:34,460 It turns out this is the active part of the molecule, 311 00:21:34,460 --> 00:21:35,900 and it works as follows. 312 00:21:35,900 --> 00:21:38,540 And so if you have a hydride ion, 313 00:21:38,540 --> 00:21:41,600 remember the way we can transfer two electrons. 314 00:21:46,390 --> 00:21:49,270 Can transfer the two electrons that way, 315 00:21:49,270 --> 00:21:50,920 and that allows it to generate. 316 00:21:50,920 --> 00:21:53,620 And I'll just draw the middle, here. 317 00:21:53,620 --> 00:21:56,320 Active part of the molecule. 318 00:22:11,180 --> 00:22:13,510 So, that would be these two nitrogens. 319 00:22:13,510 --> 00:22:15,350 We've added two electrons to it. 320 00:22:15,350 --> 00:22:20,590 And so this is abbreviated FADH2, 321 00:22:20,590 --> 00:22:24,700 or the reduced form of FAD. 322 00:22:24,700 --> 00:22:29,060 And it is colorless, because now you no longer 323 00:22:29,060 --> 00:22:32,810 have that conjugated double bond system, loses its color. 324 00:22:32,810 --> 00:22:36,590 And so you can follow whether FAD is oxidized or reduced 325 00:22:36,590 --> 00:22:40,550 by just looking at color change. 326 00:22:40,550 --> 00:22:42,500 So, that's FAD. 327 00:22:42,500 --> 00:22:45,890 It's an electron carrier, carries electrons 328 00:22:45,890 --> 00:22:49,130 by a hydride transfer, very similarly 329 00:22:49,130 --> 00:22:52,370 to what we described for NAD, but obviously 330 00:22:52,370 --> 00:22:54,890 a different molecule. 331 00:22:54,890 --> 00:22:59,000 And the last molecule, the last co-factor 332 00:22:59,000 --> 00:23:05,910 that we need for this reaction, is lipoic acid. 333 00:23:05,910 --> 00:23:08,220 Which, unlike most things in metabolism, 334 00:23:08,220 --> 00:23:12,840 doesn't have an abbreviation and also functions 335 00:23:12,840 --> 00:23:14,700 as an electron carrier. 336 00:23:14,700 --> 00:23:17,040 And so lipoic acid looks like this. 337 00:23:37,570 --> 00:23:40,810 So, that is lipoic acid. 338 00:23:40,810 --> 00:23:45,190 This is in the oxidized form. 339 00:23:45,190 --> 00:23:48,100 And so where it's oxidized is here 340 00:23:48,100 --> 00:23:49,660 at this sulfur-sulfur bond. 341 00:23:49,660 --> 00:23:53,380 So, you can think of this as the same as a disulfide bond 342 00:23:53,380 --> 00:23:57,110 that you learned about from Professor Yaffe in proteins. 343 00:23:57,110 --> 00:24:01,390 And so this is the oxidized form of the disulfide bond. 344 00:24:01,390 --> 00:24:09,230 If I take this hydride, transfer two electrons across that bond, 345 00:24:09,230 --> 00:24:13,675 then it goes to-- and I'll just draw here the active end. 346 00:24:19,370 --> 00:24:25,110 Then we go here to the reduced form of lipoic acid, 347 00:24:25,110 --> 00:24:28,020 and there's no abbreviation for oxidized 348 00:24:28,020 --> 00:24:29,700 or reduced lipoic acid. 349 00:24:29,700 --> 00:24:33,960 It's just lipoic acid, oxidized lipoic acid, reduced. 350 00:24:33,960 --> 00:24:36,210 And how they're oxidized and reduced 351 00:24:36,210 --> 00:24:41,550 is basically very similar to the disulfide bonds 352 00:24:41,550 --> 00:24:46,770 that you saw in proteins being oxidized, disulfide bond being 353 00:24:46,770 --> 00:24:52,350 oxidized, or it can be reduced to not be a disulfide bond. 354 00:24:52,350 --> 00:25:02,760 Now, it turns out that these cofactors, our TPP, FAD, 355 00:25:02,760 --> 00:25:07,680 and lipoic acid, are all stably associated 356 00:25:07,680 --> 00:25:14,060 with different subunits of a multi-protein complex 357 00:25:14,060 --> 00:25:20,300 that assembles to catalyze that reaction to convert pyruvate 358 00:25:20,300 --> 00:25:23,330 to acetyl-CoA. 359 00:25:23,330 --> 00:25:25,610 The enzyme or enzyme complex that 360 00:25:25,610 --> 00:25:29,210 carries this out is referred to as PDH, 361 00:25:29,210 --> 00:25:32,630 sometimes abbreviated PDC, which stands 362 00:25:32,630 --> 00:25:46,110 for the pyruvate dehydrogenase complex. 363 00:25:46,110 --> 00:25:50,190 So, pyruvate dehydrogenase, PDH, pyruvate dehydrogenase complex, 364 00:25:50,190 --> 00:25:51,790 PDC. 365 00:25:51,790 --> 00:25:56,460 This complex is basically three different polypeptides 366 00:25:56,460 --> 00:25:58,650 that come together to form a complex 367 00:25:58,650 --> 00:26:00,960 and catalyze that reaction. 368 00:26:00,960 --> 00:26:03,610 Now, where this complex sits-- 369 00:26:03,610 --> 00:26:05,640 so, this is the mitochondria. 370 00:26:05,640 --> 00:26:07,770 This is the matrix. 371 00:26:07,770 --> 00:26:10,530 That's where this pyruvate dehydrogenase reaction occurs. 372 00:26:10,530 --> 00:26:13,170 That's where the TCA cycle occurs. 373 00:26:13,170 --> 00:26:15,630 And this PDH complex is basically 374 00:26:15,630 --> 00:26:19,590 sitting here at the membrane on the matrix side 375 00:26:19,590 --> 00:26:22,450 of the membrane. 376 00:26:22,450 --> 00:26:24,720 Now, the three different polypeptides 377 00:26:24,720 --> 00:26:26,970 that come together to form this complex 378 00:26:26,970 --> 00:26:35,010 are creatively named E1, E2, and E3, four. 379 00:26:35,010 --> 00:26:37,680 Enzyme one, two, and three. 380 00:26:37,680 --> 00:26:41,010 And each of these, as I alluded to, 381 00:26:41,010 --> 00:26:44,567 is associated with a different co-factor. 382 00:26:49,710 --> 00:26:56,760 And so E1 is associated with thymine pyrophosphate. 383 00:26:56,760 --> 00:27:07,510 E2 is associated with lipoic acid. 384 00:27:07,510 --> 00:27:12,675 And E3 is associated with FAD. 385 00:27:17,090 --> 00:27:20,120 Now, let's go through the mechanism 386 00:27:20,120 --> 00:27:25,345 for how this pyruvate dehydrogenase reaction works. 387 00:28:22,470 --> 00:28:27,950 So remember, this is the active part of TBP plus. 388 00:28:27,950 --> 00:28:32,450 It is bound in the active site of E1. 389 00:28:32,450 --> 00:28:43,600 And it reacts with pyruvate to catalyze alpha decarboxylation, 390 00:28:43,600 --> 00:28:48,550 just like we described for conversion of pyruvate 391 00:28:48,550 --> 00:28:50,110 to alcohol. 392 00:28:50,110 --> 00:28:53,800 So, you're going to see exactly the same mechanism 393 00:28:53,800 --> 00:28:56,490 that we drew before. 394 00:29:15,980 --> 00:29:20,450 So, that decarboxylates the alpha-keto acid, 395 00:29:20,450 --> 00:29:27,440 just like we saw to generate acetaldehyde. 396 00:29:33,390 --> 00:29:37,290 The difference is rather than resolve this such 397 00:29:37,290 --> 00:29:39,780 that this carbon has the same oxidation state 398 00:29:39,780 --> 00:29:44,520 and make acetaldehyde, instead the next step of this reaction 399 00:29:44,520 --> 00:29:56,230 is going to be oxidized by reducing lipoic acid. 400 00:29:56,230 --> 00:29:59,040 So, the active part of lipoic acid 401 00:29:59,040 --> 00:30:04,530 that is in the active site of the E2 subunit. 402 00:31:20,750 --> 00:31:27,170 So this will now regenerate E1, but what we're left with, 403 00:31:27,170 --> 00:31:30,820 then, is this. 404 00:31:30,820 --> 00:31:34,810 Now, this intermediate bound to E2. 405 00:31:45,130 --> 00:31:54,170 Here's where coenzyme A can come in, which 406 00:31:54,170 --> 00:32:04,260 will then generate acetyl-CoA. 407 00:32:07,650 --> 00:32:19,890 But now we are left with E2 in the reduced state, 408 00:32:19,890 --> 00:32:22,950 rather than being in the oxidized state. 