WEBVTT 00:00:00.090 --> 00:00:02.490 The following content is provided under a Creative 00:00:02.490 --> 00:00:04.030 Commons license. 00:00:04.030 --> 00:00:06.330 Your support will help MIT OpenCourseWare 00:00:06.330 --> 00:00:10.720 continue to offer high quality educational resources for free. 00:00:10.720 --> 00:00:13.320 To make a donation or view additional materials 00:00:13.320 --> 00:00:17.280 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.280 --> 00:00:18.240 at ocw.mit.edu. 00:00:21.940 --> 00:00:24.180 PROFESSOR: Let's look at storyboard two. 00:00:24.180 --> 00:00:25.840 We're going to look in more detail 00:00:25.840 --> 00:00:28.624 at carbohydrate catabolism at this point. 00:00:28.624 --> 00:00:30.040 We're eventually going to be doing 00:00:30.040 --> 00:00:33.220 the pathway of glycolysis, but we have to get there first. 00:00:33.220 --> 00:00:34.870 And it depends on what precursors 00:00:34.870 --> 00:00:37.980 are available to enter the pathway of glycolysis. 00:00:37.980 --> 00:00:39.730 One option that we'll look at a little bit 00:00:39.730 --> 00:00:42.520 later is to take in glucose from the blood 00:00:42.520 --> 00:00:45.190 by way of a glucose carrier, and then 00:00:45.190 --> 00:00:48.560 phosphorylate the glucose using either hexokinase or 00:00:48.560 --> 00:00:49.960 glucokinase. 00:00:49.960 --> 00:00:53.530 The second option is to take glycogen, the polymeric storage 00:00:53.530 --> 00:00:57.370 form of glucose, and degrade it to glucose 1-phosphate, which 00:00:57.370 --> 00:01:00.010 will be then converted to glucose 6-phosphate which 00:01:00.010 --> 00:01:02.260 will enter the pathway of glycolysis. 00:01:02.260 --> 00:01:05.260 We're going to start with this pathway of glycogen breakdown, 00:01:05.260 --> 00:01:08.170 or glycogenolysis. 00:01:08.170 --> 00:01:10.760 Panel A shows the structure of glycogen, 00:01:10.760 --> 00:01:13.630 which consists of glucose monomeric units connected 00:01:13.630 --> 00:01:16.360 by a bond between the 1 and 4 carbons 00:01:16.360 --> 00:01:18.580 in the alpha configuration. 00:01:18.580 --> 00:01:20.140 Chemistry is going to be happening 00:01:20.140 --> 00:01:22.800 at the non-reducing end, which is to the left. 00:01:22.800 --> 00:01:25.030 The reducing end, which is to the right, 00:01:25.030 --> 00:01:28.660 is connected to a scaffolding protein called glycogenin. 00:01:28.660 --> 00:01:31.460 We'll come back to glycogenin later in the course. 00:01:31.460 --> 00:01:34.600 Let's look next at panel B. The reaction begins 00:01:34.600 --> 00:01:38.320 with the proteination of the red oxygen between the terminal two 00:01:38.320 --> 00:01:42.220 glucose moieties by a proton on the glycogen phosphorylase 00:01:42.220 --> 00:01:43.150 enzyme. 00:01:43.150 --> 00:01:46.180 The intermediate product is a resonant stabilized pair 00:01:46.180 --> 00:01:49.090 of positively charged species, or cations, 00:01:49.090 --> 00:01:52.300 an oxonium ion at the 1 prime oxygen, 00:01:52.300 --> 00:01:54.940 and a carbocation at the 1-carbon. 00:01:54.940 --> 00:01:57.220 The electrophilic carbocation is attacked 00:01:57.220 --> 00:02:00.160 by the negatively charged phosphate residue, which 00:02:00.160 --> 00:02:03.745 is non-covalently associated with glycogen phosphorylase 00:02:03.745 --> 00:02:07.070 to form glucose 1-phosphate. 00:02:07.070 --> 00:02:09.440 Note that the glucose 1-phosphate that forms 00:02:09.440 --> 00:02:12.460 has the covalently attached phosphate on the alpha, 00:02:12.460 --> 00:02:15.650 or bottom face, as it's drawn in the figure. 