WEBVTT 00:00:00.000 --> 00:00:01.936 [SQUEAKING] 00:00:01.936 --> 00:00:04.356 [RUSTLING] 00:00:04.356 --> 00:00:05.324 [CLICKING] 00:00:10.660 --> 00:00:12.370 MATTHEW VANDER HEIDEN: Hello, everybody. 00:00:12.370 --> 00:00:17.380 Last time, I introduced the idea of the TCA cycle, 00:00:17.380 --> 00:00:19.580 tricarboxylic acid cycle. 00:00:19.580 --> 00:00:22.570 Also known as the citric acid cycle, because citric acid 00:00:22.570 --> 00:00:25.690 is a tricarboxylic acid, as you'll see later today. 00:00:25.690 --> 00:00:27.700 Also known as the Krebs cycle, named 00:00:27.700 --> 00:00:31.810 after Hans Krebs, who discovered it 00:00:31.810 --> 00:00:35.540 in the early part of the last century. 00:00:35.540 --> 00:00:38.330 The TCA cycle is the series of reactions 00:00:38.330 --> 00:00:40.430 that occurs in the mitochondrial matrix 00:00:40.430 --> 00:00:43.550 and it allows the complete oxidation of two carbon 00:00:43.550 --> 00:00:47.090 units, derived from many things, including pyruvate, derived 00:00:47.090 --> 00:00:52.910 from glucose and glycolysis and enables the complete oxidation 00:00:52.910 --> 00:00:55.520 of that carbon to CO2. 00:00:55.520 --> 00:00:58.070 Now, it's a cycle because those two carbon 00:00:58.070 --> 00:01:03.050 units from pyruvate or other sources enter the cycle, 00:01:03.050 --> 00:01:05.930 combine with 4-carbon oxaloacetate, 00:01:05.930 --> 00:01:08.240 and combine to make 6-carbon citrate. 00:01:08.240 --> 00:01:11.540 Hence the citric acid cycle, or TCA 00:01:11.540 --> 00:01:15.800 cycle, that is then oxidized back to 4 carbon units, 00:01:15.800 --> 00:01:21.560 forming a cycle that allows cells 00:01:21.560 --> 00:01:23.270 to release lots of energy. 00:01:23.270 --> 00:01:25.370 Complete oxygen enables, ultimately, 00:01:25.370 --> 00:01:28.010 complete oxidation of glucose to CO2. 00:01:28.010 --> 00:01:30.560 We've talked many times how this releases energy. 00:01:30.560 --> 00:01:32.750 And it also generates lots of intermediates 00:01:32.750 --> 00:01:36.260 for the cell that can be used to make other stuff. 00:01:36.260 --> 00:01:39.290 Last time, I alluded to the fact that citrate can be 00:01:39.290 --> 00:01:42.380 used, say, to make fatty acids. 00:01:42.380 --> 00:01:44.360 Today, what I want to do is I want to dive 00:01:44.360 --> 00:01:49.010 into the details of how this TCA cycle occurs 00:01:49.010 --> 00:01:51.980 and the consequences of the way it works, 00:01:51.980 --> 00:01:55.640 and how that affects other aspects of metabolism. 00:01:55.640 --> 00:01:57.920 You'll see that it actually affects 00:01:57.920 --> 00:02:01.440 the ability of different organisms, 00:02:01.440 --> 00:02:03.050 whether or not they can make things 00:02:03.050 --> 00:02:05.930 from intermediates in this cycle, 00:02:05.930 --> 00:02:07.665 because of how the cycle works. 00:02:17.750 --> 00:02:18.395 Now, to start. 00:02:21.150 --> 00:02:24.980 Of course, if we're going to start from pyruvate. 00:02:27.870 --> 00:02:31.210 Pyruvate, of course, has three carbons. 00:02:31.210 --> 00:02:35.760 And so if we're going to generate a two-carbon acetate 00:02:35.760 --> 00:02:37.890 group, we have to lose CO2. 00:02:44.220 --> 00:02:47.280 And as I described a couple lectures ago, 00:02:47.280 --> 00:02:49.740 acetate is also the thing that we 00:02:49.740 --> 00:02:52.710 can derive from metabolism of ethanol, 00:02:52.710 --> 00:02:54.720 showing us two things-- 00:02:54.720 --> 00:02:57.750 metabolized glucose to pyruvate or ethanol itself-- 00:02:57.750 --> 00:02:59.820 can both be turned into acetate. 00:02:59.820 --> 00:03:05.940 But really, the donor is this molecule, acetyl-CoA. 00:03:05.940 --> 00:03:09.240 Which is basically also a carboxylic acid, but rather 00:03:09.240 --> 00:03:12.180 than being a carboxylic acid, instead makes this 00:03:12.180 --> 00:03:15.880 a thioester bond, which activates this carbon 00:03:15.880 --> 00:03:35.760 as a leaving group, such that it can combine with oxaloacetate, 00:03:35.760 --> 00:03:41.130 which you'll remember from gluconeogenesis, 00:03:41.130 --> 00:03:52.760 releasing that S-CoA molecule to generate six-carbon citric. 00:04:15.730 --> 00:04:16.690 So, this is citric. 00:04:16.690 --> 00:04:20.110 You can see that we've made a bond from this carbon 00:04:20.110 --> 00:04:25.700 on the acetate, losing this S-CoA group to this carbon 00:04:25.700 --> 00:04:30.290 oxaloacetate to make this six-carbon citrate molecule, 00:04:30.290 --> 00:04:32.170 which is a one, two, three-- 00:04:32.170 --> 00:04:36.460 three-carboxylic acid, or tricarboxylic acid. 00:04:36.460 --> 00:04:39.280 Hence the name citric acid cycle, 00:04:39.280 --> 00:04:41.450 tricarboxylic acid cycle. 00:04:41.450 --> 00:04:44.590 Now, these six carbons can then-- 00:04:44.590 --> 00:04:46.900 or this six-carbon citrate molecule 00:04:46.900 --> 00:04:53.740 can then be oxidized, generating two CO2 molecules that 00:04:53.740 --> 00:04:56.400 are released, and ultimately reforming 00:04:56.400 --> 00:05:01.780 oxaloacetate that can pick up another two-carbon acetyl-CoA 00:05:01.780 --> 00:05:05.110 to generate another citrate, and around and around the cycle 00:05:05.110 --> 00:05:10.030 goes, allowing in the end the net entry 00:05:10.030 --> 00:05:13.840 of two carbons, effectively from acetate, 00:05:13.840 --> 00:05:18.640 and release of two carbons as CO2. 00:05:18.640 --> 00:05:24.190 Now, as I alluded to, this can come from pyruvate. 00:05:24.190 --> 00:05:27.670 It can come from acetate itself, vinegar. 00:05:27.670 --> 00:05:29.200 It can come from alcohol. 00:05:29.200 --> 00:05:31.000 It turns out that when you break down fat, 00:05:31.000 --> 00:05:35.180 you also break it into two carbon units. 00:05:35.180 --> 00:05:38.680 And so this cycle becomes very useful for cells, 00:05:38.680 --> 00:05:43.600 because it allows the oxidation of many different molecules 00:05:43.600 --> 00:05:48.800 to completely turn that carbon into CO2. 00:05:48.800 --> 00:05:53.370 Now, if we're going to do this from glucose, 00:05:53.370 --> 00:06:05.750 however, you'll remember that pyruvate has three carbons. 00:06:05.750 --> 00:06:07.690 And so if we're going to turn pyruvate 00:06:07.690 --> 00:06:14.440 into acetate, or acetyl-CoA, we have to lose a carbon of CO2. 00:06:14.440 --> 00:06:17.930 We have to lose this carbon as CO2. 00:06:17.930 --> 00:06:21.700 Now, we saw this before, that we can do this via-- 00:06:21.700 --> 00:06:26.800 this is exactly how we generated ethanol 00:06:26.800 --> 00:06:32.830 when we did fermentation of pyruvate to ethanol, 00:06:32.830 --> 00:06:36.130 but that molecule didn't generate acetate, 00:06:36.130 --> 00:06:38.530 It generated acetaldehyde. 00:06:38.530 --> 00:06:42.190 The difference, of course, being whether or not this carbon gets 00:06:42.190 --> 00:06:44.830 oxidized in the case of acetate to the acid, 00:06:44.830 --> 00:06:47.200 or in acetaldehyde in ethanol metabolism, 00:06:47.200 --> 00:06:49.570 where it remains an aldehyde. 00:06:49.570 --> 00:06:52.330 Now obviously, you can make ethanol and then 00:06:52.330 --> 00:06:54.580 turn that ethanol into acetate. 00:06:54.580 --> 00:06:57.800 And that's a pathway that I guess certainly would work. 00:06:57.800 --> 00:07:01.420 However, that's not the way it happens in most organisms. 00:07:01.420 --> 00:07:07.180 Most organisms actually directly produce 00:07:07.180 --> 00:07:11.870 acetate, or more correctly, acetyl-CoA from pyruvate. 00:07:11.870 --> 00:07:14.200 So let's take a look at that reaction. 00:07:18.500 --> 00:07:20.370 So here once again is pyruvate. 00:07:24.490 --> 00:07:27.910 And if we turn that pyruvate into 00:07:27.910 --> 00:07:34.590 this two-carbon acetyl-CoA, let's 00:07:34.590 --> 00:07:37.390 look here what has to happen. 00:07:37.390 --> 00:07:42.060 Well, the first thing is that we have 00:07:42.060 --> 00:07:48.050 to decarboxylate this molecule. 00:07:48.050 --> 00:07:51.610 And so that generates a CO2. 00:07:51.610 --> 00:07:55.420 We have to lose that one carbon. 00:07:55.420 --> 00:08:00.010 Also, as I pointed out, if we do this 00:08:00.010 --> 00:08:02.320 as if we did it in ethanol metabolism, 00:08:02.320 --> 00:08:04.700 we'd be left with an aldehyde. 00:08:04.700 --> 00:08:10.930 But this is an acid, and so this carbon also has to be oxidized. 00:08:10.930 --> 00:08:12.040 We know how to do that. 00:08:12.040 --> 00:08:19.240 We can donate those electrons to something like NAD, 00:08:19.240 --> 00:08:23.050 so the NAD is reduced to NADH. 00:08:23.050 --> 00:08:30.770 And we had to add this S-CoA molecule, which 00:08:30.770 --> 00:08:33.450 I'll come to in a minute. 00:08:33.450 --> 00:08:39.730 And so each of these steps ends up 00:08:39.730 --> 00:08:43.720 making a fairly complicated reaction. 00:08:43.720 --> 00:08:46.798 Obviously, several co-factors are going to be involved. 00:08:46.798 --> 00:08:49.090 You should be able to guess that just by looking at it. 00:08:49.090 --> 00:08:51.400 Obviously, I already drew up there NAD. 00:08:51.400 --> 00:08:54.460 If we're going to decarboxylate, remember 00:08:54.460 --> 00:09:03.750 this is a alpha carboxylic, an alpha 00:09:03.750 --> 00:09:08.170 ketoacid-- the ketone group is alpha to the carboxylic acid. 00:09:08.170 --> 00:09:11.190 And so if we're doing alpha decarboxylation, 00:09:11.190 --> 00:09:13.500 as you might guess, we need a co-factor. 00:09:13.500 --> 00:09:15.210 That co-factor, as I told you before, 00:09:15.210 --> 00:09:17.850 is the co-factor factor thiamine pyrophosphate. 00:09:17.850 --> 00:09:21.940 And finally, there's this S-CoA group. 