409 00:32:22,950 --> 00:32:27,990 So E2 has to be re-oxidized in order for this complex 410 00:32:27,990 --> 00:32:32,820 to carry out the next catalytic cycle, and the way 411 00:32:32,820 --> 00:32:35,130 that works is as follows. 412 00:32:35,130 --> 00:32:41,130 So you have FAD bound on E3. 413 00:32:41,130 --> 00:32:54,550 And so you have a hydride ion from the oxidation of E3 414 00:32:54,550 --> 00:32:58,120 that can be transferred to FAD. 415 00:32:58,120 --> 00:33:07,500 That will generate FAD from the oxidized to the reduced form. 416 00:33:07,500 --> 00:33:16,200 So re-oxidize lipoic acid on E2, reduce FAD to FADH2, 417 00:33:16,200 --> 00:33:20,010 and then that FADH2 can be re-oxidized back 418 00:33:20,010 --> 00:33:26,420 to FAD via transferring those electrons to NAD 419 00:33:26,420 --> 00:33:31,720 plus to generate NADH. 420 00:33:31,720 --> 00:33:37,180 So in this case, FADH2 re-oxidized the FAD, NAD 421 00:33:37,180 --> 00:33:41,080 plus reduced to NADH. 422 00:33:41,080 --> 00:33:44,410 And so, effectively, what this happens 423 00:33:44,410 --> 00:33:49,090 is that enzyme E1 and E2 cooperate 424 00:33:49,090 --> 00:34:02,830 to call what's referred to as oxidative alpha decarboxylation 425 00:34:02,830 --> 00:34:07,090 of pyruvate, while adding -CoA, so that's 426 00:34:07,090 --> 00:34:15,090 where you get acetyl-CoA, with reduction of lipoic acid in E2, 427 00:34:15,090 --> 00:34:20,370 and then E3 re-oxidizes the lipoic acid in E2 428 00:34:20,370 --> 00:34:24,409 while generating NADH. 429 00:34:24,409 --> 00:34:26,677 This NADH, once it's generated, of course 430 00:34:26,677 --> 00:34:28,219 those electrons have to go somewhere, 431 00:34:28,219 --> 00:34:30,409 so they, like in glycolysis-- 432 00:34:30,409 --> 00:34:33,889 it also needs to regenerate to NAD at some point. 433 00:34:33,889 --> 00:34:38,120 This is ultimately the electrons that end up on oxygen, 434 00:34:38,120 --> 00:34:42,590 and it's really this series of electron transfers, 435 00:34:42,590 --> 00:34:45,500 with oxygen being a good electron acceptor, that 436 00:34:45,500 --> 00:34:50,570 ultimately allows controlled energy release during carbon 437 00:34:50,570 --> 00:34:51,739 oxidation. 438 00:34:51,739 --> 00:34:55,070 And cells, you'll see, can use that to make ATP or do 439 00:34:55,070 --> 00:34:58,940 other kinds of work. 440 00:34:58,940 --> 00:35:02,720 So, the net reaction and/or another way 441 00:35:02,720 --> 00:35:07,160 to draw the pyruvate dehydrogenase reaction 442 00:35:07,160 --> 00:35:15,390 would be as follows, and that's taking pyruvate to acetyl-CoA. 443 00:35:18,890 --> 00:35:27,980 And so we're going to take coenzyme A and release CO2. 444 00:35:27,980 --> 00:35:42,500 This is done by TPP plus, as part of E1. 445 00:35:42,500 --> 00:35:52,660 That involves converting the lipoic acid in E2 446 00:35:52,660 --> 00:35:56,080 from the oxidized to the reduced state. 447 00:35:56,080 --> 00:36:00,130 That lipoic acid then has to be re-oxidized. 448 00:36:00,130 --> 00:36:02,260 If something's oxidized, something else 449 00:36:02,260 --> 00:36:03,790 has to be reduced. 450 00:36:03,790 --> 00:36:10,090 That's FAD on E3, which also then cycles 451 00:36:10,090 --> 00:36:14,020 between the oxidized and reduce state. 452 00:36:14,020 --> 00:36:17,440 And ultimately, those electrons ended up 453 00:36:17,440 --> 00:36:24,010 being transferred to NAD plus to generate NADH. 454 00:36:24,010 --> 00:36:32,380 And so the PDH complex is basically a chain 455 00:36:32,380 --> 00:36:41,010 of electron transfer reactions. 456 00:36:41,010 --> 00:36:45,690 And it's the first example of a chain of electron transfer 457 00:36:45,690 --> 00:36:46,230 reactions. 458 00:36:46,230 --> 00:36:48,630 We're going to see that there is the electron transport 459 00:36:48,630 --> 00:36:51,900 chain in the mitochondria, effectively does 460 00:36:51,900 --> 00:36:52,870 the same thing. 461 00:36:52,870 --> 00:36:56,400 And by coupling oxidation and reduction reactions 462 00:36:56,400 --> 00:37:00,480 across chains of molecules like this, effectively as a preview, 463 00:37:00,480 --> 00:37:03,930 allows the stepwise energy release of these oxidation 464 00:37:03,930 --> 00:37:08,170 reactions to occur. 465 00:37:08,170 --> 00:37:11,550 So remember, if we burn glucose, completely oxidize it in one 466 00:37:11,550 --> 00:37:13,750 step, where those electrons are directly transferred 467 00:37:13,750 --> 00:37:15,510 to oxygen in combustion. 468 00:37:15,510 --> 00:37:18,720 Lots of energy released, but all in one step. 469 00:37:18,720 --> 00:37:21,210 By doing these stepwise electron transfers, 470 00:37:21,210 --> 00:37:25,110 we can then basically break up that energy release 471 00:37:25,110 --> 00:37:29,190 in a way that can be captured by cells to do work. 472 00:37:29,190 --> 00:37:31,050 Here, the way energy is captured, 473 00:37:31,050 --> 00:37:34,860 it's not so obvious in this electron transport reaction. 474 00:37:34,860 --> 00:37:38,580 But effectively, you're using oxidation 475 00:37:38,580 --> 00:37:46,770 of the ketone on pyruvate, with decarboxylation to the acid 476 00:37:46,770 --> 00:37:50,400 to generate, rather than just the acid, a thioester bond. 477 00:37:50,400 --> 00:37:52,470 And that's that thioester bond-- 478 00:37:52,470 --> 00:37:55,110 as well as NADH, that's energy as well-- but it's 479 00:37:55,110 --> 00:37:58,620 really that thioester bond that then can be recaptured later 480 00:37:58,620 --> 00:38:03,420 to drive synthesis of citrate in the TCA cycle. 481 00:38:06,480 --> 00:38:11,970 Now, next what I want to do is I want to discuss the TCA cycle 482 00:38:11,970 --> 00:38:12,600 reactions. 483 00:38:12,600 --> 00:38:15,420 Now that you see how you can get acetyl-CoA, at least 484 00:38:15,420 --> 00:38:18,450 from pyruvate, I want to discuss how you can now 485 00:38:18,450 --> 00:38:23,910 use that acetyl-CoA and oxidize it back to combine it 486 00:38:23,910 --> 00:38:27,960 with oxaloacetate, and oxidize it back 487 00:38:27,960 --> 00:38:31,350 to make citrate, and then oxidize it back to oxaloacetate 488 00:38:31,350 --> 00:38:35,490 to run the TCA cycle. 489 00:38:35,490 --> 00:38:37,960 OK, great. 490 00:38:37,960 --> 00:38:54,120 So, the first reaction of the TCA cycle. 491 00:38:54,120 --> 00:39:00,090 Here's acetyl-CoA. 492 00:39:00,090 --> 00:39:13,530 It will combine with oxaloacetate. 493 00:39:26,420 --> 00:39:58,130 This reaction is catalyzed by an enzyme called citrate synthase, 494 00:39:58,130 --> 00:40:05,480 and generates the six-carbon tricarboxylic acid citrate. 495 00:40:05,480 --> 00:40:09,340 So, how does this reaction work? 496 00:40:47,990 --> 00:40:52,920 Here's drawing acetyl-CoA in a slightly different way. 497 00:40:52,920 --> 00:41:25,620 If I redraw this as the enol, this 498 00:41:25,620 --> 00:42:26,340 will generate citrate via, effectively, that mechanism. 499 00:42:29,610 --> 00:42:42,600 Next, citrate is converted by adding water 500 00:42:42,600 --> 00:42:48,450 across this carbon-carbon, or by removing water 501 00:42:48,450 --> 00:42:56,790 across this carbon-carbon bond, to generate 502 00:42:56,790 --> 00:42:59,260 this intermediate called cis-aconitate. 503 00:43:31,989 --> 00:43:34,660 So this intermediate is called cis-aconitate. 