00:02:15.650 --> 00:02:18.020 The structure of glycogen phosphorylase 00:02:18.020 --> 00:02:21.170 allows attack of it's phosphate from the bottom, 00:02:21.170 --> 00:02:26.594 giving rise to alpha isomer only of glucose 1-phosphate. 00:02:26.594 --> 00:02:28.010 The structure of the phosphorylase 00:02:28.010 --> 00:02:30.290 precludes access of its phosphate 00:02:30.290 --> 00:02:32.360 to the top face of the sugar molecule, 00:02:32.360 --> 00:02:35.930 so you get only one stereoisomer of glucose 1-phosphate out 00:02:35.930 --> 00:02:37.320 of this reaction. 00:02:37.320 --> 00:02:39.710 The other product shown at the bottom of panel C 00:02:39.710 --> 00:02:42.650 is the glycogen chain, which is truncated or shortened 00:02:42.650 --> 00:02:44.540 by one glucose unit. 00:02:44.540 --> 00:02:47.720 Now looking at the big picture, the epinephrine molecule 00:02:47.720 --> 00:02:49.850 produced as part of the stress response 00:02:49.850 --> 00:02:53.210 interacted with the cell membrane of the muscle cell. 00:02:53.210 --> 00:02:55.700 And that interaction ultimately activated glycogen 00:02:55.700 --> 00:02:59.480 phosphorylase to enable it to degrade glycogen, the storage 00:02:59.480 --> 00:03:03.200 form of glucose, and liberate glucose 1-phosphate, which 00:03:03.200 --> 00:03:06.290 is going to then find its way into glycolysis to generate 00:03:06.290 --> 00:03:08.270 fast energy to enable the student 00:03:08.270 --> 00:03:10.670 to be able to stand up in class and avoid 00:03:10.670 --> 00:03:12.410 the stressful situation. 00:03:12.410 --> 00:03:14.600 I'm not going to go through all of the details 00:03:14.600 --> 00:03:16.790 of the chemical reactions of glycolysis. 00:03:16.790 --> 00:03:18.320 You can find those in the book which 00:03:18.320 --> 00:03:21.750 does a good job of presenting those details. 00:03:21.750 --> 00:03:23.660 In the first step of glycolysis, we 00:03:23.660 --> 00:03:27.380 see the phosphorylated hexose, glucose 6-phosphate, 00:03:27.380 --> 00:03:31.040 be converted by phosphoglucoisomerase 00:03:31.040 --> 00:03:33.800 into the furanose fructose 6-phosphate. 00:03:33.800 --> 00:03:35.930 The next step involves the phosphorylation 00:03:35.930 --> 00:03:39.950 of the fructose 6-phosphate by phosphofructokinase 1. 00:03:39.950 --> 00:03:43.070 ATP is the phosphate donor, and you form fructose 00:03:43.070 --> 00:03:46.200 1,6-bisphosphate as the product. 00:03:46.200 --> 00:03:47.930 You'll notice that phosphofructokinase 00:03:47.930 --> 00:03:51.230 catalyzes one way, a thermodynamically irreversible 00:03:51.230 --> 00:03:52.560 reaction. 00:03:52.560 --> 00:03:55.280 This step, like hexokinase we talked about earlier, 00:03:55.280 --> 00:03:57.870 are sites of regulation of the pathway. 00:03:57.870 --> 00:04:01.820 The word glycolysis comes from the Greek word sugar splitting, 00:04:01.820 --> 00:04:03.740 and we're now at the step where the splitting 00:04:03.740 --> 00:04:06.560 of the sugar from one 6-carbon compound into two 00:04:06.560 --> 00:04:08.840 3-carbon compounds occurs. 00:04:08.840 --> 00:04:12.290 The enzyme that splits the sugar in half is called aldolase. 00:04:12.290 --> 00:04:13.940 And, again, I advise you to take a look 00:04:13.940 --> 00:04:17.300 at the book to see the detail of the chemical reaction. 00:04:17.300 --> 00:04:21.589 Briefly, the enzyme is going to form a proteinated Schiff base 00:04:21.589 --> 00:04:25.340 at the number two carbon of the fructose 1,6-bisphosphate. 