00:09:21.940 --> 00:09:25.320 We need to describe what that is. 00:09:25.320 --> 00:09:29.190 However, before delving into those, and there's 00:09:29.190 --> 00:09:32.160 actually a couple other factors that are needed as well, 00:09:32.160 --> 00:09:36.150 I want to mention one other issue 00:09:36.150 --> 00:09:40.980 about this reaction, in that this reaction happens 00:09:40.980 --> 00:09:44.820 in the mitochondria, because that's also 00:09:44.820 --> 00:09:46.770 where the TCA cycle happens. 00:09:46.770 --> 00:09:50.820 So, recall that if we divide the cell into two compartments 00:09:50.820 --> 00:09:53.880 like an eukaryotic cell, here we have the cytosol 00:09:53.880 --> 00:09:56.640 and the mitochondria in a eukaryotic cell. 00:09:56.640 --> 00:09:59.640 As we've already talked about, having different compartments 00:09:59.640 --> 00:10:02.393 helps facilitate different metabolic reactions, 00:10:02.393 --> 00:10:04.560 because you can have different conditions in the two 00:10:04.560 --> 00:10:05.860 compartments. 00:10:05.860 --> 00:10:15.240 And so as we said, glycolysis occurs in the cytosol. 00:10:15.240 --> 00:10:17.090 Glucose to pyruvate. 00:10:17.090 --> 00:10:26.720 And last time I mentioned, the TCA cycle 00:10:26.720 --> 00:10:31.340 occurs in the mitochondria. 00:10:31.340 --> 00:10:34.700 And so that means if we're going to fully oxidize 00:10:34.700 --> 00:10:38.900 the pyruvate carbon to CO2 using the TCA 00:10:38.900 --> 00:10:41.510 cycle in the mitochondria, that pyruvate 00:10:41.510 --> 00:10:44.840 has to get from the cytosol to the mitochondria, or at least 00:10:44.840 --> 00:10:47.408 carbons from the pyruvate have to get there. 00:10:47.408 --> 00:10:48.950 It turns out, you'll see in a minute, 00:10:48.950 --> 00:10:50.870 acetyl-CoA is a very large group. 00:10:50.870 --> 00:10:53.750 And so pyruvate itself is transported 00:10:53.750 --> 00:10:55.640 into the mitochondria. 00:10:55.640 --> 00:10:57.260 And that's where the reaction occurs 00:10:57.260 --> 00:11:00.620 to turn it into acetyl-CoA that can then enter the TCA cycle 00:11:00.620 --> 00:11:02.540 and oxidize. 00:11:02.540 --> 00:11:07.370 However, what this means is that a transporter is actually 00:11:07.370 --> 00:11:10.610 needed to get this across the mitochondrial membranes 00:11:10.610 --> 00:11:13.220 and into the matrix of the mitochondria where the TCA 00:11:13.220 --> 00:11:15.162 cycle happens. 00:11:15.162 --> 00:11:17.120 And I like to mention this because it turns out 00:11:17.120 --> 00:11:18.290 the pyruvate carrier-- 00:11:18.290 --> 00:11:19.790 that is, the transporter-- the way 00:11:19.790 --> 00:11:22.820 that it actually gets that pyruvate from the cytosol 00:11:22.820 --> 00:11:25.220 into the mitochondria actually was 00:11:25.220 --> 00:11:27.050 an unknown thing about metabolism 00:11:27.050 --> 00:11:30.380 until 2012, so not that long ago. 00:11:30.380 --> 00:11:35.150 Sometimes one can be left with the feeling, 00:11:35.150 --> 00:11:38.690 listening to these metabolism classes or reading a textbook, 00:11:38.690 --> 00:11:42.350 that everything about metabolism has been known forever, 00:11:42.350 --> 00:11:43.940 but it's actually not true. 00:11:43.940 --> 00:11:47.510 Here's a very key, central part of the pathway that 00:11:47.510 --> 00:11:49.760 was actually just discovered, at least 00:11:49.760 --> 00:11:52.220 at the time of this lecture, less than 10 years ago. 00:11:55.040 --> 00:11:59.340 It also illustrates that not all metabolism is completely 00:11:59.340 --> 00:12:02.510 understood. 00:12:02.510 --> 00:12:06.980 Now, let's get back to this reaction, how you turn pyruvate 00:12:06.980 --> 00:12:09.590 into acetyl-CoA. 00:12:09.590 --> 00:12:11.900 And I want to go through now and point out 00:12:11.900 --> 00:12:15.000 that several co-factors are needed. 00:12:15.000 --> 00:12:19.460 And so I already mentioned one of them, S-CoA, 00:12:19.460 --> 00:12:26.490 which is shorthand for coenzyme A. So, 00:12:26.490 --> 00:12:29.870 I need to tell you what that is. 00:12:29.870 --> 00:12:34.850 You hopefully are already familiar with TPP plus, 00:12:34.850 --> 00:12:36.170 thiamine pyrophosphate. 00:12:36.170 --> 00:12:39.290 We talked about that when we talked 00:12:39.290 --> 00:12:42.890 about how you do alpha decarboxylation to generate 00:12:42.890 --> 00:12:45.440 ethanol from pyruvate, also used here 00:12:45.440 --> 00:12:48.620 for the alpha decarboxylation reaction. 00:12:48.620 --> 00:12:51.200 Redox reaction happens, and so we 00:12:51.200 --> 00:12:57.760 needed NAD plus to get converted into NADH. 00:12:57.760 --> 00:13:01.120 We talked a lot about how that serves as an electron carrier, 00:13:01.120 --> 00:13:04.120 but it turns out that there's two additional electron 00:13:04.120 --> 00:13:07.390 carriers that are involved in this reaction. 00:13:07.390 --> 00:13:11.950 One of them is called FAD and the other one 00:13:11.950 --> 00:13:14.155 is called lipoic acid. 00:13:18.120 --> 00:13:19.700 Now you might say, why do we need 00:13:19.700 --> 00:13:22.070 all these electron carriers? 00:13:22.070 --> 00:13:24.260 Well, these are just different molecules 00:13:24.260 --> 00:13:30.600 that can carry two electrons, similar to NADH. 00:13:30.600 --> 00:13:34.430 And effectively, what these can do by having multiple electron 00:13:34.430 --> 00:13:35.480 carriers-- 00:13:35.480 --> 00:13:39.680 one can build chains of oxidation and reduction 00:13:39.680 --> 00:13:40.940 reactions. 00:13:40.940 --> 00:13:44.120 And it turns out these chains of oxidation reduction reactions 00:13:44.120 --> 00:13:48.200 really become central to energy transfers in biology, 00:13:48.200 --> 00:13:50.690 because building these chains allows 00:13:50.690 --> 00:13:54.020 more easy stepwise release of energy 00:13:54.020 --> 00:13:57.660 as one moves across these oxidation reduction reactions. 00:13:57.660 --> 00:14:00.170 Which remember, as I alluded to earlier, really 00:14:00.170 --> 00:14:06.890 are at the core of bioenergetics and a lot of what allows energy 00:14:06.890 --> 00:14:09.980 release from these pathways. 00:14:09.980 --> 00:14:13.490 What I mean by this will be more explicit 00:14:13.490 --> 00:14:18.350 as we go through what some of these co-factors look like. 00:14:18.350 --> 00:14:25.550 So, I'm not going to draw TPP plus or NAD again, but let's 00:14:25.550 --> 00:14:30.090 define what some of these other cofactors look like. 00:14:30.090 --> 00:14:39.410 So, let's start with coenzyme A. So, coenzyme A, it turns out, 00:14:39.410 --> 00:14:41.210 is useful. 00:14:41.210 --> 00:14:46.360 It's actually involved in lots of acylation reactions. 00:14:46.360 --> 00:14:48.070 What's an acylation reaction? 00:14:48.070 --> 00:14:52.750 Well, that's basically if you're making a carbon-carbon bond 00:14:52.750 --> 00:14:56.200 by adding a molecule of greater than one carbon, so two carbons 00:14:56.200 --> 00:14:58.030 or greater, to something else. 00:14:58.030 --> 00:15:00.070 That's an acylation reaction, as we 00:15:00.070 --> 00:15:04.810 did with adding the two-carbon acetate to oxaloacetate 00:15:04.810 --> 00:15:06.460 to make citrate. 00:15:06.460 --> 00:15:08.680 And you'll actually see coenzyme A will come up 00:15:08.680 --> 00:15:11.110 in this in many, many lectures throughout the rest 00:15:11.110 --> 00:15:12.400 of the course. 00:15:12.400 --> 00:15:16.750 Why this becomes useful is because it activates 00:15:16.750 --> 00:15:25.530 this basically carbon to the left of, in this case, 00:15:25.530 --> 00:15:26.450 the ketone. 00:15:26.450 --> 00:15:29.330 Because, by having that thioester there, 00:15:29.330 --> 00:15:33.620 ends up activating that carbon and allows it to carry out 00:15:33.620 --> 00:15:37.070 these acylation reactions. 00:15:37.070 --> 00:15:41.000 Coenzyme A itself is derived from a vitamin 00:15:41.000 --> 00:15:44.285 called pantothenic acid. 00:15:48.670 --> 00:15:52.720 And as a vitamin, this is something 00:15:52.720 --> 00:15:55.370 that you have to get from the diet. 00:15:55.370 --> 00:15:58.690 Now, when we abbreviate coenzyme A, 00:15:58.690 --> 00:16:02.440 which is often abbreviated S-CoA, 00:16:02.440 --> 00:16:04.960 you get the sense that it's this little tiny molecule 00:16:04.960 --> 00:16:06.700 that's just stuck on sulfur-- 00:16:06.700 --> 00:16:09.610 stuck on to the end to make this thioester bond. 00:16:09.610 --> 00:16:12.730 But it turns out coenzyme A is actually a giant molecule, one 00:16:12.730 --> 00:16:19.390 of the reasons why you actually synthesize acetyl-CoA 00:16:19.390 --> 00:16:20.800 in the mitochondria, because it's 00:16:20.800 --> 00:16:24.170 hard to transport this giant molecule. 00:16:24.170 --> 00:16:28.990 And so this is what pantothenic acid looks like. 00:16:47.920 --> 00:16:53.400 So, if I put an acid group here and a hydroxyl group there, 00:16:53.400 --> 00:16:55.530 that would be pantothenic acid, the thing 00:16:55.530 --> 00:17:00.075 that's in your cereal, on the side of your cereal box. 00:17:05.470 --> 00:17:07.690 Then there's these additional pieces. 00:17:07.690 --> 00:17:17.380 This is the active end of the molecule, 00:17:17.380 --> 00:17:19.839 that's that sulfur from the S-CoA, 00:17:19.839 --> 00:17:25.180 and then this side of the molecule is esterified to two 00:17:25.180 --> 00:17:28.810 phosphates, which are esterified to-- 00:17:31.477 --> 00:17:33.060 I'm not going to draw it out, but this 00:17:33.060 --> 00:17:39.200 would have an adenine base and a phosphate there. 