504 00:43:38,260 --> 00:43:41,440 so all I did was dehydrate across that bond 505 00:43:41,440 --> 00:43:43,340 so there's a double bond there. 506 00:43:43,340 --> 00:43:56,180 And then if I re-add water across that bond, 507 00:43:56,180 --> 00:44:19,640 I generate this molecule called isocitrate. 508 00:44:22,240 --> 00:44:26,500 So effectively, to convert citrate to isocitrate, 509 00:44:26,500 --> 00:44:29,980 I'm moving the hydroxyl group from that carbon 510 00:44:29,980 --> 00:44:31,630 to this carbon. 511 00:44:31,630 --> 00:44:35,760 To do that, I basically dehydrate, make a double bond, 512 00:44:35,760 --> 00:44:38,710 remove water, re-add water across that bond 513 00:44:38,710 --> 00:44:41,950 in the opposite direction to generate this molecule, 514 00:44:41,950 --> 00:44:43,550 isocitrate. 515 00:44:43,550 --> 00:45:00,210 This reaction is carried out by an enzyme called aconitase, 516 00:45:00,210 --> 00:45:05,510 and converts citrate to isocitrate. 517 00:45:05,510 --> 00:45:10,820 I think it's a little easier to see this reaction 518 00:45:10,820 --> 00:45:13,090 if I draw it a slightly different way. 519 00:46:12,590 --> 00:46:14,890 So, this here is just drawing citrate 520 00:46:14,890 --> 00:46:21,560 by just slightly rotating the molecule to look like that. 521 00:46:21,560 --> 00:46:24,490 And so I'm basically removing water here. 522 00:46:51,040 --> 00:46:57,100 And then I'm now just adding water back 523 00:46:57,100 --> 00:47:20,850 in the opposite orientation to generate isocitrate. 524 00:47:28,830 --> 00:47:33,300 Now, you'll notice when I drew this-- 525 00:47:33,300 --> 00:47:36,900 if you look at citrate, this is actually a symmetrical 526 00:47:36,900 --> 00:47:43,380 molecule, so the top half and the bottom half of citrate 527 00:47:43,380 --> 00:47:45,700 are identical. 528 00:47:45,700 --> 00:47:49,410 And so what's interesting about nature is 529 00:47:49,410 --> 00:47:51,510 that it treats these carbons-- 530 00:47:51,510 --> 00:47:54,060 the green carbons that came from acetyl-CoA-- 531 00:47:54,060 --> 00:47:57,270 different from the side of the molecule that 532 00:47:57,270 --> 00:48:00,210 comes from oxaloacetate. 533 00:48:00,210 --> 00:48:05,370 And effectively, nature always moves the hydroxyl group 534 00:48:05,370 --> 00:48:08,820 to this carbon that came from oxaloacetate, 535 00:48:08,820 --> 00:48:11,670 and never moves it to this carbon 536 00:48:11,670 --> 00:48:15,270 that came from acetyl-CoA. 537 00:48:15,270 --> 00:48:18,240 This is an example where enzymes-- 538 00:48:18,240 --> 00:48:22,950 nature treats a symmetrical molecule like citrate 539 00:48:22,950 --> 00:48:25,080 in an asymmetrical way. 540 00:48:25,080 --> 00:48:29,910 And this has consequences for how carbon is actually traced 541 00:48:29,910 --> 00:48:32,400 through the entire TCA cycle. 542 00:48:32,400 --> 00:48:34,590 Because even though you might think things could get 543 00:48:34,590 --> 00:48:37,230 scrambled at citrate, they never do. 544 00:48:37,230 --> 00:48:41,580 Meaning an isocitrate-- it's always these green carbons 545 00:48:41,580 --> 00:48:43,320 that came from acetyl-CoA. 546 00:48:43,320 --> 00:48:45,180 You never get those green carbons 547 00:48:45,180 --> 00:48:48,960 on the other side of isocitrate. 548 00:48:48,960 --> 00:48:50,970 And so when we go through the TCA cycle, 549 00:48:50,970 --> 00:48:54,030 I'll keep these carbons green until the point where 550 00:48:54,030 --> 00:48:58,290 you can no longer distinguish which carbon came from 551 00:48:58,290 --> 00:48:59,585 came from which reaction. 552 00:49:02,160 --> 00:49:07,740 The next reaction is we're going to oxidize 553 00:49:07,740 --> 00:49:12,030 this carbon of isocitrate. 554 00:49:12,030 --> 00:49:14,550 So if we're going to oxidize that carbon, 555 00:49:14,550 --> 00:49:17,370 those electrons have to go somewhere. 556 00:49:36,310 --> 00:49:40,150 And so if we oxidize the carbon, we 557 00:49:40,150 --> 00:49:49,240 can use NAD plus as an electron acceptor, reduce it to NADH. 558 00:49:49,240 --> 00:49:58,250 That generates this intermediate. 559 00:50:20,460 --> 00:50:23,940 So hopefully this is clear to everybody at this point, 560 00:50:23,940 --> 00:50:26,150 but just in case. 561 00:50:29,230 --> 00:50:34,560 So, this carbon here and isocitrate. 562 00:50:34,560 --> 00:50:49,570 If I oxidize that alcohol to the ketone, 563 00:50:49,570 --> 00:50:51,610 now I generate a hydride ion. 564 00:50:51,610 --> 00:50:54,280 Those two electrons and the hydrogen can go to NAD plus 565 00:50:54,280 --> 00:50:57,070 and reduce it to NADH. 566 00:50:57,070 --> 00:50:59,590 That generates this intermediate. 567 00:51:02,430 --> 00:51:06,220 It's called oxalosuccinate. 568 00:51:06,220 --> 00:51:11,860 Which then, if you notice, the oxalosuccinate 569 00:51:11,860 --> 00:51:16,420 is now a beta keto acid. 570 00:51:16,420 --> 00:51:22,420 So alpha, beta. 571 00:51:22,420 --> 00:51:25,730 The acid group is beta to the ketone, 572 00:51:25,730 --> 00:51:27,680 so it's a beta keto acid. 573 00:51:27,680 --> 00:51:32,270 Remember, beta decarboxylation is favorable, 574 00:51:32,270 --> 00:51:35,860 and so I can lose that CO2. 575 00:51:35,860 --> 00:51:55,170 And what I'm left with is this molecule, which 576 00:51:55,170 --> 00:52:06,100 is called alpha keto glutarate. 577 00:52:06,100 --> 00:52:14,370 So this whole reaction here, the oxidation 578 00:52:14,370 --> 00:52:16,590 of the alcohol to the ketone, followed 579 00:52:16,590 --> 00:52:20,010 by beta decarboxylation of oxalosuccinate 580 00:52:20,010 --> 00:52:22,560 to alpha ketoglutarate, is carried out 581 00:52:22,560 --> 00:52:28,680 by an enzyme called isocitrate dehydrogenase. 582 00:52:34,310 --> 00:52:50,662 And of course, just to remind you, 583 00:52:50,662 --> 00:52:58,660 here's that beta keto acid in oxalosuccinate. 584 00:53:04,510 --> 00:53:20,570 And so that can decarboxylate, leading this enol. 585 00:53:20,570 --> 00:53:35,800 Which can, of course, rearrange back to the ketone that we see, 586 00:53:35,800 --> 00:53:42,250 an alpha ketoglutarate. 587 00:53:42,250 --> 00:53:49,710 Now, if you look at alpha ketoglutarate, 588 00:53:49,710 --> 00:53:56,865 you'll notice that-- and I will redraw over here. 589 00:54:11,940 --> 00:54:18,630 So, this here is just me redrawing alpha ketoglutarate, 590 00:54:18,630 --> 00:54:23,960 which is often abbreviated alpha kG. 591 00:54:23,960 --> 00:54:26,370 Just drew it as a straight line. 592 00:54:26,370 --> 00:54:29,300 Now, if you'll notice, alpha keto glutarate 593 00:54:29,300 --> 00:54:32,750 is a alpha ketoacid. 594 00:54:32,750 --> 00:54:37,620 And so here the acid group is alpha to the ketone. 595 00:54:37,620 --> 00:54:39,620 So, an alpha keto acid. 596 00:54:39,620 --> 00:54:42,170 It's effectively just like pyruvate, 597 00:54:42,170 --> 00:54:45,110 but has this additional pieces on it. 598 00:54:45,110 --> 00:54:48,740 And it turns out the next step in the TCA cycle 599 00:54:48,740 --> 00:54:52,820 is the exact reaction that we saw 600 00:54:52,820 --> 00:54:56,240 with pyruvate dehydrogenase. 601 00:54:56,240 --> 00:55:01,250 It's alpha decarboxylation, oxidative alpha 602 00:55:01,250 --> 00:55:22,280 keto acid decarboxylation, as follows. 