00:04:25.340 --> 00:04:28.820 That proteinated Schiff base is going to draw electrons all 00:04:28.820 --> 00:04:32.660 the way over from the hydroxyl group on carbon 4 of fructose 00:04:32.660 --> 00:04:34.430 1,6-bisphosphate. 00:04:34.430 --> 00:04:36.110 That movement of electrons is going 00:04:36.110 --> 00:04:38.570 to result in cleavage of the molecule 00:04:38.570 --> 00:04:42.010 into two parts at the broken line shown in the figure. 00:04:42.010 --> 00:04:44.880 Carbons 1, 2, and 3 are going to form 00:04:44.880 --> 00:04:48.520 DHAP, or dihydroxyacetone phosphate, 00:04:48.520 --> 00:04:50.570 and carbons 4, 5, and 6 are going 00:04:50.570 --> 00:04:54.110 to form GAP, or glyceraldehyde 3-phosphate. 00:04:54.110 --> 00:04:58.720 GAP and DHAP are the products of the aldolase reaction. 00:04:58.720 --> 00:05:02.420 As an aside, this is also a branch point in the pathway. 00:05:02.420 --> 00:05:05.350 Dihydroxyacetone phosphate is an opportunity 00:05:05.350 --> 00:05:08.470 for the cell to make the glycerol backbones of lipids 00:05:08.470 --> 00:05:10.550 that we'll come to later. 00:05:10.550 --> 00:05:12.940 So we see here that glycolysis is indeed 00:05:12.940 --> 00:05:14.860 a resource that can be used for things 00:05:14.860 --> 00:05:17.810 other than energy generation. 00:05:17.810 --> 00:05:20.000 Now let's look at panel B. 00:05:20.000 --> 00:05:23.510 Imagine that a cell is committed to make as much energy 00:05:23.510 --> 00:05:24.500 as it can. 00:05:24.500 --> 00:05:26.210 For example, the stand up, sit down 00:05:26.210 --> 00:05:28.740 scenario we talked about earlier. 00:05:28.740 --> 00:05:33.470 In that case, the enzyme TIM, or triosephosphate isomerase, 00:05:33.470 --> 00:05:36.680 is going to interconvert very quickly dihydroxyacetone 00:05:36.680 --> 00:05:39.590 phosphate and glyceraldehyde 3-phosphate, 00:05:39.590 --> 00:05:41.230 and glyceraldehyde 2-phosphate is 00:05:41.230 --> 00:05:43.370 going to be the molecule that progresses 00:05:43.370 --> 00:05:46.270 along the glycolysis pathway. 00:05:46.270 --> 00:05:49.900 In the next step of glycolysis, glyceraldehyde 3-phosphate 00:05:49.900 --> 00:05:52.900 will be oxidized by glyceraldehyde 3-phosphate 00:05:52.900 --> 00:05:55.960 dehydrogenase, or GAPDH. 00:05:55.960 --> 00:05:58.330 Again, take a look at the mechanism in the book. 00:05:58.330 --> 00:06:03.380 In brief, it involves attack by a thiol residue of a GAPDH 00:06:03.380 --> 00:06:08.260 cysteine on the aldehydic carbon of glyceraldehyde 3-phosphate. 00:06:08.260 --> 00:06:12.270 The product is a thiohemiacetal which is then oxidized. 00:06:12.270 --> 00:06:16.950 Its electrons are transferred as hydride to NAD+ on the enzyme 00:06:16.950 --> 00:06:18.500 to form NADH. 00:06:18.500 --> 00:06:22.400 This is the only oxidation step in the pathway of glycolysis. 00:06:22.400 --> 00:06:25.780 The oxidation of the hemiacetal produces the thioester, 00:06:25.780 --> 00:06:28.810 and that thioester is a very high energy compound. 00:06:28.810 --> 00:06:32.080 The thioester is attacked by inorganic phosphate to form 00:06:32.080 --> 00:06:35.410 an acetylphosphate, another high energy compound, 00:06:35.410 --> 00:06:38.710 which is called 1,3-bisphosphoglycerate. 00:06:38.710 --> 00:06:42.170 The acetylphosphate of 1,3-bisphosphoglycerate then 00:06:42.170 --> 00:06:47.230 phosphorylates ADP to form ATP in the first ATP forming step 00:06:47.230 --> 00:06:48.610 of glycolysis. 