00:17:39.200 --> 00:17:47.540 So basically, this is ADP, with a phosphate 00:17:47.540 --> 00:17:51.740 added to the 3 prime position of the ADP molecule, 00:17:51.740 --> 00:17:56.000 added to pantothenic acid, added to this short chain 00:17:56.000 --> 00:17:57.560 with the sulfur on the end. 00:17:57.560 --> 00:18:01.550 And this whole molecule together is coenzyme A. 00:18:01.550 --> 00:18:03.530 And so when we say acetyl-CoA, it's 00:18:03.530 --> 00:18:12.630 this giant molecule, S-thioester to the carbonyl, 00:18:12.630 --> 00:18:16.950 the acid on acetate, to CH3. 00:18:16.950 --> 00:18:20.680 And so that would be, basically, acetyl-CoA. 00:18:20.680 --> 00:18:24.030 And so one convenient thing about this 00:18:24.030 --> 00:18:26.610 is it's much easier than drawing that big molecule, 00:18:26.610 --> 00:18:30.930 but it is a little bit misleading in terms 00:18:30.930 --> 00:18:32.670 of its size. 00:18:32.670 --> 00:18:35.970 So, that's coenzyme A. 00:18:35.970 --> 00:18:42.240 Next one I want to talk about is FAD 00:18:42.240 --> 00:18:51.120 which stands for flavin adenine dinucleotide. 00:18:54.600 --> 00:18:58.110 So, flavin adenine dinucleotide is an electron carrier, 00:18:58.110 --> 00:18:59.835 just like NAD plus. 00:19:05.330 --> 00:19:11.480 And it's derived from the vitamin riboflavin, 00:19:11.480 --> 00:19:14.480 also referred to as vitamin B2. 00:19:17.240 --> 00:19:21.410 Another thing from the side of your cereal box, and basically 00:19:21.410 --> 00:19:23.440 looks like this. 00:19:23.440 --> 00:19:31.670 So, like NAD, it's a dinucleotide. 00:19:40.140 --> 00:19:45.270 And so here's ADP, just like we do for CoA, or just 00:19:45.270 --> 00:19:47.430 like one end of NAD. 00:19:47.430 --> 00:19:51.330 One difference is that the other nucleotide down here 00:19:51.330 --> 00:19:54.010 actually isn't technically a sugar. 00:19:54.010 --> 00:19:57.240 It's ribitol instead of ribose. 00:19:57.240 --> 00:19:58.240 What does that mean? 00:19:58.240 --> 00:19:59.940 It doesn't have an aldehyde. 00:19:59.940 --> 00:20:04.710 Instead, it is just a five-carbon chain 00:20:04.710 --> 00:20:08.010 where all of the carbons are alcohols. 00:20:14.450 --> 00:20:16.250 And so since there's no aldehyde, 00:20:16.250 --> 00:20:18.200 it doesn't form a ring. 00:20:45.150 --> 00:20:49.680 And the base on this end, as a nicotinamide, 00:20:49.680 --> 00:21:07.180 is this flavin group, which looks like this. 00:21:07.180 --> 00:21:15.740 And so this is FAD, which is in the oxidized form. 00:21:15.740 --> 00:21:19.250 Turns out, in the oxidized form, FAD 00:21:19.250 --> 00:21:25.520 is yellow, hence riboflavin-- "flavin", yellow. 00:21:25.520 --> 00:21:27.410 And the reason it's yellow-- you see 00:21:27.410 --> 00:21:30.770 there's a conjugated double bond here across this part. 00:21:30.770 --> 00:21:34.460 It turns out this is the active part of the molecule, 00:21:34.460 --> 00:21:35.900 and it works as follows. 00:21:35.900 --> 00:21:38.540 And so if you have a hydride ion, 00:21:38.540 --> 00:21:41.600 remember the way we can transfer two electrons. 00:21:46.390 --> 00:21:49.270 Can transfer the two electrons that way, 00:21:49.270 --> 00:21:50.920 and that allows it to generate. 00:21:50.920 --> 00:21:53.620 And I'll just draw the middle, here. 00:21:53.620 --> 00:21:56.320 Active part of the molecule. 00:22:11.180 --> 00:22:13.510 So, that would be these two nitrogens. 00:22:13.510 --> 00:22:15.350 We've added two electrons to it. 00:22:15.350 --> 00:22:20.590 And so this is abbreviated FADH2, 00:22:20.590 --> 00:22:24.700 or the reduced form of FAD. 00:22:24.700 --> 00:22:29.060 And it is colorless, because now you no longer 00:22:29.060 --> 00:22:32.810 have that conjugated double bond system, loses its color. 00:22:32.810 --> 00:22:36.590 And so you can follow whether FAD is oxidized or reduced 00:22:36.590 --> 00:22:40.550 by just looking at color change. 00:22:40.550 --> 00:22:42.500 So, that's FAD. 00:22:42.500 --> 00:22:45.890 It's an electron carrier, carries electrons 00:22:45.890 --> 00:22:49.130 by a hydride transfer, very similarly 00:22:49.130 --> 00:22:52.370 to what we described for NAD, but obviously 00:22:52.370 --> 00:22:54.890 a different molecule. 00:22:54.890 --> 00:22:59.000 And the last molecule, the last co-factor 00:22:59.000 --> 00:23:05.910 that we need for this reaction, is lipoic acid. 00:23:05.910 --> 00:23:08.220 Which, unlike most things in metabolism, 00:23:08.220 --> 00:23:12.840 doesn't have an abbreviation and also functions 00:23:12.840 --> 00:23:14.700 as an electron carrier. 00:23:14.700 --> 00:23:17.040 And so lipoic acid looks like this. 00:23:37.570 --> 00:23:40.810 So, that is lipoic acid. 00:23:40.810 --> 00:23:45.190 This is in the oxidized form. 00:23:45.190 --> 00:23:48.100 And so where it's oxidized is here 00:23:48.100 --> 00:23:49.660 at this sulfur-sulfur bond. 00:23:49.660 --> 00:23:53.380 So, you can think of this as the same as a disulfide bond 00:23:53.380 --> 00:23:57.110 that you learned about from Professor Yaffe in proteins. 00:23:57.110 --> 00:24:01.390 And so this is the oxidized form of the disulfide bond. 00:24:01.390 --> 00:24:09.230 If I take this hydride, transfer two electrons across that bond, 00:24:09.230 --> 00:24:13.675 then it goes to-- and I'll just draw here the active end. 00:24:19.370 --> 00:24:25.110 Then we go here to the reduced form of lipoic acid, 00:24:25.110 --> 00:24:28.020 and there's no abbreviation for oxidized 00:24:28.020 --> 00:24:29.700 or reduced lipoic acid. 00:24:29.700 --> 00:24:33.960 It's just lipoic acid, oxidized lipoic acid, reduced. 00:24:33.960 --> 00:24:36.210 And how they're oxidized and reduced 00:24:36.210 --> 00:24:41.550 is basically very similar to the disulfide bonds 00:24:41.550 --> 00:24:46.770 that you saw in proteins being oxidized, disulfide bond being 00:24:46.770 --> 00:24:52.350 oxidized, or it can be reduced to not be a disulfide bond. 00:24:52.350 --> 00:25:02.760 Now, it turns out that these cofactors, our TPP, FAD, 00:25:02.760 --> 00:25:07.680 and lipoic acid, are all stably associated 00:25:07.680 --> 00:25:14.060 with different subunits of a multi-protein complex 00:25:14.060 --> 00:25:20.300 that assembles to catalyze that reaction to convert pyruvate 00:25:20.300 --> 00:25:23.330 to acetyl-CoA. 00:25:23.330 --> 00:25:25.610 The enzyme or enzyme complex that 00:25:25.610 --> 00:25:29.210 carries this out is referred to as PDH, 00:25:29.210 --> 00:25:32.630 sometimes abbreviated PDC, which stands 00:25:32.630 --> 00:25:46.110 for the pyruvate dehydrogenase complex. 00:25:46.110 --> 00:25:50.190 So, pyruvate dehydrogenase, PDH, pyruvate dehydrogenase complex, 00:25:50.190 --> 00:25:51.790 PDC. 00:25:51.790 --> 00:25:56.460 This complex is basically three different polypeptides 00:25:56.460 --> 00:25:58.650 that come together to form a complex 00:25:58.650 --> 00:26:00.960 and catalyze that reaction. 00:26:00.960 --> 00:26:03.610 Now, where this complex sits-- 00:26:03.610 --> 00:26:05.640 so, this is the mitochondria. 00:26:05.640 --> 00:26:07.770 This is the matrix. 00:26:07.770 --> 00:26:10.530 That's where this pyruvate dehydrogenase reaction occurs. 00:26:10.530 --> 00:26:13.170 That's where the TCA cycle occurs. 00:26:13.170 --> 00:26:15.630 And this PDH complex is basically 00:26:15.630 --> 00:26:19.590 sitting here at the membrane on the matrix side 00:26:19.590 --> 00:26:22.450 of the membrane. 00:26:22.450 --> 00:26:24.720 Now, the three different polypeptides 00:26:24.720 --> 00:26:26.970 that come together to form this complex 00:26:26.970 --> 00:26:35.010 are creatively named E1, E2, and E3, four. 00:26:35.010 --> 00:26:37.680 Enzyme one, two, and three. 00:26:37.680 --> 00:26:41.010 And each of these, as I alluded to, 00:26:41.010 --> 00:26:44.567 is associated with a different co-factor. 00:26:49.710 --> 00:26:56.760 And so E1 is associated with thymine pyrophosphate. 00:26:56.760 --> 00:27:07.510 E2 is associated with lipoic acid. 00:27:07.510 --> 00:27:12.675 And E3 is associated with FAD. 00:27:17.090 --> 00:27:20.120 Now, let's go through the mechanism 00:27:20.120 --> 00:27:25.345 for how this pyruvate dehydrogenase reaction works. 00:28:22.470 --> 00:28:27.950 So remember, this is the active part of TBP plus. 00:28:27.950 --> 00:28:32.450 It is bound in the active site of E1. 00:28:32.450 --> 00:28:43.600 And it reacts with pyruvate to catalyze alpha decarboxylation, 00:28:43.600 --> 00:28:48.550 just like we described for conversion of pyruvate 00:28:48.550 --> 00:28:50.110 to alcohol. 00:28:50.110 --> 00:28:53.800 So, you're going to see exactly the same mechanism 00:28:53.800 --> 00:28:56.490 that we drew before. 00:29:15.980 --> 00:29:20.450 So, that decarboxylates the alpha-keto acid, 00:29:20.450 --> 00:29:27.440 just like we saw to generate acetaldehyde. 00:29:33.390 --> 00:29:37.290 The difference is rather than resolve this such 00:29:37.290 --> 00:29:39.780 that this carbon has the same oxidation state 00:29:39.780 --> 00:29:44.520 and make acetaldehyde, instead the next step of this reaction 00:29:44.520 --> 00:29:56.230 is going to be oxidized by reducing lipoic acid. 00:29:56.230 --> 00:29:59.040 So, the active part of lipoic acid 00:29:59.040 --> 00:30:04.530 that is in the active site of the E2 subunit. 00:31:20.750 --> 00:31:27.170 So this will now regenerate E1, but what we're left with, 00:31:27.170 --> 00:31:30.820 then, is this. 00:31:30.820 --> 00:31:34.810 Now, this intermediate bound to E2. 00:31:45.130 --> 00:31:54.170 Here's where coenzyme A can come in, which 00:31:54.170 --> 00:32:04.260 will then generate acetyl-CoA. 00:32:07.650 --> 00:32:19.890 But now we are left with E2 in the reduced state, 00:32:19.890 --> 00:32:22.950 rather than being in the oxidized state. 