603 00:55:22,280 --> 00:55:25,090 So, we carry out. 604 00:55:57,150 --> 00:56:04,710 This is a molecule called succinyl-CoA. 605 00:56:04,710 --> 00:56:10,830 And so, you'll see what happened there is decarboxylated here, 606 00:56:10,830 --> 00:56:18,030 this carbon, while oxidizing this ketone to the acid 607 00:56:18,030 --> 00:56:19,800 and adding -CoA. 608 00:56:19,800 --> 00:56:21,400 That's a redox reaction. 609 00:56:21,400 --> 00:56:28,830 So NAD, NADH, it turns out this is exactly the same mechanism-- 610 00:56:28,830 --> 00:56:30,360 so I don't need to draw it again-- 611 00:56:30,360 --> 00:56:34,740 that I just showed you for pyruvate dehydrogenase. 612 00:56:34,740 --> 00:56:36,720 So it needs all the same cofactors-- 613 00:56:36,720 --> 00:56:40,770 TPP plus, lipoic acid, FAD. 614 00:56:40,770 --> 00:56:47,010 And in fact, it even shares some of the same enzyme complexes, 615 00:56:47,010 --> 00:56:51,720 subunits, as pyruvate dehydrogenase. 616 00:56:51,720 --> 00:56:57,330 And so this is a reaction that's catalyzed by alpha 617 00:56:57,330 --> 00:56:59,460 ketoglutarate dehydrogenase. 618 00:57:02,520 --> 00:57:06,210 Like pyruvate dehydrogenase, this is a complex, 619 00:57:06,210 --> 00:57:09,650 and so it has a unique E1, which makes sense. 620 00:57:09,650 --> 00:57:12,000 Remember, E1 of pyruvate dehydrogenase 621 00:57:12,000 --> 00:57:15,030 was actually the subunit that bound the pyruvate. 622 00:57:15,030 --> 00:57:17,820 E1 of alpha ketoglutarate dehydrogenase is unique. 623 00:57:17,820 --> 00:57:20,400 It binds alpha ketoglutarate instead of pyruvate. 624 00:57:20,400 --> 00:57:24,750 However, they share the same E2 and E3s, 625 00:57:24,750 --> 00:57:28,140 and so the E2s and E3s would carry out 626 00:57:28,140 --> 00:57:32,340 exactly the same reaction and play the same part 627 00:57:32,340 --> 00:57:38,400 in the mechanism of how you do this alpha oxidative alpha 628 00:57:38,400 --> 00:57:41,100 decarboxylation to turn alpha ketoglutarate 629 00:57:41,100 --> 00:57:45,330 into succinyl-CoA. 630 00:57:45,330 --> 00:57:47,340 Now, remember in pyruvate dehydrogenase, 631 00:57:47,340 --> 00:57:49,110 once we got that acetyl-CoA, we then 632 00:57:49,110 --> 00:57:52,680 use this CoA group to drive condensation with citrate. 633 00:57:52,680 --> 00:57:54,540 Well, in this case, what happens is 634 00:57:54,540 --> 00:57:58,110 you don't want to combine condensation here. 635 00:57:58,110 --> 00:58:00,690 Instead, what is going to happen is 636 00:58:00,690 --> 00:58:08,160 you want to use this CoA group to now generate ATP. 637 00:58:08,160 --> 00:58:15,210 And so this is going to couple release of the CoA 638 00:58:15,210 --> 00:58:17,430 to generate an ATP equivalent. 639 00:58:17,430 --> 00:58:22,750 It's actually GTP that's generated by the TCA cycle. 640 00:58:22,750 --> 00:58:34,630 And so, this is going to generate 641 00:58:34,630 --> 00:58:39,490 this molecule, succinate. 642 00:58:39,490 --> 00:58:47,790 And the enzyme that does this is called succinic bio-kinase. 643 00:58:56,930 --> 00:59:01,205 So, let's go through over here how this enzyme works. 644 00:59:12,800 --> 00:59:24,950 So here's succinyl-CoA, basically using favorable loss 645 00:59:24,950 --> 00:59:40,920 of the CoA breaking the thioester bond to generate 646 00:59:40,920 --> 00:59:42,750 this acid anhydride. 647 00:59:42,750 --> 00:59:48,150 We saw an acid anhydride before in glycolysis. 648 00:59:48,150 --> 00:59:51,060 Remember, we made 1-3-bisphosphoglycerate, 649 00:59:51,060 --> 00:59:54,180 so that's a good phosphate donor. 650 00:59:54,180 --> 00:59:59,110 And then that can be used to generate succinate, 651 00:59:59,110 --> 01:00:05,560 and transferring that phosphate to GDP to make GTP, 652 01:00:05,560 --> 01:00:08,620 just like 1-3-bisphosphoglycerate, 653 01:00:08,620 --> 01:00:10,420 was able to transfer the phosphate 654 01:00:10,420 --> 01:00:18,370 from the acid anhydride to ADP to make ATP in glycolysis. 655 01:00:20,950 --> 01:00:34,490 So, the next step is to oxidize this carbon-carbon bond 656 01:00:34,490 --> 01:00:37,400 in succinate. 657 01:00:37,400 --> 01:00:41,690 So if we're going to oxidize a carbon-carbon bond, 658 01:00:41,690 --> 01:00:44,090 those electrons have to go somewhere. 659 01:00:50,670 --> 01:00:53,790 So there's our carbon-carbon bond. 660 01:01:01,670 --> 01:01:13,200 And so if we generate hydride ion 661 01:01:13,200 --> 01:01:18,270 just like we did in other oxidation reactions, 662 01:01:18,270 --> 01:01:23,880 but this hydride ion is transferred not to NAD, 663 01:01:23,880 --> 01:01:30,510 but instead to FAD, a different electron carrier, 664 01:01:30,510 --> 01:01:34,680 to generate if FADH2. 665 01:01:34,680 --> 01:01:38,370 Now, I should point out succinate, like citrate, is 666 01:01:38,370 --> 01:01:40,050 a symmetrical molecule. 667 01:01:40,050 --> 01:01:43,680 But at this point, nature doesn't tell the difference. 668 01:01:43,680 --> 01:01:47,440 Once it generates succinate, this molecule 669 01:01:47,440 --> 01:01:48,750 now gets scrambled. 670 01:01:48,750 --> 01:01:51,330 And so everything downstream of succinate, 671 01:01:51,330 --> 01:01:54,750 you no longer know which carbons came from acetyl-CoA. 672 01:02:05,770 --> 01:02:10,540 This generates this molecule called fumarate, 673 01:02:10,540 --> 01:02:19,020 and this reaction is carried out by an enzyme called 674 01:02:19,020 --> 01:02:32,020 succinate dehydrogenase, often abbreviated SDH 675 01:02:32,020 --> 01:02:34,570 for succinate dehydrogenase. 676 01:02:37,850 --> 01:02:44,900 Now, the next reaction is, we're going 677 01:02:44,900 --> 01:02:50,520 to add water across this double bond of fumarate. 678 01:03:28,270 --> 01:03:33,695 And that generates this intermediate, malate. 679 01:03:37,950 --> 01:03:42,630 This reaction is carried out by an enzyme 680 01:03:42,630 --> 01:03:51,930 called fumarate hydratase. 681 01:03:51,930 --> 01:03:55,320 And it's simply adding a water molecule 682 01:03:55,320 --> 01:03:58,470 across that double bond. 683 01:03:58,470 --> 01:04:01,710 Once we have malate, if you look what's 684 01:04:01,710 --> 01:04:06,450 the difference between malate and oxaloacetate, 685 01:04:06,450 --> 01:04:11,550 the difference is that in oxaloacetate, this carbon 686 01:04:11,550 --> 01:04:12,960 is a ketone. 687 01:04:12,960 --> 01:04:15,390 Whereas in malate, it's an alcohol. 688 01:04:15,390 --> 01:04:18,150 And so if we want to turn this carbon from the alcohol 689 01:04:18,150 --> 01:04:24,490 into the ketone, that is, of course, a oxidation reaction, 690 01:04:24,490 --> 01:04:32,470 and so those electrons have to go somewhere. 691 01:04:32,470 --> 01:04:36,790 Don't need to draw the mechanism again. 692 01:04:36,790 --> 01:04:41,080 It's basically just the hydride transfer 693 01:04:41,080 --> 01:04:45,720 to oxidize this to an alcohol, to the ketone. 694 01:04:45,720 --> 01:04:51,180 That oxidation couples to a reduction of NAD plus to NADH. 695 01:04:51,180 --> 01:04:58,093 This is carried out by an enzyme called malate dehydrogenase. 696 01:05:07,190 --> 01:05:12,980 And doing this completes the TCA cycle, 697 01:05:12,980 --> 01:05:16,160 regenerating oxaloacetate, that can then 698 01:05:16,160 --> 01:05:18,680 recombine with another acetyl-CoA 699 01:05:18,680 --> 01:05:24,200 to go through another round of the cycle. 