00:06:48.610 --> 00:06:50.590 Keep in mind that two molecules of GAP 00:06:50.590 --> 00:06:53.646 have been formed in the upstream part of the pathway. 00:06:53.646 --> 00:06:55.270 So for each molecule of glucose, you're 00:06:55.270 --> 00:06:58.030 getting two molecules of ATP at this step. 00:06:58.030 --> 00:07:00.100 The enzyme that does this phosphorylation 00:07:00.100 --> 00:07:02.950 is phosphoglycerate kinase. 00:07:02.950 --> 00:07:06.670 After 1,3-bisphosphoglycerate has lost its terminal phosphate 00:07:06.670 --> 00:07:10.750 from the 1-carbon, it forms the acid 3-phosphoglycerate. 00:07:10.750 --> 00:07:13.060 As a reminder, mutases are enzymes 00:07:13.060 --> 00:07:15.280 that move a functional group from one atom 00:07:15.280 --> 00:07:18.230 to another on the same molecule. 00:07:18.230 --> 00:07:21.520 The next enzyme in the pathway is phosphoglycerate mutase, 00:07:21.520 --> 00:07:23.950 which in effect moves the phosphate from the 3 00:07:23.950 --> 00:07:27.220 to the 2-carbon, forming 2-phosphoglycerate, which 00:07:27.220 --> 00:07:30.850 is the next intermediate in the glycolysis pathway. 00:07:30.850 --> 00:07:33.040 Although it's easy to say that the phosphate is 00:07:33.040 --> 00:07:36.326 quote unquote "moved," that is somewhat inaccurate. 00:07:36.326 --> 00:07:38.450 If you look at the details of the step in the book, 00:07:38.450 --> 00:07:41.020 you'll see that the reaction starts with the transfer 00:07:41.020 --> 00:07:44.050 of a phosphate from the mutase protein to the substrate 00:07:44.050 --> 00:07:48.400 3-phosphoglycerate, forming an intermediate bis-phosphorylated 00:07:48.400 --> 00:07:51.850 product 2,3-bisphosphoglycerate. 00:07:51.850 --> 00:07:55.710 As an aside, this is the same powerful allosteric effector 00:07:55.710 --> 00:07:58.810 that JoAnne described when she taught us about how small 00:07:58.810 --> 00:08:01.840 molecules can dramatically reduce the affinity 00:08:01.840 --> 00:08:05.770 of hemoglobin for oxygen. In the case of the mutase, however, 00:08:05.770 --> 00:08:09.640 the enzyme will take the phosphate off of the 3-hydroxyl 00:08:09.640 --> 00:08:12.430 of 2,3-bisphosphoglycerate and the enzyme will 00:08:12.430 --> 00:08:14.900 re-phosphorylate itself. 00:08:14.900 --> 00:08:17.740 So the final product of the phosphoglycerate mutase 00:08:17.740 --> 00:08:21.150 reaction is 2-phosphoglycerate. 00:08:21.150 --> 00:08:23.390 Now let's turn to storyboard four. 00:08:23.390 --> 00:08:26.160 In panel A, we see the enzyme enolase, 00:08:26.160 --> 00:08:28.560 which removes the hydrogen from the 2-carbon 00:08:28.560 --> 00:08:30.210 of 2-phosphoglycerate. 00:08:30.210 --> 00:08:31.950 This is not an easy task. 00:08:31.950 --> 00:08:35.130 The PK of that hydrogen is about 30. 00:08:35.130 --> 00:08:38.820 Nevertheless, the reaction does occur and liberates water. 00:08:38.820 --> 00:08:41.520 The product is phosphoenolpyruvate, 00:08:41.520 --> 00:08:46.770 usually abbreviated PEP, or PEP, a very high energy compound. 00:08:46.770 --> 00:08:50.160 We're now almost at the end of the pathway of glycolysis. 00:08:50.160 --> 00:08:52.170 And as I mentioned earlier, one usually 00:08:52.170 --> 00:08:55.650 looks for highly exergonic steps near the beginnings 00:08:55.650 --> 00:08:59.460 or ends of pathways to see where the pathway is regulated. 00:08:59.460 --> 00:09:03.270 Pyruvate kinase, or PK, the last step in the pathway 00:09:03.270 --> 00:09:05.980 is such a regulation point. 00:09:05.980 --> 00:09:09.280 The pyruvate kinase reaction occurs in two steps. 