00:32:22.950 --> 00:32:27.990 So E2 has to be re-oxidized in order for this complex 00:32:27.990 --> 00:32:32.820 to carry out the next catalytic cycle, and the way 00:32:32.820 --> 00:32:35.130 that works is as follows. 00:32:35.130 --> 00:32:41.130 So you have FAD bound on E3. 00:32:41.130 --> 00:32:54.550 And so you have a hydride ion from the oxidation of E3 00:32:54.550 --> 00:32:58.120 that can be transferred to FAD. 00:32:58.120 --> 00:33:07.500 That will generate FAD from the oxidized to the reduced form. 00:33:07.500 --> 00:33:16.200 So re-oxidize lipoic acid on E2, reduce FAD to FADH2, 00:33:16.200 --> 00:33:20.010 and then that FADH2 can be re-oxidized back 00:33:20.010 --> 00:33:26.420 to FAD via transferring those electrons to NAD 00:33:26.420 --> 00:33:31.720 plus to generate NADH. 00:33:31.720 --> 00:33:37.180 So in this case, FADH2 re-oxidized the FAD, NAD 00:33:37.180 --> 00:33:41.080 plus reduced to NADH. 00:33:41.080 --> 00:33:44.410 And so, effectively, what this happens 00:33:44.410 --> 00:33:49.090 is that enzyme E1 and E2 cooperate 00:33:49.090 --> 00:34:02.830 to call what's referred to as oxidative alpha decarboxylation 00:34:02.830 --> 00:34:07.090 of pyruvate, while adding -CoA, so that's 00:34:07.090 --> 00:34:15.090 where you get acetyl-CoA, with reduction of lipoic acid in E2, 00:34:15.090 --> 00:34:20.370 and then E3 re-oxidizes the lipoic acid in E2 00:34:20.370 --> 00:34:24.409 while generating NADH. 00:34:24.409 --> 00:34:26.677 This NADH, once it's generated, of course 00:34:26.677 --> 00:34:28.219 those electrons have to go somewhere, 00:34:28.219 --> 00:34:30.409 so they, like in glycolysis-- 00:34:30.409 --> 00:34:33.889 it also needs to regenerate to NAD at some point. 00:34:33.889 --> 00:34:38.120 This is ultimately the electrons that end up on oxygen, 00:34:38.120 --> 00:34:42.590 and it's really this series of electron transfers, 00:34:42.590 --> 00:34:45.500 with oxygen being a good electron acceptor, that 00:34:45.500 --> 00:34:50.570 ultimately allows controlled energy release during carbon 00:34:50.570 --> 00:34:51.739 oxidation. 00:34:51.739 --> 00:34:55.070 And cells, you'll see, can use that to make ATP or do 00:34:55.070 --> 00:34:58.940 other kinds of work. 00:34:58.940 --> 00:35:02.720 So, the net reaction and/or another way 00:35:02.720 --> 00:35:07.160 to draw the pyruvate dehydrogenase reaction 00:35:07.160 --> 00:35:15.390 would be as follows, and that's taking pyruvate to acetyl-CoA. 00:35:18.890 --> 00:35:27.980 And so we're going to take coenzyme A and release CO2. 00:35:27.980 --> 00:35:42.500 This is done by TPP plus, as part of E1. 00:35:42.500 --> 00:35:52.660 That involves converting the lipoic acid in E2 00:35:52.660 --> 00:35:56.080 from the oxidized to the reduced state. 00:35:56.080 --> 00:36:00.130 That lipoic acid then has to be re-oxidized. 00:36:00.130 --> 00:36:02.260 If something's oxidized, something else 00:36:02.260 --> 00:36:03.790 has to be reduced. 00:36:03.790 --> 00:36:10.090 That's FAD on E3, which also then cycles 00:36:10.090 --> 00:36:14.020 between the oxidized and reduce state. 00:36:14.020 --> 00:36:17.440 And ultimately, those electrons ended up 00:36:17.440 --> 00:36:24.010 being transferred to NAD plus to generate NADH. 00:36:24.010 --> 00:36:32.380 And so the PDH complex is basically a chain 00:36:32.380 --> 00:36:41.010 of electron transfer reactions. 00:36:41.010 --> 00:36:45.690 And it's the first example of a chain of electron transfer 00:36:45.690 --> 00:36:46.230 reactions. 00:36:46.230 --> 00:36:48.630 We're going to see that there is the electron transport 00:36:48.630 --> 00:36:51.900 chain in the mitochondria, effectively does 00:36:51.900 --> 00:36:52.870 the same thing. 00:36:52.870 --> 00:36:56.400 And by coupling oxidation and reduction reactions 00:36:56.400 --> 00:37:00.480 across chains of molecules like this, effectively as a preview, 00:37:00.480 --> 00:37:03.930 allows the stepwise energy release of these oxidation 00:37:03.930 --> 00:37:08.170 reactions to occur. 00:37:08.170 --> 00:37:11.550 So remember, if we burn glucose, completely oxidize it in one 00:37:11.550 --> 00:37:13.750 step, where those electrons are directly transferred 00:37:13.750 --> 00:37:15.510 to oxygen in combustion. 00:37:15.510 --> 00:37:18.720 Lots of energy released, but all in one step. 00:37:18.720 --> 00:37:21.210 By doing these stepwise electron transfers, 00:37:21.210 --> 00:37:25.110 we can then basically break up that energy release 00:37:25.110 --> 00:37:29.190 in a way that can be captured by cells to do work. 00:37:29.190 --> 00:37:31.050 Here, the way energy is captured, 00:37:31.050 --> 00:37:34.860 it's not so obvious in this electron transport reaction. 00:37:34.860 --> 00:37:38.580 But effectively, you're using oxidation 00:37:38.580 --> 00:37:46.770 of the ketone on pyruvate, with decarboxylation to the acid 00:37:46.770 --> 00:37:50.400 to generate, rather than just the acid, a thioester bond. 00:37:50.400 --> 00:37:52.470 And that's that thioester bond-- 00:37:52.470 --> 00:37:55.110 as well as NADH, that's energy as well-- but it's 00:37:55.110 --> 00:37:58.620 really that thioester bond that then can be recaptured later 00:37:58.620 --> 00:38:03.420 to drive synthesis of citrate in the TCA cycle. 00:38:06.480 --> 00:38:11.970 Now, next what I want to do is I want to discuss the TCA cycle 00:38:11.970 --> 00:38:12.600 reactions. 00:38:12.600 --> 00:38:15.420 Now that you see how you can get acetyl-CoA, at least 00:38:15.420 --> 00:38:18.450 from pyruvate, I want to discuss how you can now 00:38:18.450 --> 00:38:23.910 use that acetyl-CoA and oxidize it back to combine it 00:38:23.910 --> 00:38:27.960 with oxaloacetate, and oxidize it back 00:38:27.960 --> 00:38:31.350 to make citrate, and then oxidize it back to oxaloacetate 00:38:31.350 --> 00:38:35.490 to run the TCA cycle. 00:38:35.490 --> 00:38:37.960 OK, great. 00:38:37.960 --> 00:38:54.120 So, the first reaction of the TCA cycle. 00:38:54.120 --> 00:39:00.090 Here's acetyl-CoA. 00:39:00.090 --> 00:39:13.530 It will combine with oxaloacetate. 00:39:26.420 --> 00:39:58.130 This reaction is catalyzed by an enzyme called citrate synthase, 00:39:58.130 --> 00:40:05.480 and generates the six-carbon tricarboxylic acid citrate. 00:40:05.480 --> 00:40:09.340 So, how does this reaction work? 00:40:47.990 --> 00:40:52.920 Here's drawing acetyl-CoA in a slightly different way. 00:40:52.920 --> 00:41:25.620 If I redraw this as the enol, this 00:41:25.620 --> 00:42:26.340 will generate citrate via, effectively, that mechanism. 00:42:29.610 --> 00:42:42.600 Next, citrate is converted by adding water 00:42:42.600 --> 00:42:48.450 across this carbon-carbon, or by removing water 00:42:48.450 --> 00:42:56.790 across this carbon-carbon bond, to generate 00:42:56.790 --> 00:42:59.260 this intermediate called cis-aconitate. 00:43:31.989 --> 00:43:34.660 So this intermediate is called cis-aconitate. 00:43:38.260 --> 00:43:41.440 so all I did was dehydrate across that bond 00:43:41.440 --> 00:43:43.340 so there's a double bond there. 00:43:43.340 --> 00:43:56.180 And then if I re-add water across that bond, 00:43:56.180 --> 00:44:19.640 I generate this molecule called isocitrate. 00:44:22.240 --> 00:44:26.500 So effectively, to convert citrate to isocitrate, 00:44:26.500 --> 00:44:29.980 I'm moving the hydroxyl group from that carbon 00:44:29.980 --> 00:44:31.630 to this carbon. 00:44:31.630 --> 00:44:35.760 To do that, I basically dehydrate, make a double bond, 00:44:35.760 --> 00:44:38.710 remove water, re-add water across that bond 00:44:38.710 --> 00:44:41.950 in the opposite direction to generate this molecule, 00:44:41.950 --> 00:44:43.550 isocitrate. 00:44:43.550 --> 00:45:00.210 This reaction is carried out by an enzyme called aconitase, 00:45:00.210 --> 00:45:05.510 and converts citrate to isocitrate. 00:45:05.510 --> 00:45:10.820 I think it's a little easier to see this reaction 00:45:10.820 --> 00:45:13.090 if I draw it a slightly different way. 00:46:12.590 --> 00:46:14.890 So, this here is just drawing citrate 00:46:14.890 --> 00:46:21.560 by just slightly rotating the molecule to look like that. 00:46:21.560 --> 00:46:24.490 And so I'm basically removing water here. 00:46:51.040 --> 00:46:57.100 And then I'm now just adding water back 00:46:57.100 --> 00:47:20.850 in the opposite orientation to generate isocitrate. 00:47:28.830 --> 00:47:33.300 Now, you'll notice when I drew this-- 00:47:33.300 --> 00:47:36.900 if you look at citrate, this is actually a symmetrical 00:47:36.900 --> 00:47:43.380 molecule, so the top half and the bottom half of citrate 00:47:43.380 --> 00:47:45.700 are identical. 00:47:45.700 --> 00:47:49.410 And so what's interesting about nature is 00:47:49.410 --> 00:47:51.510 that it treats these carbons-- 00:47:51.510 --> 00:47:54.060 the green carbons that came from acetyl-CoA-- 00:47:54.060 --> 00:47:57.270 different from the side of the molecule that 00:47:57.270 --> 00:48:00.210 comes from oxaloacetate. 00:48:00.210 --> 00:48:05.370 And effectively, nature always moves the hydroxyl group 00:48:05.370 --> 00:48:08.820 to this carbon that came from oxaloacetate, 00:48:08.820 --> 00:48:11.670 and never moves it to this carbon 00:48:11.670 --> 00:48:15.270 that came from acetyl-CoA. 00:48:15.270 --> 00:48:18.240 This is an example where enzymes-- 00:48:18.240 --> 00:48:22.950 nature treats a symmetrical molecule like citrate 00:48:22.950 --> 00:48:25.080 in an asymmetrical way. 00:48:25.080 --> 00:48:29.910 And this has consequences for how carbon is actually traced 00:48:29.910 --> 00:48:32.400 through the entire TCA cycle. 