700 01:05:24,200 --> 01:05:28,700 You'll notice that going through this cycle, 701 01:05:28,700 --> 01:05:32,090 there's two CO2s lost. 702 01:05:32,090 --> 01:05:39,020 One of them is lost here at the isocitrate to alpha 703 01:05:39,020 --> 01:05:40,860 ketoglutarate reaction. 704 01:05:40,860 --> 01:05:45,415 So, this decarboxylation from oxaloacetate 705 01:05:45,415 --> 01:05:46,670 to alpha ketoglutarate. 706 01:05:46,670 --> 01:05:50,000 That beta decarboxylation, that's the first CO2. 707 01:05:50,000 --> 01:05:53,405 The other one is lost here at the alpha ketoglutarate. 708 01:05:57,370 --> 01:06:00,100 The alpha ketoglutarate dehydrogenase step, 709 01:06:00,100 --> 01:06:03,970 where you have this alpha decarboxylation 710 01:06:03,970 --> 01:06:07,540 to take alpha ketoglutarate to succinyl-CoA 711 01:06:07,540 --> 01:06:12,280 oxidative alpha decarboxylation. 712 01:06:12,280 --> 01:06:15,670 Now, what's cool about this, as you noticed, 713 01:06:15,670 --> 01:06:20,770 we just discussed all the reactions of the TCA cycle. 714 01:06:20,770 --> 01:06:23,460 And I showed you, reminded you, of some chemistry 715 01:06:23,460 --> 01:06:26,010 that you've already seen, but unless you 716 01:06:26,010 --> 01:06:29,475 count the chemistry we showed you earlier for how the PDH 717 01:06:29,475 --> 01:06:31,350 and alpha ketoglutarate dehydrogenase 718 01:06:31,350 --> 01:06:35,610 reactions work with E1, E2 and E3, with lipoic acid, FAD. 719 01:06:35,610 --> 01:06:37,300 That was obviously new for today. 720 01:06:37,300 --> 01:06:39,600 But other than that, everything else 721 01:06:39,600 --> 01:06:42,240 was chemistry that you've already seen. 722 01:06:42,240 --> 01:06:43,890 And this really points out the point 723 01:06:43,890 --> 01:06:47,460 that I made earlier, that metabolism is really variations 724 01:06:47,460 --> 01:06:49,230 on relatively few reactions. 725 01:06:49,230 --> 01:06:52,630 We've just repurposed some of the same tricks, if you will, 726 01:06:52,630 --> 01:06:55,700 that we're used in glycolysis, and allowed 727 01:06:55,700 --> 01:07:00,090 it to now do an entire different pathway, the TCA cycle. 728 01:07:00,090 --> 01:07:02,870 It also points out how Hans Krebs was-- well, is still-- 729 01:07:02,870 --> 01:07:06,530 remarkable, able to figure out from chemistry alone, 730 01:07:06,530 --> 01:07:09,410 because there's actually quite a bit of logic to the way 731 01:07:09,410 --> 01:07:12,030 metabolism works. 732 01:07:12,030 --> 01:07:13,280 Now, I want to say this again. 733 01:07:13,280 --> 01:07:16,280 Note there were two carbons that entered acetyl-CoA, 734 01:07:16,280 --> 01:07:20,050 and two carbons that were lost to CO2. 735 01:07:20,050 --> 01:07:22,690 But if you look, the green carbons 736 01:07:22,690 --> 01:07:27,190 remain in the same places until they get to succinate. 737 01:07:27,190 --> 01:07:29,320 and so the two carbons that enter 738 01:07:29,320 --> 01:07:32,350 are not lost on the first turn of the cycle. 739 01:07:32,350 --> 01:07:37,810 It's actually two carbons that came from oxaloacetate 740 01:07:37,810 --> 01:07:42,220 that are converted to CO2 as that acetyl-CoA goes 741 01:07:42,220 --> 01:07:43,340 through the cycle. 742 01:07:43,340 --> 01:07:47,920 And so to oxidize the exact carbons from acetyl-CoA to CO2 743 01:07:47,920 --> 01:07:53,220 requires more than one turn of the cycle. 744 01:07:53,220 --> 01:07:57,660 You'll also notice that the cycle is oxidation. 745 01:07:57,660 --> 01:08:01,670 And so oxidation reactions, of course, release energy. 746 01:08:01,670 --> 01:08:02,670 We've talked about that. 747 01:08:02,670 --> 01:08:05,070 And so it's favorable. 748 01:08:05,070 --> 01:08:12,035 And the products, if you will, are three NADH molecules. 749 01:08:12,035 --> 01:08:16,500 You can say plus one more NADH if we're going all the way 750 01:08:16,500 --> 01:08:21,029 from glucose or from pyruvate. 751 01:08:21,029 --> 01:08:24,120 Glucose derived pyruvate because the pyruvate dehydrogenase 752 01:08:24,120 --> 01:08:29,649 reaction also generates an NADH to make that a acetyl-CoA. 753 01:08:29,649 --> 01:08:38,359 One FADH2, as well as one GTP molecule. 754 01:08:38,359 --> 01:08:41,569 And so lots of oxidation going on here. 755 01:08:41,569 --> 01:08:44,350 We completely oxidized two carbons to CO2, 756 01:08:44,350 --> 01:08:47,229 so that's energy release. 757 01:08:47,229 --> 01:08:52,279 But you notice you only get one GTP from the molecule. 758 01:08:52,279 --> 01:08:55,510 Now, this GTP, of course-- that reaction, 759 01:08:55,510 --> 01:08:57,939 the succinic thiokinase reaction, 760 01:08:57,939 --> 01:09:00,010 like the reactions we saw on glycolysis, 761 01:09:00,010 --> 01:09:04,540 is such that it can generate GTP at a high DGP-GDP 762 01:09:04,540 --> 01:09:06,218 ratio, or ATP-ADP ratio. 763 01:09:06,218 --> 01:09:07,510 Remember, those are equivalent. 764 01:09:07,510 --> 01:09:10,899 Those energy charge are similar, and so that makes sense. 765 01:09:10,899 --> 01:09:15,250 But most of the energy released is actually 766 01:09:15,250 --> 01:09:19,960 reducing NAD and FAD to NADH and FADH2. 767 01:09:19,960 --> 01:09:25,300 And of course, these need to transfer their electrons 768 01:09:25,300 --> 01:09:28,420 somewhere else, and that's the role of oxygen. 769 01:09:28,420 --> 01:09:31,250 Oxygen, remember, is a very good electron donor, 770 01:09:31,250 --> 01:09:34,479 and so it's the ultimate transfer of those electrons 771 01:09:34,479 --> 01:09:40,750 from these molecules to oxygen that also provide energy 772 01:09:40,750 --> 01:09:44,740 that the cell can use to do work, but it does so, in a way, 773 01:09:44,740 --> 01:09:49,910 by charging up different ratios in the cell. 774 01:09:49,910 --> 01:09:56,830 So the NAD-NADH or the FADH2-FAD ratios. 775 01:09:56,830 --> 01:10:02,020 And just like we talked about, the ratio or the energy of ATP 776 01:10:02,020 --> 01:10:05,170 is in the interconversion between ATP and ADP. 777 01:10:05,170 --> 01:10:09,190 It's the ratio that drives the free energy change. 778 01:10:09,190 --> 01:10:13,780 The same thing exists for an NADH and NAD, FADH2 and FAD. 779 01:10:13,780 --> 01:10:16,990 And so charging up these ratios while passing through the TCA 780 01:10:16,990 --> 01:10:19,810 cycle, and the ultimate downstream transfer 781 01:10:19,810 --> 01:10:22,540 of those electrons to oxygen, really 782 01:10:22,540 --> 01:10:26,650 is where most of the energy is captured as carbon 783 01:10:26,650 --> 01:10:30,220 is oxidized through the TCA cycle. 784 01:10:30,220 --> 01:10:35,080 And exactly how that works and how it can be related to ATP 785 01:10:35,080 --> 01:10:37,750 will be something that'll be more explicit in the coming 786 01:10:37,750 --> 01:10:39,300 lectures. 787 01:10:39,300 --> 01:10:43,200 Now, I want to point out apart from the oxidation, 788 01:10:43,200 --> 01:10:46,360 there's actually lots of intermediates made here. 789 01:10:46,360 --> 01:10:48,450 And it turns out, a bunch of these intermediates 790 01:10:48,450 --> 01:10:51,610 are useful for cells to make stuff. 791 01:10:51,610 --> 01:10:53,250 So we talked about gluconeogenesis. 