00:09:09.280 --> 00:09:11.470 In the first step, phosphoenolpyruvate 00:09:11.470 --> 00:09:14.560 phosphorylates ADP to form ATP. 00:09:14.560 --> 00:09:18.430 The enol product then undergoes enol-keto tautomerization, 00:09:18.430 --> 00:09:20.740 yielding the ketone pyruvate. 00:09:20.740 --> 00:09:24.040 And pyruvate is the end of the pathway. 00:09:24.040 --> 00:09:26.450 Let's turn now to story board five, 00:09:26.450 --> 00:09:29.130 panel A. Let's take a look at this pathway 00:09:29.130 --> 00:09:31.110 from a higher altitude. 00:09:31.110 --> 00:09:32.760 First, as I just mentioned, the pathway 00:09:32.760 --> 00:09:34.920 is regulated at the top and bottom 00:09:34.920 --> 00:09:37.290 specifically at the hexokinase step, 00:09:37.290 --> 00:09:41.100 the glycogen phosphorylase step, and the pyruvate kinase step. 00:09:41.100 --> 00:09:43.620 It's also regulated in the middle, specifically 00:09:43.620 --> 00:09:47.010 at the phosphofructokinase one step. 00:09:47.010 --> 00:09:49.320 Regulation can be allosteric, which we 00:09:49.320 --> 00:09:51.960 shall see as the case with PFK. 00:09:51.960 --> 00:09:53.115 It also can be covalent. 00:09:53.115 --> 00:09:55.740 We saw that this is the case with glycogen phosphorylase, 00:09:55.740 --> 00:09:57.342 or GP. 00:09:57.342 --> 00:09:58.800 The last lecture, we'll take a look 00:09:58.800 --> 00:10:01.680 at regulation of these enzymes in great detail. 00:10:01.680 --> 00:10:04.500 As a second issue, let's now look at the pathway 00:10:04.500 --> 00:10:08.490 as drawn in summary form in panel B of this storyboard. 00:10:08.490 --> 00:10:12.600 We start with a single molecule of the 6-carbon sugar glucose. 00:10:12.600 --> 00:10:15.600 Hexokinase or glucokinase will utilize 00:10:15.600 --> 00:10:19.140 one ATP to form a phosphorylated intermediate. 00:10:19.140 --> 00:10:23.040 Phosphofructokinase-1 will use a second ATP to form a doubly 00:10:23.040 --> 00:10:26.670 phosphorylated hexose, fructose 1,6-bisphosphate. 00:10:26.670 --> 00:10:29.490 Bis-phosphorylated hexose will split 00:10:29.490 --> 00:10:33.210 into two trioses, glyceraldehyde 3-phosphate and 00:10:33.210 --> 00:10:35.310 dihydroxyacetone phosphate. 00:10:35.310 --> 00:10:37.320 These are inter-convertible. 00:10:37.320 --> 00:10:40.590 The chemical species glyceraldehyde 3-phosphate 00:10:40.590 --> 00:10:42.630 is subjected to oxidation. 00:10:42.630 --> 00:10:46.200 Because we get two molecules of GAP per molecule of glucose, 00:10:46.200 --> 00:10:51.030 GAP oxidation will produce two molecules of NADH. 00:10:51.030 --> 00:10:52.680 In the next step, we're going to make 00:10:52.680 --> 00:10:56.730 two ATP's using the enzyme phosphoglycerate kinase. 00:10:56.730 --> 00:10:59.000 At this point, we're ATP neutral. 00:10:59.000 --> 00:11:03.210 We've consumed two ATP's and we've generated two ATP's. 00:11:03.210 --> 00:11:07.050 And lastly, the enzyme pyruvate kinase is going to generate 00:11:07.050 --> 00:11:09.210 an additional two ATP's. 00:11:09.210 --> 00:11:13.140 I put those in a box, because these are the two net ATP's 00:11:13.140 --> 00:11:14.410 for the whole pathway. 00:11:14.410 --> 00:11:17.101 So that's the pathway of glycolysis. 00:11:17.101 --> 00:11:19.350 I want to give you a little bit of a preview of coming 00:11:19.350 --> 00:11:21.180 attractions at this point. 00:11:21.180 --> 00:11:23.940 What you'll notice is that the pathway involves an oxidation 00:11:23.940 --> 00:11:28.800 step in which we consumed two NAD+ molecules and generated 00:11:28.800 --> 00:11:30.630 two NADH's. 00:11:30.630 --> 00:11:33.000 NAD+ is derived from a vitamin. 