00:48:32.400 --> 00:48:34.590 Because even though you might think things could get 00:48:34.590 --> 00:48:37.230 scrambled at citrate, they never do. 00:48:37.230 --> 00:48:41.580 Meaning an isocitrate-- it's always these green carbons 00:48:41.580 --> 00:48:43.320 that came from acetyl-CoA. 00:48:43.320 --> 00:48:45.180 You never get those green carbons 00:48:45.180 --> 00:48:48.960 on the other side of isocitrate. 00:48:48.960 --> 00:48:50.970 And so when we go through the TCA cycle, 00:48:50.970 --> 00:48:54.030 I'll keep these carbons green until the point where 00:48:54.030 --> 00:48:58.290 you can no longer distinguish which carbon came from 00:48:58.290 --> 00:48:59.585 came from which reaction. 00:49:02.160 --> 00:49:07.740 The next reaction is we're going to oxidize 00:49:07.740 --> 00:49:12.030 this carbon of isocitrate. 00:49:12.030 --> 00:49:14.550 So if we're going to oxidize that carbon, 00:49:14.550 --> 00:49:17.370 those electrons have to go somewhere. 00:49:36.310 --> 00:49:40.150 And so if we oxidize the carbon, we 00:49:40.150 --> 00:49:49.240 can use NAD plus as an electron acceptor, reduce it to NADH. 00:49:49.240 --> 00:49:58.250 That generates this intermediate. 00:50:20.460 --> 00:50:23.940 So hopefully this is clear to everybody at this point, 00:50:23.940 --> 00:50:26.150 but just in case. 00:50:29.230 --> 00:50:34.560 So, this carbon here and isocitrate. 00:50:34.560 --> 00:50:49.570 If I oxidize that alcohol to the ketone, 00:50:49.570 --> 00:50:51.610 now I generate a hydride ion. 00:50:51.610 --> 00:50:54.280 Those two electrons and the hydrogen can go to NAD plus 00:50:54.280 --> 00:50:57.070 and reduce it to NADH. 00:50:57.070 --> 00:50:59.590 That generates this intermediate. 00:51:02.430 --> 00:51:06.220 It's called oxalosuccinate. 00:51:06.220 --> 00:51:11.860 Which then, if you notice, the oxalosuccinate 00:51:11.860 --> 00:51:16.420 is now a beta keto acid. 00:51:16.420 --> 00:51:22.420 So alpha, beta. 00:51:22.420 --> 00:51:25.730 The acid group is beta to the ketone, 00:51:25.730 --> 00:51:27.680 so it's a beta keto acid. 00:51:27.680 --> 00:51:32.270 Remember, beta decarboxylation is favorable, 00:51:32.270 --> 00:51:35.860 and so I can lose that CO2. 00:51:35.860 --> 00:51:55.170 And what I'm left with is this molecule, which 00:51:55.170 --> 00:52:06.100 is called alpha keto glutarate. 00:52:06.100 --> 00:52:14.370 So this whole reaction here, the oxidation 00:52:14.370 --> 00:52:16.590 of the alcohol to the ketone, followed 00:52:16.590 --> 00:52:20.010 by beta decarboxylation of oxalosuccinate 00:52:20.010 --> 00:52:22.560 to alpha ketoglutarate, is carried out 00:52:22.560 --> 00:52:28.680 by an enzyme called isocitrate dehydrogenase. 00:52:34.310 --> 00:52:50.662 And of course, just to remind you, 00:52:50.662 --> 00:52:58.660 here's that beta keto acid in oxalosuccinate. 00:53:04.510 --> 00:53:20.570 And so that can decarboxylate, leading this enol. 00:53:20.570 --> 00:53:35.800 Which can, of course, rearrange back to the ketone that we see, 00:53:35.800 --> 00:53:42.250 an alpha ketoglutarate. 00:53:42.250 --> 00:53:49.710 Now, if you look at alpha ketoglutarate, 00:53:49.710 --> 00:53:56.865 you'll notice that-- and I will redraw over here. 00:54:11.940 --> 00:54:18.630 So, this here is just me redrawing alpha ketoglutarate, 00:54:18.630 --> 00:54:23.960 which is often abbreviated alpha kG. 00:54:23.960 --> 00:54:26.370 Just drew it as a straight line. 00:54:26.370 --> 00:54:29.300 Now, if you'll notice, alpha keto glutarate 00:54:29.300 --> 00:54:32.750 is a alpha ketoacid. 00:54:32.750 --> 00:54:37.620 And so here the acid group is alpha to the ketone. 00:54:37.620 --> 00:54:39.620 So, an alpha keto acid. 00:54:39.620 --> 00:54:42.170 It's effectively just like pyruvate, 00:54:42.170 --> 00:54:45.110 but has this additional pieces on it. 00:54:45.110 --> 00:54:48.740 And it turns out the next step in the TCA cycle 00:54:48.740 --> 00:54:52.820 is the exact reaction that we saw 00:54:52.820 --> 00:54:56.240 with pyruvate dehydrogenase. 00:54:56.240 --> 00:55:01.250 It's alpha decarboxylation, oxidative alpha 00:55:01.250 --> 00:55:22.280 keto acid decarboxylation, as follows. 00:55:22.280 --> 00:55:25.090 So, we carry out. 00:55:57.150 --> 00:56:04.710 This is a molecule called succinyl-CoA. 00:56:04.710 --> 00:56:10.830 And so, you'll see what happened there is decarboxylated here, 00:56:10.830 --> 00:56:18.030 this carbon, while oxidizing this ketone to the acid 00:56:18.030 --> 00:56:19.800 and adding -CoA. 00:56:19.800 --> 00:56:21.400 That's a redox reaction. 00:56:21.400 --> 00:56:28.830 So NAD, NADH, it turns out this is exactly the same mechanism-- 00:56:28.830 --> 00:56:30.360 so I don't need to draw it again-- 00:56:30.360 --> 00:56:34.740 that I just showed you for pyruvate dehydrogenase. 00:56:34.740 --> 00:56:36.720 So it needs all the same cofactors-- 00:56:36.720 --> 00:56:40.770 TPP plus, lipoic acid, FAD. 00:56:40.770 --> 00:56:47.010 And in fact, it even shares some of the same enzyme complexes, 00:56:47.010 --> 00:56:51.720 subunits, as pyruvate dehydrogenase. 00:56:51.720 --> 00:56:57.330 And so this is a reaction that's catalyzed by alpha 00:56:57.330 --> 00:56:59.460 ketoglutarate dehydrogenase. 00:57:02.520 --> 00:57:06.210 Like pyruvate dehydrogenase, this is a complex, 00:57:06.210 --> 00:57:09.650 and so it has a unique E1, which makes sense. 00:57:09.650 --> 00:57:12.000 Remember, E1 of pyruvate dehydrogenase 00:57:12.000 --> 00:57:15.030 was actually the subunit that bound the pyruvate. 00:57:15.030 --> 00:57:17.820 E1 of alpha ketoglutarate dehydrogenase is unique. 00:57:17.820 --> 00:57:20.400 It binds alpha ketoglutarate instead of pyruvate. 00:57:20.400 --> 00:57:24.750 However, they share the same E2 and E3s, 00:57:24.750 --> 00:57:28.140 and so the E2s and E3s would carry out 00:57:28.140 --> 00:57:32.340 exactly the same reaction and play the same part 00:57:32.340 --> 00:57:38.400 in the mechanism of how you do this alpha oxidative alpha 00:57:38.400 --> 00:57:41.100 decarboxylation to turn alpha ketoglutarate 00:57:41.100 --> 00:57:45.330 into succinyl-CoA. 00:57:45.330 --> 00:57:47.340 Now, remember in pyruvate dehydrogenase, 00:57:47.340 --> 00:57:49.110 once we got that acetyl-CoA, we then 00:57:49.110 --> 00:57:52.680 use this CoA group to drive condensation with citrate. 00:57:52.680 --> 00:57:54.540 Well, in this case, what happens is 00:57:54.540 --> 00:57:58.110 you don't want to combine condensation here. 00:57:58.110 --> 00:58:00.690 Instead, what is going to happen is 00:58:00.690 --> 00:58:08.160 you want to use this CoA group to now generate ATP. 00:58:08.160 --> 00:58:15.210 And so this is going to couple release of the CoA 00:58:15.210 --> 00:58:17.430 to generate an ATP equivalent. 00:58:17.430 --> 00:58:22.750 It's actually GTP that's generated by the TCA cycle. 00:58:22.750 --> 00:58:34.630 And so, this is going to generate 00:58:34.630 --> 00:58:39.490 this molecule, succinate. 00:58:39.490 --> 00:58:47.790 And the enzyme that does this is called succinic bio-kinase. 00:58:56.930 --> 00:59:01.205 So, let's go through over here how this enzyme works. 00:59:12.800 --> 00:59:24.950 So here's succinyl-CoA, basically using favorable loss 00:59:24.950 --> 00:59:40.920 of the CoA breaking the thioester bond to generate 00:59:40.920 --> 00:59:42.750 this acid anhydride. 00:59:42.750 --> 00:59:48.150 We saw an acid anhydride before in glycolysis. 00:59:48.150 --> 00:59:51.060 Remember, we made 1-3-bisphosphoglycerate, 00:59:51.060 --> 00:59:54.180 so that's a good phosphate donor. 00:59:54.180 --> 00:59:59.110 And then that can be used to generate succinate, 00:59:59.110 --> 01:00:05.560 and transferring that phosphate to GDP to make GTP, 01:00:05.560 --> 01:00:08.620 just like 1-3-bisphosphoglycerate, 01:00:08.620 --> 01:00:10.420 was able to transfer the phosphate 01:00:10.420 --> 01:00:18.370 from the acid anhydride to ADP to make ATP in glycolysis. 01:00:20.950 --> 01:00:34.490 So, the next step is to oxidize this carbon-carbon bond 01:00:34.490 --> 01:00:37.400 in succinate. 01:00:37.400 --> 01:00:41.690 So if we're going to oxidize a carbon-carbon bond, 01:00:41.690 --> 01:00:44.090 those electrons have to go somewhere. 01:00:50.670 --> 01:00:53.790 So there's our carbon-carbon bond. 01:01:01.670 --> 01:01:13.200 And so if we generate hydride ion 01:01:13.200 --> 01:01:18.270 just like we did in other oxidation reactions, 01:01:18.270 --> 01:01:23.880 but this hydride ion is transferred not to NAD, 01:01:23.880 --> 01:01:30.510 but instead to FAD, a different electron carrier, 01:01:30.510 --> 01:01:34.680 to generate if FADH2. 01:01:34.680 --> 01:01:38.370 Now, I should point out succinate, like citrate, is 01:01:38.370 --> 01:01:40.050 a symmetrical molecule. 01:01:40.050 --> 01:01:43.680 But at this point, nature doesn't tell the difference. 01:01:43.680 --> 01:01:47.440 Once it generates succinate, this molecule 01:01:47.440 --> 01:01:48.750 now gets scrambled. 01:01:48.750 --> 01:01:51.330 And so everything downstream of succinate, 01:01:51.330 --> 01:01:54.750 you no longer know which carbons came from acetyl-CoA. 01:02:05.770 --> 01:02:10.540 This generates this molecule called fumarate, 01:02:10.540 --> 01:02:19.020 and this reaction is carried out by an enzyme called 01:02:19.020 --> 01:02:32.020 succinate dehydrogenase, often abbreviated SDH 01:02:32.020 --> 01:02:34.570 for succinate dehydrogenase. 01:02:37.850 --> 01:02:44.900 Now, the next reaction is, we're going 01:02:44.900 --> 01:02:50.520 to add water across this double bond of fumarate. 01:03:28.270 --> 01:03:33.695 And that generates this intermediate, malate. 