792 01:10:53,250 --> 01:10:55,170 Gluconeogenesis needs electron balance. 793 01:10:55,170 --> 01:10:58,140 We get NADH from the TCA cycle, and so you 794 01:10:58,140 --> 01:11:01,290 can think of gluconeogenesis as an alternative 795 01:11:01,290 --> 01:11:03,440 to fermentation to dispose of electrons. 796 01:11:03,440 --> 01:11:06,000 Well, you can use the NADH from the TCA cycle 797 01:11:06,000 --> 01:11:08,980 to run gluconeogenesis as well. 798 01:11:08,980 --> 01:11:11,620 But beyond the cofactors, the carbon itself. 799 01:11:11,620 --> 01:11:14,460 So citrate, I've alluded to now a few times, 800 01:11:14,460 --> 01:11:17,130 is important as a precursor to make fat. 801 01:11:17,130 --> 01:11:19,620 We'll discuss that in later lectures, too. 802 01:11:19,620 --> 01:11:22,710 But other intermediates in this pathway 803 01:11:22,710 --> 01:11:26,710 are useful for various amino acids and nucleic acids. 804 01:11:26,710 --> 01:11:29,340 And so there's lots of things that 805 01:11:29,340 --> 01:11:32,490 can come from the TCA cycle that cells 806 01:11:32,490 --> 01:11:37,840 can find useful to do, not just catabolism, 807 01:11:37,840 --> 01:11:41,880 but also anabolic processes. 808 01:11:41,880 --> 01:11:46,090 Now, the way the TCA cycle works, though, 809 01:11:46,090 --> 01:11:48,660 is that there's actually an issue 810 01:11:48,660 --> 01:11:52,140 if you want to use the intermediates from the TCA 811 01:11:52,140 --> 01:11:55,120 cycle to make stuff. 812 01:11:55,120 --> 01:11:59,890 And so what is that issue? 813 01:11:59,890 --> 01:12:05,418 Well, the TCA cycle functions at a site as a cycle. 814 01:12:05,418 --> 01:12:07,710 And so, if we're going to take things in and out of it, 815 01:12:07,710 --> 01:12:11,730 that has consequences for how the cycle runs. 816 01:12:11,730 --> 01:12:14,740 Now there's a couple words for this 817 01:12:14,740 --> 01:12:17,200 that I want to just introduce to you. 818 01:12:17,200 --> 01:12:20,370 The first one is cataplerosis and the second one 819 01:12:20,370 --> 01:12:23,200 is anaplerosis. 820 01:12:23,200 --> 01:12:32,680 And so cataplerosis is the act of removing stuff 821 01:12:32,680 --> 01:12:34,225 from a metabolic cycle. 822 01:12:38,030 --> 01:12:39,680 So, we're going to remove citrate 823 01:12:39,680 --> 01:12:41,120 from the cycle to make fat. 824 01:12:41,120 --> 01:12:42,860 That's cataplerosis. 825 01:12:42,860 --> 01:12:57,540 And anaplerosis is adding stuff back to a metabolic cycle 826 01:12:57,540 --> 01:13:01,380 so it can continue to function. 827 01:13:01,380 --> 01:13:05,130 Viewing this is really evident if you think of the TCA cycle 828 01:13:05,130 --> 01:13:07,330 as a chicken and egg problem. 829 01:13:07,330 --> 01:13:09,670 So, the very first time acetyl-CoA 830 01:13:09,670 --> 01:13:12,720 was generated, how do you start the TCA 831 01:13:12,720 --> 01:13:14,490 cycle in the first place? 832 01:13:14,490 --> 01:13:16,800 You can't add it to the TCA cycle 833 01:13:16,800 --> 01:13:19,920 unless you have oxaloacetate to combine 834 01:13:19,920 --> 01:13:22,050 with the acetyl-CoA, which can then 835 01:13:22,050 --> 01:13:25,170 generate another oxaloacetate. 836 01:13:25,170 --> 01:13:29,500 So where does the first oxaloacetate come from? 837 01:13:29,500 --> 01:13:31,560 Well, we already talked about one reaction. 838 01:13:31,560 --> 01:13:35,040 We talked about it in the context of gluconeogenesis. 839 01:13:35,040 --> 01:13:36,400 That can solve this problem. 840 01:13:36,400 --> 01:13:38,220 And so we have pyruvate. 841 01:13:38,220 --> 01:13:40,380 And so we talked earlier today how 842 01:13:40,380 --> 01:13:45,720 we can do oxidative decarboxylation of pyruvate 843 01:13:45,720 --> 01:13:48,450 to give acetyl-CoA. 844 01:13:48,450 --> 01:13:50,160 But we talked to in the gluconeogenesis 845 01:13:50,160 --> 01:13:55,470 lecture how we can add a CO2 pyruvate to generate 846 01:13:55,470 --> 01:13:57,270 oxaloacetate. 847 01:13:57,270 --> 01:13:59,910 So if I do those two reactions, now I 848 01:13:59,910 --> 01:14:06,400 have all the carbon I need to generate a citrate 849 01:14:06,400 --> 01:14:11,190 and start off the TCA cycle. 850 01:14:11,190 --> 01:14:16,130 Now obviously, if I do cataplerosis 851 01:14:16,130 --> 01:14:19,220 and I remove that citrate I made to make fat, 852 01:14:19,220 --> 01:14:21,200 well, now I need two pyruvate again 853 01:14:21,200 --> 01:14:25,940 to generate the next citrate if I'm using this pathway, 854 01:14:25,940 --> 01:14:29,420 because every time I bring an acetyl-CoA into the cycle, 855 01:14:29,420 --> 01:14:32,660 I need an oxaloacetate to combine it with. 856 01:14:32,660 --> 01:14:35,840 And so if I remove something, I have to add something back. 857 01:14:35,840 --> 01:14:39,740 And so pyruvate to oxaloacetate, the pyruvate carboxylase 858 01:14:39,740 --> 01:14:45,850 reaction is an example of an anaplerotic reaction. 859 01:14:45,850 --> 01:14:49,900 Now what this means, though, is that in order 860 01:14:49,900 --> 01:14:52,600 to do an anaplerosis, you have to be 861 01:14:52,600 --> 01:14:56,950 able to generate four-carbon oxaloacetate, 862 01:14:56,950 --> 01:14:59,780 or a four-carbon molecule. 863 01:14:59,780 --> 01:15:02,590 Now, pyruvate carboxylase, pyruvate to oxaloacetate, 864 01:15:02,590 --> 01:15:03,802 allows you to do that. 865 01:15:03,802 --> 01:15:05,260 We can take a three-carbon molecule 866 01:15:05,260 --> 01:15:08,080 and generate four-carbon oxaloacetate. 867 01:15:08,080 --> 01:15:12,940 However, if we start from a two-carbon molecule 868 01:15:12,940 --> 01:15:20,210 like acetate or acetyl-CoA that enters the cycle, 869 01:15:20,210 --> 01:15:24,410 it's actually not so simple to take that two-carbon molecule 870 01:15:24,410 --> 01:15:27,350 and turn it into four-carbon oxaloacetate. 871 01:15:27,350 --> 01:15:31,070 And in fact, humans lack any enzymes 872 01:15:31,070 --> 01:15:33,440 that allow them to take a two-carbon unit, 873 01:15:33,440 --> 01:15:36,680 to take acetate or acetyl-CoA, and turn it 874 01:15:36,680 --> 01:15:41,000 into anything that's longer than net-- turn 875 01:15:41,000 --> 01:15:44,270 it into anything that's longer than two carbons. 876 01:15:44,270 --> 01:15:46,100 And this has important implications 877 01:15:46,100 --> 01:15:49,070 for human physiology, because what it says 878 01:15:49,070 --> 01:15:52,370 is that we can't make glucose from anything 879 01:15:52,370 --> 01:15:56,580 that starts with something less than three carbons long. 880 01:15:56,580 --> 01:15:59,750 So if you drink alcohol and you metabolize that alcohol 881 01:15:59,750 --> 01:16:02,510 to acetate, it turns out you take fat 882 01:16:02,510 --> 01:16:05,330 and you break down fat also to acetyl-CoA, 883 01:16:05,330 --> 01:16:07,520 to acetate two carbons long. 884 01:16:07,520 --> 01:16:13,460 There is no way to turn those molecules into glucose, 885 01:16:13,460 --> 01:16:16,610 because you cannot generate the oxaloacetate to do 886 01:16:16,610 --> 01:16:21,480 the anaplerosis that's necessary to get it there. 887 01:16:24,230 --> 01:16:30,020 What this means is that our body can only 888 01:16:30,020 --> 01:16:33,200 store calories that come from two carbon units, 889 01:16:33,200 --> 01:16:35,600 fat or alcohol, as fat. 890 01:16:35,600 --> 01:16:41,202 We can never turn them back into glucose or make glycogen. 