00:11:33.000 --> 00:11:35.400 We'll only have limited amounts of it. 00:11:35.400 --> 00:11:38.490 We need to find a way to regenerate the NAD+ in order 00:11:38.490 --> 00:11:40.950 to process the next molecule of glucose, 00:11:40.950 --> 00:11:43.440 and we'll see that nature has several ways to solve that 00:11:43.440 --> 00:11:44.940 problem. 00:11:44.940 --> 00:11:48.960 Nature actually has three ways to regenerate NAD+. 00:11:48.960 --> 00:11:52.140 The first we'll call alcoholic fermentation. 00:11:52.140 --> 00:11:54.780 The second is homolactic fermentation, 00:11:54.780 --> 00:11:56.940 and the third is respiration. 00:11:56.940 --> 00:12:00.060 Alcoholic fermentation and homolactic fermentation 00:12:00.060 --> 00:12:03.390 occur in the absence of oxygen. That is, anaerobically. 00:12:03.390 --> 00:12:07.050 Respiration by definition is an aerobic process. 00:12:07.050 --> 00:12:10.470 Now we'll look at each of these mechanisms of regeneration 00:12:10.470 --> 00:12:15.090 of NAD+ in some detail, but also, in addition to NAD+, 00:12:15.090 --> 00:12:17.910 you're going to have to generate a number of other products that 00:12:17.910 --> 00:12:19.980 can be useful to the cell. 00:12:19.980 --> 00:12:21.450 We'll see those later. 00:12:21.450 --> 00:12:25.110 Let's look at panel C. Under anaerobic conditions, 00:12:25.110 --> 00:12:27.840 yeast will take pyruvate and convert it initially 00:12:27.840 --> 00:12:32.610 to acetaldehyde, and then reduce the acetaldehyde to ethanol. 00:12:32.610 --> 00:12:35.610 These are the reactions of alcoholic fermentation. 00:12:35.610 --> 00:12:39.240 Yeast uses an enzyme called pyruvate decarboxylase 00:12:39.240 --> 00:12:40.850 to process the pyruvate. 00:12:40.850 --> 00:12:44.730 Pyruvate decarboxylase, or PDC, has on it 00:12:44.730 --> 00:12:48.240 a covalently attached thiamin pyrophosphate. 00:12:48.240 --> 00:12:51.750 Thiamin is derived from vitamin B1. 00:12:51.750 --> 00:12:55.650 As we go through the pyruvate decarboxylase reactions, 00:12:55.650 --> 00:12:57.540 at the outset I want you to keep in mind 00:12:57.540 --> 00:13:00.120 that PDC, pyruvate decarboxylase, 00:13:00.120 --> 00:13:03.090 is very similar to the front end of the chemical reaction 00:13:03.090 --> 00:13:05.910 series that's conducted by an enzyme present in mammals 00:13:05.910 --> 00:13:06.840 like us. 00:13:06.840 --> 00:13:10.200 That enzyme complex has pyruvate dehydrogenase, 00:13:10.200 --> 00:13:12.120 which we'll come to a little later when we 00:13:12.120 --> 00:13:14.100 talk about respiration. 00:13:14.100 --> 00:13:17.490 The thiazole ring in TPP forms an ylide. 00:13:17.490 --> 00:13:21.750 That means that despite the fact that the PKA of the thiamin 00:13:21.750 --> 00:13:23.880 pyrophosphate is about 19, you are 00:13:23.880 --> 00:13:27.970 able to form a carbanion at the carbon of the thiazolium ring 00:13:27.970 --> 00:13:28.470 system. 00:13:28.470 --> 00:13:31.470 That carbanion attacks the middle carbon of pyruvate, 00:13:31.470 --> 00:13:35.140 converting it from a ketone to an alcohol. 00:13:35.140 --> 00:13:38.710 Now you have the thiazolium ring system with the positive charge 00:13:38.710 --> 00:13:41.590 beta to the carboxylate of pyruvate. 00:13:41.590 --> 00:13:43.660 That system readily decarboxylates 00:13:43.660 --> 00:13:47.080 as shown, liberating thiamin pyrophosphate and the product, 00:13:47.080 --> 00:13:48.820 acetaldehyde. 