01:03:37.950 --> 01:03:42.630 This reaction is carried out by an enzyme 01:03:42.630 --> 01:03:51.930 called fumarate hydratase. 01:03:51.930 --> 01:03:55.320 And it's simply adding a water molecule 01:03:55.320 --> 01:03:58.470 across that double bond. 01:03:58.470 --> 01:04:01.710 Once we have malate, if you look what's 01:04:01.710 --> 01:04:06.450 the difference between malate and oxaloacetate, 01:04:06.450 --> 01:04:11.550 the difference is that in oxaloacetate, this carbon 01:04:11.550 --> 01:04:12.960 is a ketone. 01:04:12.960 --> 01:04:15.390 Whereas in malate, it's an alcohol. 01:04:15.390 --> 01:04:18.150 And so if we want to turn this carbon from the alcohol 01:04:18.150 --> 01:04:24.490 into the ketone, that is, of course, a oxidation reaction, 01:04:24.490 --> 01:04:32.470 and so those electrons have to go somewhere. 01:04:32.470 --> 01:04:36.790 Don't need to draw the mechanism again. 01:04:36.790 --> 01:04:41.080 It's basically just the hydride transfer 01:04:41.080 --> 01:04:45.720 to oxidize this to an alcohol, to the ketone. 01:04:45.720 --> 01:04:51.180 That oxidation couples to a reduction of NAD plus to NADH. 01:04:51.180 --> 01:04:58.093 This is carried out by an enzyme called malate dehydrogenase. 01:05:07.190 --> 01:05:12.980 And doing this completes the TCA cycle, 01:05:12.980 --> 01:05:16.160 regenerating oxaloacetate, that can then 01:05:16.160 --> 01:05:18.680 recombine with another acetyl-CoA 01:05:18.680 --> 01:05:24.200 to go through another round of the cycle. 01:05:24.200 --> 01:05:28.700 You'll notice that going through this cycle, 01:05:28.700 --> 01:05:32.090 there's two CO2s lost. 01:05:32.090 --> 01:05:39.020 One of them is lost here at the isocitrate to alpha 01:05:39.020 --> 01:05:40.860 ketoglutarate reaction. 01:05:40.860 --> 01:05:45.415 So, this decarboxylation from oxaloacetate 01:05:45.415 --> 01:05:46.670 to alpha ketoglutarate. 01:05:46.670 --> 01:05:50.000 That beta decarboxylation, that's the first CO2. 01:05:50.000 --> 01:05:53.405 The other one is lost here at the alpha ketoglutarate. 01:05:57.370 --> 01:06:00.100 The alpha ketoglutarate dehydrogenase step, 01:06:00.100 --> 01:06:03.970 where you have this alpha decarboxylation 01:06:03.970 --> 01:06:07.540 to take alpha ketoglutarate to succinyl-CoA 01:06:07.540 --> 01:06:12.280 oxidative alpha decarboxylation. 01:06:12.280 --> 01:06:15.670 Now, what's cool about this, as you noticed, 01:06:15.670 --> 01:06:20.770 we just discussed all the reactions of the TCA cycle. 01:06:20.770 --> 01:06:23.460 And I showed you, reminded you, of some chemistry 01:06:23.460 --> 01:06:26.010 that you've already seen, but unless you 01:06:26.010 --> 01:06:29.475 count the chemistry we showed you earlier for how the PDH 01:06:29.475 --> 01:06:31.350 and alpha ketoglutarate dehydrogenase 01:06:31.350 --> 01:06:35.610 reactions work with E1, E2 and E3, with lipoic acid, FAD. 01:06:35.610 --> 01:06:37.300 That was obviously new for today. 01:06:37.300 --> 01:06:39.600 But other than that, everything else 01:06:39.600 --> 01:06:42.240 was chemistry that you've already seen. 01:06:42.240 --> 01:06:43.890 And this really points out the point 01:06:43.890 --> 01:06:47.460 that I made earlier, that metabolism is really variations 01:06:47.460 --> 01:06:49.230 on relatively few reactions. 01:06:49.230 --> 01:06:52.630 We've just repurposed some of the same tricks, if you will, 01:06:52.630 --> 01:06:55.700 that we're used in glycolysis, and allowed 01:06:55.700 --> 01:07:00.090 it to now do an entire different pathway, the TCA cycle. 01:07:00.090 --> 01:07:02.870 It also points out how Hans Krebs was-- well, is still-- 01:07:02.870 --> 01:07:06.530 remarkable, able to figure out from chemistry alone, 01:07:06.530 --> 01:07:09.410 because there's actually quite a bit of logic to the way 01:07:09.410 --> 01:07:12.030 metabolism works. 01:07:12.030 --> 01:07:13.280 Now, I want to say this again. 01:07:13.280 --> 01:07:16.280 Note there were two carbons that entered acetyl-CoA, 01:07:16.280 --> 01:07:20.050 and two carbons that were lost to CO2. 01:07:20.050 --> 01:07:22.690 But if you look, the green carbons 01:07:22.690 --> 01:07:27.190 remain in the same places until they get to succinate. 01:07:27.190 --> 01:07:29.320 and so the two carbons that enter 01:07:29.320 --> 01:07:32.350 are not lost on the first turn of the cycle. 01:07:32.350 --> 01:07:37.810 It's actually two carbons that came from oxaloacetate 01:07:37.810 --> 01:07:42.220 that are converted to CO2 as that acetyl-CoA goes 01:07:42.220 --> 01:07:43.340 through the cycle. 01:07:43.340 --> 01:07:47.920 And so to oxidize the exact carbons from acetyl-CoA to CO2 01:07:47.920 --> 01:07:53.220 requires more than one turn of the cycle. 01:07:53.220 --> 01:07:57.660 You'll also notice that the cycle is oxidation. 01:07:57.660 --> 01:08:01.670 And so oxidation reactions, of course, release energy. 01:08:01.670 --> 01:08:02.670 We've talked about that. 01:08:02.670 --> 01:08:05.070 And so it's favorable. 01:08:05.070 --> 01:08:12.035 And the products, if you will, are three NADH molecules. 01:08:12.035 --> 01:08:16.500 You can say plus one more NADH if we're going all the way 01:08:16.500 --> 01:08:21.029 from glucose or from pyruvate. 01:08:21.029 --> 01:08:24.120 Glucose derived pyruvate because the pyruvate dehydrogenase 01:08:24.120 --> 01:08:29.649 reaction also generates an NADH to make that a acetyl-CoA. 01:08:29.649 --> 01:08:38.359 One FADH2, as well as one GTP molecule. 01:08:38.359 --> 01:08:41.569 And so lots of oxidation going on here. 01:08:41.569 --> 01:08:44.350 We completely oxidized two carbons to CO2, 01:08:44.350 --> 01:08:47.229 so that's energy release. 01:08:47.229 --> 01:08:52.279 But you notice you only get one GTP from the molecule. 01:08:52.279 --> 01:08:55.510 Now, this GTP, of course-- that reaction, 01:08:55.510 --> 01:08:57.939 the succinic thiokinase reaction, 01:08:57.939 --> 01:09:00.010 like the reactions we saw on glycolysis, 01:09:00.010 --> 01:09:04.540 is such that it can generate GTP at a high DGP-GDP 01:09:04.540 --> 01:09:06.218 ratio, or ATP-ADP ratio. 01:09:06.218 --> 01:09:07.510 Remember, those are equivalent. 01:09:07.510 --> 01:09:10.899 Those energy charge are similar, and so that makes sense. 01:09:10.899 --> 01:09:15.250 But most of the energy released is actually 01:09:15.250 --> 01:09:19.960 reducing NAD and FAD to NADH and FADH2. 01:09:19.960 --> 01:09:25.300 And of course, these need to transfer their electrons 01:09:25.300 --> 01:09:28.420 somewhere else, and that's the role of oxygen. 01:09:28.420 --> 01:09:31.250 Oxygen, remember, is a very good electron donor, 01:09:31.250 --> 01:09:34.479 and so it's the ultimate transfer of those electrons 01:09:34.479 --> 01:09:40.750 from these molecules to oxygen that also provide energy 01:09:40.750 --> 01:09:44.740 that the cell can use to do work, but it does so, in a way, 01:09:44.740 --> 01:09:49.910 by charging up different ratios in the cell. 01:09:49.910 --> 01:09:56.830 So the NAD-NADH or the FADH2-FAD ratios. 01:09:56.830 --> 01:10:02.020 And just like we talked about, the ratio or the energy of ATP 01:10:02.020 --> 01:10:05.170 is in the interconversion between ATP and ADP. 01:10:05.170 --> 01:10:09.190 It's the ratio that drives the free energy change. 01:10:09.190 --> 01:10:13.780 The same thing exists for an NADH and NAD, FADH2 and FAD. 01:10:13.780 --> 01:10:16.990 And so charging up these ratios while passing through the TCA 01:10:16.990 --> 01:10:19.810 cycle, and the ultimate downstream transfer 01:10:19.810 --> 01:10:22.540 of those electrons to oxygen, really 01:10:22.540 --> 01:10:26.650 is where most of the energy is captured as carbon 01:10:26.650 --> 01:10:30.220 is oxidized through the TCA cycle. 01:10:30.220 --> 01:10:35.080 And exactly how that works and how it can be related to ATP 01:10:35.080 --> 01:10:37.750 will be something that'll be more explicit in the coming 01:10:37.750 --> 01:10:39.300 lectures. 01:10:39.300 --> 01:10:43.200 Now, I want to point out apart from the oxidation, 01:10:43.200 --> 01:10:46.360 there's actually lots of intermediates made here. 01:10:46.360 --> 01:10:48.450 And it turns out, a bunch of these intermediates 01:10:48.450 --> 01:10:51.610 are useful for cells to make stuff. 01:10:51.610 --> 01:10:53.250 So we talked about gluconeogenesis. 01:10:53.250 --> 01:10:55.170 Gluconeogenesis needs electron balance. 01:10:55.170 --> 01:10:58.140 We get NADH from the TCA cycle, and so you 01:10:58.140 --> 01:11:01.290 can think of gluconeogenesis as an alternative 01:11:01.290 --> 01:11:03.440 to fermentation to dispose of electrons. 01:11:03.440 --> 01:11:06.000 Well, you can use the NADH from the TCA cycle 01:11:06.000 --> 01:11:08.980 to run gluconeogenesis as well. 01:11:08.980 --> 01:11:11.620 But beyond the cofactors, the carbon itself. 01:11:11.620 --> 01:11:14.460 So citrate, I've alluded to now a few times, 01:11:14.460 --> 01:11:17.130 is important as a precursor to make fat. 01:11:17.130 --> 01:11:19.620 We'll discuss that in later lectures, too. 01:11:19.620 --> 01:11:22.710 But other intermediates in this pathway 01:11:22.710 --> 01:11:26.710 are useful for various amino acids and nucleic acids. 01:11:26.710 --> 01:11:29.340 And so there's lots of things that 01:11:29.340 --> 01:11:32.490 can come from the TCA cycle that cells 01:11:32.490 --> 01:11:37.840 can find useful to do, not just catabolism, 01:11:37.840 --> 01:11:41.880 but also anabolic processes. 01:11:41.880 --> 01:11:46.090 Now, the way the TCA cycle works, though, 01:11:46.090 --> 01:11:48.660 is that there's actually an issue 01:11:48.660 --> 01:11:52.140 if you want to use the intermediates from the TCA 01:11:52.140 --> 01:11:55.120 cycle to make stuff. 