891 01:16:41,202 --> 01:16:43,160 And this is very relevant for those of you that 892 01:16:43,160 --> 01:16:44,577 go to medical school, because it's 893 01:16:44,577 --> 01:16:46,670 relevant to our physiology. 894 01:16:46,670 --> 01:16:48,710 And that is, when our bodies exhaust 895 01:16:48,710 --> 01:16:51,320 all of our stores of glucose, what happens? 896 01:16:51,320 --> 01:16:54,140 Our liver can no longer do gluconeogenesis. 897 01:16:54,140 --> 01:16:55,130 And so what happens? 898 01:16:55,130 --> 01:16:57,450 Now it has to switch over to doing something else. 899 01:16:57,450 --> 01:16:59,370 It has to work with two carbon units. 900 01:16:59,370 --> 01:17:02,150 And ultimately, this is ketone metabolism, which we'll 901 01:17:02,150 --> 01:17:05,600 talk about in a few lectures. 902 01:17:05,600 --> 01:17:07,655 It also said that the body-- 903 01:17:11,310 --> 01:17:15,060 that this is also the basis of a very old adage that's 904 01:17:15,060 --> 01:17:17,280 out there that some of you may have heard-- 905 01:17:17,280 --> 01:17:20,220 that you need to have some other fuel if you're 906 01:17:20,220 --> 01:17:22,260 going to burn fat. 907 01:17:22,260 --> 01:17:25,740 The basis for that is that fat is turned into two carbon 908 01:17:25,740 --> 01:17:27,840 units, acetyl-CoA units. 909 01:17:27,840 --> 01:17:30,180 And so if you're going to take those acetyl-CoA units 910 01:17:30,180 --> 01:17:33,090 and ultimately burn them away, turn them into CO2, 911 01:17:33,090 --> 01:17:35,190 you need a source of oxaloacetate, 912 01:17:35,190 --> 01:17:37,590 or your TCA cycle won't work. 913 01:17:37,590 --> 01:17:40,290 And you don't need a lot of something, but it is true. 914 01:17:40,290 --> 01:17:45,000 You can't start just with acetyl-CoA as a human and turn 915 01:17:45,000 --> 01:17:46,860 it into CO2, so you need some-- 916 01:17:46,860 --> 01:17:49,200 at least a little bit of oxaloacetate 917 01:17:49,200 --> 01:17:52,410 to get your TCA cycle started. 918 01:17:52,410 --> 01:17:57,335 Now, that's a problem that we as humans and other mammals face, 919 01:17:57,335 --> 01:17:59,460 but it turns out there's lots of microbes out there 920 01:17:59,460 --> 01:18:02,880 that grow just fine on acetate or on alcohol, 921 01:18:02,880 --> 01:18:04,980 even if it's only carbon source. 922 01:18:04,980 --> 01:18:08,550 And so those organisms must have some way 923 01:18:08,550 --> 01:18:10,830 to build stuff from two carbon units. 924 01:18:10,830 --> 01:18:15,280 That is a way to use two carbon units and do an anaplerosis. 925 01:18:15,280 --> 01:18:17,610 And it turns out the way they do this is via something 926 01:18:17,610 --> 01:18:20,410 called the glyoxylate cycle. 927 01:18:23,050 --> 01:18:26,220 And so the glyoxylate cycle is an alternative version 928 01:18:26,220 --> 01:18:29,610 of the TCA cycle that effectively uses two enzymes 929 01:18:29,610 --> 01:18:32,010 that we lack as mammals. 930 01:18:32,010 --> 01:18:34,230 And so I'll quickly tell you about it here. 931 01:18:47,920 --> 01:18:57,420 So, this is isocitrate from the TCA cycle. 932 01:18:57,420 --> 01:19:08,270 And some microbes have an enzyme called isocitrate lyase. 933 01:19:08,270 --> 01:19:18,430 And what isocitrate lyase does is basically splits citrate 934 01:19:18,430 --> 01:19:23,260 in half, such that the top portion of the molecule 935 01:19:23,260 --> 01:19:30,190 is another TCA cycle intermediate, succinate. 936 01:19:30,190 --> 01:19:34,150 And the bottom portion of a molecule 937 01:19:34,150 --> 01:19:42,850 is this two carbon aldehyde called glyoxylate. 938 01:19:42,850 --> 01:20:00,890 Glyoxylate can react with acetyl-CoA by another enzyme 939 01:20:00,890 --> 01:20:08,390 that we lack as humans called malate synthase. 940 01:20:11,030 --> 01:20:14,880 And I don't have time to show the mechanism again, 941 01:20:14,880 --> 01:20:19,160 but malate synthase basically adds the two carbons 942 01:20:19,160 --> 01:20:26,180 from acetyl-CoA to the aldehyde, the carbonyl, 943 01:20:26,180 --> 01:20:29,900 the aldehyde carbonyl of glyoxylate in a reaction that 944 01:20:29,900 --> 01:20:33,620 is, for all intents and purposes, exactly what happens 945 01:20:33,620 --> 01:20:36,050 in citrate synthase. 946 01:20:36,050 --> 01:20:51,930 That will generate this molecule, which is malate, also 947 01:20:51,930 --> 01:20:54,370 in the TCA cycle. 948 01:20:54,370 --> 01:20:57,990 And so having these two extra reactions, isocitrate lyase 949 01:20:57,990 --> 01:21:01,920 and malate synthase, gives microbes 950 01:21:01,920 --> 01:21:08,040 the ability to have acetyl-CoA be anaplerotic. 951 01:21:08,040 --> 01:21:09,450 And so how does that work? 952 01:21:09,450 --> 01:21:15,840 Well, that's because if we start with one oxaloacetate, four 953 01:21:15,840 --> 01:21:26,400 carbons, and acetyl-CoA, two carbons, can run the TCA cycle 954 01:21:26,400 --> 01:21:31,110 and make citrate, six carbons. 955 01:21:31,110 --> 01:21:33,884 Turn that citrate into isocitrate. 956 01:21:37,950 --> 01:21:49,090 Use isocitrate lyase to generate glyoxylate, two carbons. 957 01:21:49,090 --> 01:21:51,550 Plus succinate. 958 01:21:55,310 --> 01:22:02,070 That succinate can run through succinate dehydrogenase 959 01:22:02,070 --> 01:22:04,050 to generate malate, which you can 960 01:22:04,050 --> 01:22:08,190 go through malate dehydrogenase to generate oxaloacetate. 961 01:22:08,190 --> 01:22:15,930 This glyoxylate can start with a second acetyl-CoA. 962 01:22:15,930 --> 01:22:19,890 Two carbons come together, generate malate. 963 01:22:19,890 --> 01:22:22,770 That generates a second malate molecule, 964 01:22:22,770 --> 01:22:30,180 which can then exit the cycle as malate or oxaloacetate 965 01:22:30,180 --> 01:22:32,130 or whatever you want. 966 01:22:32,130 --> 01:22:36,420 And so basically, it allows two acetyl-CoAs to net 967 01:22:36,420 --> 01:22:41,040 generate an oxaloacetate, and so net generates a way 968 01:22:41,040 --> 01:22:45,090 to do an anaplerosis from two carbon units 969 01:22:45,090 --> 01:22:51,160 by having this malate synthase reaction 970 01:22:51,160 --> 01:22:57,360 and this isocitrate lyase reaction 971 01:22:57,360 --> 01:23:00,930 and run this alternative version of the TCA cycle, 972 01:23:00,930 --> 01:23:03,990 called the glyoxylate cycle. 973 01:23:03,990 --> 01:23:08,100 And it's a nice way how life-- again, no new chemistry here, 974 01:23:08,100 --> 01:23:10,740 just variations on what we've already shown-- 975 01:23:10,740 --> 01:23:12,690 repurposed the similar chemistries 976 01:23:12,690 --> 01:23:16,620 that it's already using as a way to live off 977 01:23:16,620 --> 01:23:24,540 of carbon sources that contain only two carbons, like ethanol 978 01:23:24,540 --> 01:23:27,350 or acetate. 979 01:23:27,350 --> 01:23:31,400 So in closing today, the last thing 980 01:23:31,400 --> 01:23:34,430 I want to talk about, very briefly, 981 01:23:34,430 --> 01:23:40,610 is how the TCA cycle is regulated. 982 01:23:40,610 --> 01:23:42,620 And we don't need to spend a ton of time 983 01:23:42,620 --> 01:23:48,710 on this, because it really follows principles that 984 01:23:48,710 --> 01:23:54,340 make sense, particularly when we think about things that we've 985 01:23:54,340 --> 01:23:55,820 already talk about. 