00:13:48.820 --> 00:13:50.800 The next enzyme in this small pathway 00:13:50.800 --> 00:13:53.770 is alcohol dehydrogenase which utilizes 00:13:53.770 --> 00:13:57.550 NADH, which came in, in principle, from the GAPDH 00:13:57.550 --> 00:13:59.740 step of glycolysis. 00:13:59.740 --> 00:14:04.690 Alcohol dehydrogenase uses the glycolysis-derived NADH 00:14:04.690 --> 00:14:08.650 to reduce the aldehyde functionality of acetaldehyde 00:14:08.650 --> 00:14:12.130 to form the product of this pathway, ethanol. 00:14:12.130 --> 00:14:17.240 Ethanol is an alcohol, hence the name alcoholic fermentation. 00:14:17.240 --> 00:14:21.070 So looking at this small pathway in total, what you see 00:14:21.070 --> 00:14:24.820 is that you form CO2 as a first product, which 00:14:24.820 --> 00:14:27.040 could be the bubbles in a carbonated beverage 00:14:27.040 --> 00:14:30.250 or what makes bread rise, and form ethanol 00:14:30.250 --> 00:14:32.200 as the other major product. 00:14:32.200 --> 00:14:34.630 And, of course, you get your NAD+ back, 00:14:34.630 --> 00:14:37.030 which you can then return to glycolysis, 00:14:37.030 --> 00:14:41.200 specifically the GAPDH step of glycolysis, 00:14:41.200 --> 00:14:43.780 to enable metabolic processing of the next molecule 00:14:43.780 --> 00:14:45.320 of glucose. 00:14:45.320 --> 00:14:47.050 So this is the pathway that yeast 00:14:47.050 --> 00:14:49.450 and other alcohol-forming organisms 00:14:49.450 --> 00:14:53.300 use to maintain redox neutrality within the cell. 00:14:53.300 --> 00:14:57.412 I'm on storyboard six, and we're going to start with panel D. 00:14:57.412 --> 00:14:59.620 The second general mechanism that we're going to look 00:14:59.620 --> 00:15:02.170 at that concerns the regeneration of NAD+ 00:15:02.170 --> 00:15:05.950 for glycolysis is called homolactic fermentation. 00:15:05.950 --> 00:15:08.800 This occurs in mammals and in lactic acid bacteria. 00:15:08.800 --> 00:15:11.170 And like alcoholic fermentation, it 00:15:11.170 --> 00:15:13.640 is also a process that occurs anaerobically. 00:15:13.640 --> 00:15:16.300 That is, in the absence of oxygen. 00:15:16.300 --> 00:15:19.570 As you can see, pyruvate is a keto acid, 00:15:19.570 --> 00:15:23.470 and the ketone at the number 2 carbon can be easily reduced. 00:15:23.470 --> 00:15:27.310 In this case, NADH will transfer hydride to the ketone 00:15:27.310 --> 00:15:31.000 in order to reduce it to the alcohol lactate. 00:15:31.000 --> 00:15:34.600 The net reaction here involves consumption of one NADH 00:15:34.600 --> 00:15:37.110 and the production of one NAD+. 00:15:37.110 --> 00:15:40.570 And this NAD+ of course, can go back and be utilized to enable 00:15:40.570 --> 00:15:43.390 oxidation of the next molecule of glucose passing through 00:15:43.390 --> 00:15:45.130 the glycolytic pathway. 00:15:45.130 --> 00:15:46.870 When a mammal is running hard, this 00:15:46.870 --> 00:15:49.120 is the pathway by which we achieve redox 00:15:49.120 --> 00:15:51.280 neutrality and glycolysis. 00:15:51.280 --> 00:15:54.100 When we exercise intensely, lactate 00:15:54.100 --> 00:15:58.040 is produced in excess to keep the glycolytic pathway active. 00:15:58.040 --> 00:16:01.820 The lactate causes the blood pH to go down. 00:16:01.820 --> 00:16:05.110 That is, the blood becomes more acidic because lactic acid has 00:16:05.110 --> 00:16:07.030 a low PKA. 00:16:07.030 --> 00:16:10.750 I also want to point out that this anaerobic pathway is also 00:16:10.750 --> 00:16:14.050 the basis for production of lactate by lactic acid 00:16:14.050 --> 00:16:18.580 bacteria, which is critical to the manufacturing of yogurt. 