01:11:55.120 --> 01:11:59.890 And so what is that issue? 01:11:59.890 --> 01:12:05.418 Well, the TCA cycle functions at a site as a cycle. 01:12:05.418 --> 01:12:07.710 And so, if we're going to take things in and out of it, 01:12:07.710 --> 01:12:11.730 that has consequences for how the cycle runs. 01:12:11.730 --> 01:12:14.740 Now there's a couple words for this 01:12:14.740 --> 01:12:17.200 that I want to just introduce to you. 01:12:17.200 --> 01:12:20.370 The first one is cataplerosis and the second one 01:12:20.370 --> 01:12:23.200 is anaplerosis. 01:12:23.200 --> 01:12:32.680 And so cataplerosis is the act of removing stuff 01:12:32.680 --> 01:12:34.225 from a metabolic cycle. 01:12:38.030 --> 01:12:39.680 So, we're going to remove citrate 01:12:39.680 --> 01:12:41.120 from the cycle to make fat. 01:12:41.120 --> 01:12:42.860 That's cataplerosis. 01:12:42.860 --> 01:12:57.540 And anaplerosis is adding stuff back to a metabolic cycle 01:12:57.540 --> 01:13:01.380 so it can continue to function. 01:13:01.380 --> 01:13:05.130 Viewing this is really evident if you think of the TCA cycle 01:13:05.130 --> 01:13:07.330 as a chicken and egg problem. 01:13:07.330 --> 01:13:09.670 So, the very first time acetyl-CoA 01:13:09.670 --> 01:13:12.720 was generated, how do you start the TCA 01:13:12.720 --> 01:13:14.490 cycle in the first place? 01:13:14.490 --> 01:13:16.800 You can't add it to the TCA cycle 01:13:16.800 --> 01:13:19.920 unless you have oxaloacetate to combine 01:13:19.920 --> 01:13:22.050 with the acetyl-CoA, which can then 01:13:22.050 --> 01:13:25.170 generate another oxaloacetate. 01:13:25.170 --> 01:13:29.500 So where does the first oxaloacetate come from? 01:13:29.500 --> 01:13:31.560 Well, we already talked about one reaction. 01:13:31.560 --> 01:13:35.040 We talked about it in the context of gluconeogenesis. 01:13:35.040 --> 01:13:36.400 That can solve this problem. 01:13:36.400 --> 01:13:38.220 And so we have pyruvate. 01:13:38.220 --> 01:13:40.380 And so we talked earlier today how 01:13:40.380 --> 01:13:45.720 we can do oxidative decarboxylation of pyruvate 01:13:45.720 --> 01:13:48.450 to give acetyl-CoA. 01:13:48.450 --> 01:13:50.160 But we talked to in the gluconeogenesis 01:13:50.160 --> 01:13:55.470 lecture how we can add a CO2 pyruvate to generate 01:13:55.470 --> 01:13:57.270 oxaloacetate. 01:13:57.270 --> 01:13:59.910 So if I do those two reactions, now I 01:13:59.910 --> 01:14:06.400 have all the carbon I need to generate a citrate 01:14:06.400 --> 01:14:11.190 and start off the TCA cycle. 01:14:11.190 --> 01:14:16.130 Now obviously, if I do cataplerosis 01:14:16.130 --> 01:14:19.220 and I remove that citrate I made to make fat, 01:14:19.220 --> 01:14:21.200 well, now I need two pyruvate again 01:14:21.200 --> 01:14:25.940 to generate the next citrate if I'm using this pathway, 01:14:25.940 --> 01:14:29.420 because every time I bring an acetyl-CoA into the cycle, 01:14:29.420 --> 01:14:32.660 I need an oxaloacetate to combine it with. 01:14:32.660 --> 01:14:35.840 And so if I remove something, I have to add something back. 01:14:35.840 --> 01:14:39.740 And so pyruvate to oxaloacetate, the pyruvate carboxylase 01:14:39.740 --> 01:14:45.850 reaction is an example of an anaplerotic reaction. 01:14:45.850 --> 01:14:49.900 Now what this means, though, is that in order 01:14:49.900 --> 01:14:52.600 to do an anaplerosis, you have to be 01:14:52.600 --> 01:14:56.950 able to generate four-carbon oxaloacetate, 01:14:56.950 --> 01:14:59.780 or a four-carbon molecule. 01:14:59.780 --> 01:15:02.590 Now, pyruvate carboxylase, pyruvate to oxaloacetate, 01:15:02.590 --> 01:15:03.802 allows you to do that. 01:15:03.802 --> 01:15:05.260 We can take a three-carbon molecule 01:15:05.260 --> 01:15:08.080 and generate four-carbon oxaloacetate. 01:15:08.080 --> 01:15:12.940 However, if we start from a two-carbon molecule 01:15:12.940 --> 01:15:20.210 like acetate or acetyl-CoA that enters the cycle, 01:15:20.210 --> 01:15:24.410 it's actually not so simple to take that two-carbon molecule 01:15:24.410 --> 01:15:27.350 and turn it into four-carbon oxaloacetate. 01:15:27.350 --> 01:15:31.070 And in fact, humans lack any enzymes 01:15:31.070 --> 01:15:33.440 that allow them to take a two-carbon unit, 01:15:33.440 --> 01:15:36.680 to take acetate or acetyl-CoA, and turn it 01:15:36.680 --> 01:15:41.000 into anything that's longer than net-- turn 01:15:41.000 --> 01:15:44.270 it into anything that's longer than two carbons. 01:15:44.270 --> 01:15:46.100 And this has important implications 01:15:46.100 --> 01:15:49.070 for human physiology, because what it says 01:15:49.070 --> 01:15:52.370 is that we can't make glucose from anything 01:15:52.370 --> 01:15:56.580 that starts with something less than three carbons long. 01:15:56.580 --> 01:15:59.750 So if you drink alcohol and you metabolize that alcohol 01:15:59.750 --> 01:16:02.510 to acetate, it turns out you take fat 01:16:02.510 --> 01:16:05.330 and you break down fat also to acetyl-CoA, 01:16:05.330 --> 01:16:07.520 to acetate two carbons long. 01:16:07.520 --> 01:16:13.460 There is no way to turn those molecules into glucose, 01:16:13.460 --> 01:16:16.610 because you cannot generate the oxaloacetate to do 01:16:16.610 --> 01:16:21.480 the anaplerosis that's necessary to get it there. 01:16:24.230 --> 01:16:30.020 What this means is that our body can only 01:16:30.020 --> 01:16:33.200 store calories that come from two carbon units, 01:16:33.200 --> 01:16:35.600 fat or alcohol, as fat. 01:16:35.600 --> 01:16:41.202 We can never turn them back into glucose or make glycogen. 01:16:41.202 --> 01:16:43.160 And this is very relevant for those of you that 01:16:43.160 --> 01:16:44.577 go to medical school, because it's 01:16:44.577 --> 01:16:46.670 relevant to our physiology. 01:16:46.670 --> 01:16:48.710 And that is, when our bodies exhaust 01:16:48.710 --> 01:16:51.320 all of our stores of glucose, what happens? 01:16:51.320 --> 01:16:54.140 Our liver can no longer do gluconeogenesis. 01:16:54.140 --> 01:16:55.130 And so what happens? 01:16:55.130 --> 01:16:57.450 Now it has to switch over to doing something else. 01:16:57.450 --> 01:16:59.370 It has to work with two carbon units. 01:16:59.370 --> 01:17:02.150 And ultimately, this is ketone metabolism, which we'll 01:17:02.150 --> 01:17:05.600 talk about in a few lectures. 01:17:05.600 --> 01:17:07.655 It also said that the body-- 01:17:11.310 --> 01:17:15.060 that this is also the basis of a very old adage that's 01:17:15.060 --> 01:17:17.280 out there that some of you may have heard-- 01:17:17.280 --> 01:17:20.220 that you need to have some other fuel if you're 01:17:20.220 --> 01:17:22.260 going to burn fat. 01:17:22.260 --> 01:17:25.740 The basis for that is that fat is turned into two carbon 01:17:25.740 --> 01:17:27.840 units, acetyl-CoA units. 01:17:27.840 --> 01:17:30.180 And so if you're going to take those acetyl-CoA units 01:17:30.180 --> 01:17:33.090 and ultimately burn them away, turn them into CO2, 01:17:33.090 --> 01:17:35.190 you need a source of oxaloacetate, 01:17:35.190 --> 01:17:37.590 or your TCA cycle won't work. 01:17:37.590 --> 01:17:40.290 And you don't need a lot of something, but it is true. 01:17:40.290 --> 01:17:45.000 You can't start just with acetyl-CoA as a human and turn 01:17:45.000 --> 01:17:46.860 it into CO2, so you need some-- 01:17:46.860 --> 01:17:49.200 at least a little bit of oxaloacetate 01:17:49.200 --> 01:17:52.410 to get your TCA cycle started. 01:17:52.410 --> 01:17:57.335 Now, that's a problem that we as humans and other mammals face, 01:17:57.335 --> 01:17:59.460 but it turns out there's lots of microbes out there 01:17:59.460 --> 01:18:02.880 that grow just fine on acetate or on alcohol, 01:18:02.880 --> 01:18:04.980 even if it's only carbon source. 01:18:04.980 --> 01:18:08.550 And so those organisms must have some way 01:18:08.550 --> 01:18:10.830 to build stuff from two carbon units. 01:18:10.830 --> 01:18:15.280 That is a way to use two carbon units and do an anaplerosis. 01:18:15.280 --> 01:18:17.610 And it turns out the way they do this is via something 01:18:17.610 --> 01:18:20.410 called the glyoxylate cycle. 01:18:23.050 --> 01:18:26.220 And so the glyoxylate cycle is an alternative version 01:18:26.220 --> 01:18:29.610 of the TCA cycle that effectively uses two enzymes 01:18:29.610 --> 01:18:32.010 that we lack as mammals. 01:18:32.010 --> 01:18:34.230 And so I'll quickly tell you about it here. 01:18:47.920 --> 01:18:57.420 So, this is isocitrate from the TCA cycle. 01:18:57.420 --> 01:19:08.270 And some microbes have an enzyme called isocitrate lyase. 01:19:08.270 --> 01:19:18.430 And what isocitrate lyase does is basically splits citrate 01:19:18.430 --> 01:19:23.260 in half, such that the top portion of the molecule 01:19:23.260 --> 01:19:30.190 is another TCA cycle intermediate, succinate. 01:19:30.190 --> 01:19:34.150 And the bottom portion of a molecule 01:19:34.150 --> 01:19:42.850 is this two carbon aldehyde called glyoxylate. 01:19:42.850 --> 01:20:00.890 Glyoxylate can react with acetyl-CoA by another enzyme 01:20:00.890 --> 01:20:08.390 that we lack as humans called malate synthase. 01:20:11.030 --> 01:20:14.880 And I don't have time to show the mechanism again, 01:20:14.880 --> 01:20:19.160 but malate synthase basically adds the two carbons 01:20:19.160 --> 01:20:26.180 from acetyl-CoA to the aldehyde, the carbonyl, 01:20:26.180 --> 01:20:29.900 the aldehyde carbonyl of glyoxylate in a reaction that 01:20:29.900 --> 01:20:33.620 is, for all intents and purposes, exactly what happens 01:20:33.620 --> 01:20:36.050 in citrate synthase. 