986 01:23:55,820 --> 01:23:59,800 And so, regulation of the TCA cycles is of course important, 987 01:23:59,800 --> 01:24:03,430 and it's really a critical hub, both for anabolic and catabolic 988 01:24:03,430 --> 01:24:03,950 pathways. 989 01:24:03,950 --> 01:24:06,940 So, you needed to get energy to fully oxidize carbon, 990 01:24:06,940 --> 01:24:10,252 but it's also a useful place to get stuff. 991 01:24:10,252 --> 01:24:11,710 And before I talk about regulation, 992 01:24:11,710 --> 01:24:14,590 I just want to point out that many of the enzymes in the TCA 993 01:24:14,590 --> 01:24:17,650 cycle, even though we talk about it in the mitochondria 994 01:24:17,650 --> 01:24:19,990 and we're about to talk about regulation in terms 995 01:24:19,990 --> 01:24:22,660 of catabolism-- that is oxidation, 996 01:24:22,660 --> 01:24:25,060 ways to release energy-- 997 01:24:25,060 --> 01:24:27,700 many of these enzymes are also in other locations 998 01:24:27,700 --> 01:24:32,170 in the cell, because there's functions for them in making 999 01:24:32,170 --> 01:24:35,020 stuff that is very different than what goes on in the TCA 1000 01:24:35,020 --> 01:24:36,010 cycle. 1001 01:24:36,010 --> 01:24:39,460 And the regulation that I'll tell you about, 1002 01:24:39,460 --> 01:24:42,280 and that comes up on MCAT exams and stuff 1003 01:24:42,280 --> 01:24:45,460 like that, usually talks about this pathway 1004 01:24:45,460 --> 01:24:49,940 as a catabolic pathway, as a way to make CO2, to make ATP. 1005 01:24:49,940 --> 01:24:53,380 However, recognize that there's also 1006 01:24:53,380 --> 01:24:57,610 variations on this pathway that can use in anabolic things, 1007 01:24:57,610 --> 01:24:58,720 making stuff. 1008 01:24:58,720 --> 01:25:03,700 And that regulation is something that really is not necessarily 1009 01:25:03,700 --> 01:25:05,590 what we're going to talk about here 1010 01:25:05,590 --> 01:25:09,790 and is a little bit less well-understood. 1011 01:25:09,790 --> 01:25:11,530 But at least the regulation, in terms 1012 01:25:11,530 --> 01:25:18,920 of catabolism, if we put it in context of glucose metabolism-- 1013 01:25:18,920 --> 01:25:25,890 so here's glycolysis, turning glucose into pyruvate. 1014 01:25:32,340 --> 01:25:35,550 And then that pyruvate can operate 1015 01:25:35,550 --> 01:25:40,290 through the pyruvate dehydrogenase reaction 1016 01:25:40,290 --> 01:25:44,430 to generate acetyl-CoA. 1017 01:25:44,430 --> 01:25:50,190 That acetyl-CoA can combine with oxaloacetate 1018 01:25:50,190 --> 01:25:53,782 to generate citrate. 1019 01:25:53,782 --> 01:25:57,530 That citrate, of course, can be used to generate fat, 1020 01:25:57,530 --> 01:26:00,400 as we've talked about. 1021 01:26:00,400 --> 01:26:02,080 That can go to isocitrate. 1022 01:26:04,940 --> 01:26:09,270 Isocitrate to alpha ketoglutarate. 1023 01:26:09,270 --> 01:26:13,500 That's catalyzed by isocitrate dehydrogenase, 1024 01:26:13,500 --> 01:26:15,630 which I will abbreviate IDH. 1025 01:26:15,630 --> 01:26:19,750 Alpha ketoglutarate to succinate. 1026 01:26:19,750 --> 01:26:30,830 More correctly, to succinyl-CoA by alpha ketoglutarate 1027 01:26:30,830 --> 01:26:35,240 dehydrogenase, Alpha KGDH. 1028 01:26:35,240 --> 01:26:38,150 And then this back to oxaloacetate. 1029 01:26:38,150 --> 01:26:41,210 And so the main enzymes that are typically 1030 01:26:41,210 --> 01:26:45,130 discussed as being regulated are alpha ketoglutarate 1031 01:26:45,130 --> 01:26:48,110 dehydrogenase, isocitrate dehydrogenase, 1032 01:26:48,110 --> 01:26:50,180 and pyruvate dehydrogenase. 1033 01:26:50,180 --> 01:26:53,190 I also drew glycolysis. 1034 01:26:53,190 --> 01:26:58,260 And I'll just write over here, gluconeogenesis up here, 1035 01:26:58,260 --> 01:27:04,570 because we can now see more fully how this plays 1036 01:27:04,570 --> 01:27:09,760 in with citrate acting as a positive regulator 1037 01:27:09,760 --> 01:27:15,830 with gluconeogenesis and an inhibitor of glycolysis. 1038 01:27:15,830 --> 01:27:21,410 Again, we're full in citrate, stop running glycolysis, 1039 01:27:21,410 --> 01:27:24,950 start running gluconeogenesis. 1040 01:27:24,950 --> 01:27:26,690 Now, the other regulation. 1041 01:27:26,690 --> 01:27:30,080 If you have a lot of acetyl-CoA, stop making it 1042 01:27:30,080 --> 01:27:34,820 from pyruvate. acetyl-CoA is a negative regulator of PDH. 1043 01:27:34,820 --> 01:27:37,550 This is carbon oxidation. 1044 01:27:37,550 --> 01:27:41,030 Releases a lot of energy, generates a lot of ATP, 1045 01:27:41,030 --> 01:27:44,150 generates a lot of NADH. 1046 01:27:44,150 --> 01:27:46,010 If you have lots of those things, 1047 01:27:46,010 --> 01:27:49,390 no reason to keep sending carbon into 1048 01:27:49,390 --> 01:27:52,960 acetyl-CoA to go into the TCA cycle. 1049 01:27:52,960 --> 01:27:56,733 Ultimately, we have to send those electrons somewhere. 1050 01:27:56,733 --> 01:27:58,150 So if there's nowhere to put them, 1051 01:27:58,150 --> 01:28:02,500 low oxygen also inhibits pyruvate dehydrogenase. 1052 01:28:02,500 --> 01:28:06,670 And of course, if you need more energy, if ADP is high, 1053 01:28:06,670 --> 01:28:10,000 that activates pyruvate dehydrogenase. 1054 01:28:10,000 --> 01:28:14,320 So at least in terms of glucose, complete glucose oxidation, 1055 01:28:14,320 --> 01:28:18,280 a lot of regulation happens at pyruvate dehydrogenase. 1056 01:28:18,280 --> 01:28:22,050 And a lot of it really makes sense. 1057 01:28:22,050 --> 01:28:27,450 High levels of ATP, high levels of NADH, 1058 01:28:27,450 --> 01:28:34,170 also inhibit isocitrate dehydrogenase and alpha 1059 01:28:34,170 --> 01:28:38,690 ketoglutarate dehydrogenase. 1060 01:28:38,690 --> 01:28:39,840 Makes sense. 1061 01:28:39,840 --> 01:28:44,990 Succinol-CoA high, inhibit alpha ketoglutarate dehydrogenase. 1062 01:28:44,990 --> 01:28:52,220 High levels of ADP, need to release more energy, 1063 01:28:52,220 --> 01:28:56,300 activate alpha ketoglutarate dehydrogenase. 1064 01:28:59,180 --> 01:29:02,030 Again, these are the feedbacks that people talk about 1065 01:29:02,030 --> 01:29:03,590 on board exams. 1066 01:29:03,590 --> 01:29:06,230 Good to know, but you can almost guess what they 1067 01:29:06,230 --> 01:29:08,210 would be from first principles. 1068 01:29:08,210 --> 01:29:09,800 Because remember, this is a pathway 1069 01:29:09,800 --> 01:29:11,660 that releases a lot of energy. 1070 01:29:11,660 --> 01:29:14,870 High energy high ATP, high NADH-- 1071 01:29:14,870 --> 01:29:18,170 don't run the cycle, don't enter carbon in the cycle. 1072 01:29:18,170 --> 01:29:23,900 Low energy, high ADP, put carbon in the cycle, 1073 01:29:23,900 --> 01:29:25,760 run the cycle faster. 1074 01:29:25,760 --> 01:29:26,900 Makes sense. 1075 01:29:26,900 --> 01:29:29,480 Also makes sense of the reciprocal regulation 1076 01:29:29,480 --> 01:29:33,060 of glycolysis and gluconeogenesis. 1077 01:29:33,060 --> 01:29:33,560 Great. 1078 01:29:33,560 --> 01:29:38,120 Next time, we will talk more about 1079 01:29:38,120 --> 01:29:46,760 how we can oxidize fatty acids, fat, by accessing acetyl-CoA 1080 01:29:46,760 --> 01:29:48,380 and entering it into the cycle. 1081 01:29:48,380 --> 01:29:49,930 Thanks.