01:20:36.050 --> 01:20:51.930 That will generate this molecule, which is malate, also 01:20:51.930 --> 01:20:54.370 in the TCA cycle. 01:20:54.370 --> 01:20:57.990 And so having these two extra reactions, isocitrate lyase 01:20:57.990 --> 01:21:01.920 and malate synthase, gives microbes 01:21:01.920 --> 01:21:08.040 the ability to have acetyl-CoA be anaplerotic. 01:21:08.040 --> 01:21:09.450 And so how does that work? 01:21:09.450 --> 01:21:15.840 Well, that's because if we start with one oxaloacetate, four 01:21:15.840 --> 01:21:26.400 carbons, and acetyl-CoA, two carbons, can run the TCA cycle 01:21:26.400 --> 01:21:31.110 and make citrate, six carbons. 01:21:31.110 --> 01:21:33.884 Turn that citrate into isocitrate. 01:21:37.950 --> 01:21:49.090 Use isocitrate lyase to generate glyoxylate, two carbons. 01:21:49.090 --> 01:21:51.550 Plus succinate. 01:21:55.310 --> 01:22:02.070 That succinate can run through succinate dehydrogenase 01:22:02.070 --> 01:22:04.050 to generate malate, which you can 01:22:04.050 --> 01:22:08.190 go through malate dehydrogenase to generate oxaloacetate. 01:22:08.190 --> 01:22:15.930 This glyoxylate can start with a second acetyl-CoA. 01:22:15.930 --> 01:22:19.890 Two carbons come together, generate malate. 01:22:19.890 --> 01:22:22.770 That generates a second malate molecule, 01:22:22.770 --> 01:22:30.180 which can then exit the cycle as malate or oxaloacetate 01:22:30.180 --> 01:22:32.130 or whatever you want. 01:22:32.130 --> 01:22:36.420 And so basically, it allows two acetyl-CoAs to net 01:22:36.420 --> 01:22:41.040 generate an oxaloacetate, and so net generates a way 01:22:41.040 --> 01:22:45.090 to do an anaplerosis from two carbon units 01:22:45.090 --> 01:22:51.160 by having this malate synthase reaction 01:22:51.160 --> 01:22:57.360 and this isocitrate lyase reaction 01:22:57.360 --> 01:23:00.930 and run this alternative version of the TCA cycle, 01:23:00.930 --> 01:23:03.990 called the glyoxylate cycle. 01:23:03.990 --> 01:23:08.100 And it's a nice way how life-- again, no new chemistry here, 01:23:08.100 --> 01:23:10.740 just variations on what we've already shown-- 01:23:10.740 --> 01:23:12.690 repurposed the similar chemistries 01:23:12.690 --> 01:23:16.620 that it's already using as a way to live off 01:23:16.620 --> 01:23:24.540 of carbon sources that contain only two carbons, like ethanol 01:23:24.540 --> 01:23:27.350 or acetate. 01:23:27.350 --> 01:23:31.400 So in closing today, the last thing 01:23:31.400 --> 01:23:34.430 I want to talk about, very briefly, 01:23:34.430 --> 01:23:40.610 is how the TCA cycle is regulated. 01:23:40.610 --> 01:23:42.620 And we don't need to spend a ton of time 01:23:42.620 --> 01:23:48.710 on this, because it really follows principles that 01:23:48.710 --> 01:23:54.340 make sense, particularly when we think about things that we've 01:23:54.340 --> 01:23:55.820 already talk about. 01:23:55.820 --> 01:23:59.800 And so, regulation of the TCA cycles is of course important, 01:23:59.800 --> 01:24:03.430 and it's really a critical hub, both for anabolic and catabolic 01:24:03.430 --> 01:24:03.950 pathways. 01:24:03.950 --> 01:24:06.940 So, you needed to get energy to fully oxidize carbon, 01:24:06.940 --> 01:24:10.252 but it's also a useful place to get stuff. 01:24:10.252 --> 01:24:11.710 And before I talk about regulation, 01:24:11.710 --> 01:24:14.590 I just want to point out that many of the enzymes in the TCA 01:24:14.590 --> 01:24:17.650 cycle, even though we talk about it in the mitochondria 01:24:17.650 --> 01:24:19.990 and we're about to talk about regulation in terms 01:24:19.990 --> 01:24:22.660 of catabolism-- that is oxidation, 01:24:22.660 --> 01:24:25.060 ways to release energy-- 01:24:25.060 --> 01:24:27.700 many of these enzymes are also in other locations 01:24:27.700 --> 01:24:32.170 in the cell, because there's functions for them in making 01:24:32.170 --> 01:24:35.020 stuff that is very different than what goes on in the TCA 01:24:35.020 --> 01:24:36.010 cycle. 01:24:36.010 --> 01:24:39.460 And the regulation that I'll tell you about, 01:24:39.460 --> 01:24:42.280 and that comes up on MCAT exams and stuff 01:24:42.280 --> 01:24:45.460 like that, usually talks about this pathway 01:24:45.460 --> 01:24:49.940 as a catabolic pathway, as a way to make CO2, to make ATP. 01:24:49.940 --> 01:24:53.380 However, recognize that there's also 01:24:53.380 --> 01:24:57.610 variations on this pathway that can use in anabolic things, 01:24:57.610 --> 01:24:58.720 making stuff. 01:24:58.720 --> 01:25:03.700 And that regulation is something that really is not necessarily 01:25:03.700 --> 01:25:05.590 what we're going to talk about here 01:25:05.590 --> 01:25:09.790 and is a little bit less well-understood. 01:25:09.790 --> 01:25:11.530 But at least the regulation, in terms 01:25:11.530 --> 01:25:18.920 of catabolism, if we put it in context of glucose metabolism-- 01:25:18.920 --> 01:25:25.890 so here's glycolysis, turning glucose into pyruvate. 01:25:32.340 --> 01:25:35.550 And then that pyruvate can operate 01:25:35.550 --> 01:25:40.290 through the pyruvate dehydrogenase reaction 01:25:40.290 --> 01:25:44.430 to generate acetyl-CoA. 01:25:44.430 --> 01:25:50.190 That acetyl-CoA can combine with oxaloacetate 01:25:50.190 --> 01:25:53.782 to generate citrate. 01:25:53.782 --> 01:25:57.530 That citrate, of course, can be used to generate fat, 01:25:57.530 --> 01:26:00.400 as we've talked about. 01:26:00.400 --> 01:26:02.080 That can go to isocitrate. 01:26:04.940 --> 01:26:09.270 Isocitrate to alpha ketoglutarate. 01:26:09.270 --> 01:26:13.500 That's catalyzed by isocitrate dehydrogenase, 01:26:13.500 --> 01:26:15.630 which I will abbreviate IDH. 01:26:15.630 --> 01:26:19.750 Alpha ketoglutarate to succinate. 01:26:19.750 --> 01:26:30.830 More correctly, to succinyl-CoA by alpha ketoglutarate 01:26:30.830 --> 01:26:35.240 dehydrogenase, Alpha KGDH. 01:26:35.240 --> 01:26:38.150 And then this back to oxaloacetate. 01:26:38.150 --> 01:26:41.210 And so the main enzymes that are typically 01:26:41.210 --> 01:26:45.130 discussed as being regulated are alpha ketoglutarate 01:26:45.130 --> 01:26:48.110 dehydrogenase, isocitrate dehydrogenase, 01:26:48.110 --> 01:26:50.180 and pyruvate dehydrogenase. 01:26:50.180 --> 01:26:53.190 I also drew glycolysis. 01:26:53.190 --> 01:26:58.260 And I'll just write over here, gluconeogenesis up here, 01:26:58.260 --> 01:27:04.570 because we can now see more fully how this plays 01:27:04.570 --> 01:27:09.760 in with citrate acting as a positive regulator 01:27:09.760 --> 01:27:15.830 with gluconeogenesis and an inhibitor of glycolysis. 01:27:15.830 --> 01:27:21.410 Again, we're full in citrate, stop running glycolysis, 01:27:21.410 --> 01:27:24.950 start running gluconeogenesis. 01:27:24.950 --> 01:27:26.690 Now, the other regulation. 01:27:26.690 --> 01:27:30.080 If you have a lot of acetyl-CoA, stop making it 01:27:30.080 --> 01:27:34.820 from pyruvate. acetyl-CoA is a negative regulator of PDH. 01:27:34.820 --> 01:27:37.550 This is carbon oxidation. 01:27:37.550 --> 01:27:41.030 Releases a lot of energy, generates a lot of ATP, 01:27:41.030 --> 01:27:44.150 generates a lot of NADH. 01:27:44.150 --> 01:27:46.010 If you have lots of those things, 01:27:46.010 --> 01:27:49.390 no reason to keep sending carbon into 01:27:49.390 --> 01:27:52.960 acetyl-CoA to go into the TCA cycle. 01:27:52.960 --> 01:27:56.733 Ultimately, we have to send those electrons somewhere. 01:27:56.733 --> 01:27:58.150 So if there's nowhere to put them, 01:27:58.150 --> 01:28:02.500 low oxygen also inhibits pyruvate dehydrogenase. 01:28:02.500 --> 01:28:06.670 And of course, if you need more energy, if ADP is high, 01:28:06.670 --> 01:28:10.000 that activates pyruvate dehydrogenase. 01:28:10.000 --> 01:28:14.320 So at least in terms of glucose, complete glucose oxidation, 01:28:14.320 --> 01:28:18.280 a lot of regulation happens at pyruvate dehydrogenase. 01:28:18.280 --> 01:28:22.050 And a lot of it really makes sense. 01:28:22.050 --> 01:28:27.450 High levels of ATP, high levels of NADH, 01:28:27.450 --> 01:28:34.170 also inhibit isocitrate dehydrogenase and alpha 01:28:34.170 --> 01:28:38.690 ketoglutarate dehydrogenase. 01:28:38.690 --> 01:28:39.840 Makes sense. 01:28:39.840 --> 01:28:44.990 Succinol-CoA high, inhibit alpha ketoglutarate dehydrogenase. 01:28:44.990 --> 01:28:52.220 High levels of ADP, need to release more energy, 01:28:52.220 --> 01:28:56.300 activate alpha ketoglutarate dehydrogenase. 01:28:59.180 --> 01:29:02.030 Again, these are the feedbacks that people talk about 01:29:02.030 --> 01:29:03.590 on board exams. 01:29:03.590 --> 01:29:06.230 Good to know, but you can almost guess what they 01:29:06.230 --> 01:29:08.210 would be from first principles. 01:29:08.210 --> 01:29:09.800 Because remember, this is a pathway 01:29:09.800 --> 01:29:11.660 that releases a lot of energy. 01:29:11.660 --> 01:29:14.870 High energy high ATP, high NADH-- 01:29:14.870 --> 01:29:18.170 don't run the cycle, don't enter carbon in the cycle. 01:29:18.170 --> 01:29:23.900 Low energy, high ADP, put carbon in the cycle, 01:29:23.900 --> 01:29:25.760 run the cycle faster. 01:29:25.760 --> 01:29:26.900 Makes sense. 01:29:26.900 --> 01:29:29.480 Also makes sense of the reciprocal regulation 01:29:29.480 --> 01:29:33.060 of glycolysis and gluconeogenesis. 01:29:33.060 --> 01:29:33.560 Great. 01:29:33.560 --> 01:29:38.120 Next time, we will talk more about 01:29:38.120 --> 01:29:46.760 how we can oxidize fatty acids, fat, by accessing acetyl-CoA 01:29:46.760 --> 01:29:48.380 and entering it into the cycle. 01:29:48.380 --> 01:29:49.930 Thanks.