00:16:18.580 --> 00:16:22.690 Let's look now at panel E. The third pathway to regenerate 00:16:22.690 --> 00:16:25.660 NAD+ for glycolysis is respiration. 00:16:25.660 --> 00:16:28.540 We're going to be going through respiration in some detail 00:16:28.540 --> 00:16:30.670 later, but right now I'm going to give you 00:16:30.670 --> 00:16:33.160 a very high level view of it. 00:16:33.160 --> 00:16:36.280 In the way of an introduction, the mitochondrial intermembrane 00:16:36.280 --> 00:16:40.030 is very well equipped to be able to transport electrons. 00:16:40.030 --> 00:16:41.770 Those electrons will travel along 00:16:41.770 --> 00:16:45.700 in an electron transport chain to oxygen, reducing the oxygen 00:16:45.700 --> 00:16:47.710 we breathe into water. 00:16:47.710 --> 00:16:50.660 This is a highly energy generating process, 00:16:50.660 --> 00:16:53.110 and the energy that's generated is part of the driving 00:16:53.110 --> 00:16:55.670 force for the synthesis of ATP. 00:16:55.670 --> 00:16:58.780 The details of how a respiring organism generates ATP 00:16:58.780 --> 00:16:59.930 is covered later. 00:16:59.930 --> 00:17:03.130 For right now, however, let's just say that the mitochondrial 00:17:03.130 --> 00:17:07.660 membrane oxidizes NADH to regenerate the NAD+ needed 00:17:07.660 --> 00:17:09.130 to sustain glycolysis. 00:17:09.130 --> 00:17:10.810 And, again, we'll see the details 00:17:10.810 --> 00:17:12.510 of how this happens later. 00:17:12.510 --> 00:17:16.089 Later, I'll also cover the ways that redox neutrality is 00:17:16.089 --> 00:17:18.190 maintained in a mammalian cell. 00:17:18.190 --> 00:17:23.500 In brief, NAD+ is generated from NADH in aerobes by a series 00:17:23.500 --> 00:17:26.560 of reactions that I call quote unquote "the shuttles," 00:17:26.560 --> 00:17:28.870 which will be covered in section 12. 00:17:28.870 --> 00:17:32.525 Before we go on, let me give you a little recap of where we are. 00:17:32.525 --> 00:17:34.900 We've seen that there are a couple of optional beginnings 00:17:34.900 --> 00:17:36.650 for glycolysis. 00:17:36.650 --> 00:17:39.250 It can begin with intake of glucose from the blood, 00:17:39.250 --> 00:17:41.560 or it can begin with the breakdown of glycogen 00:17:41.560 --> 00:17:42.340 by glycogenolysis. 00:17:42.340 --> 00:17:47.950 The formal pathway takes glucose as glucose 6-phosphate down 00:17:47.950 --> 00:17:49.390 to pyruvate. 00:17:49.390 --> 00:17:52.300 We get a total of two ATP's in that process, 00:17:52.300 --> 00:17:55.720 and we produce two NADH's. 00:17:55.720 --> 00:17:59.530 Now, however, we've got to have a way to be able to regenerate 00:17:59.530 --> 00:18:03.970 our NAD+ from those NADH's in order to be able to make 00:18:03.970 --> 00:18:07.870 the pathway ready to process the next molecule of glucose. 00:18:07.870 --> 00:18:11.980 Accordingly, nature developed three endings to the pathway 00:18:11.980 --> 00:18:15.370 that result in the regeneration of NAD+. 00:18:15.370 --> 00:18:18.160 These endings are alcoholic fermentation, 00:18:18.160 --> 00:18:21.280 homolactic fermentation, and respiration. 00:18:21.280 --> 00:18:24.670 Looking at that picture in panel E once again, in us, 00:18:24.670 --> 00:18:27.020 respiration happens in the mitochondria 00:18:27.020 --> 00:18:29.740 and primarily in the mitochondrial intermembrane 00:18:29.740 --> 00:18:33.550 and in the jelly-like mitochondrial matrix. 00:18:33.550 --> 00:18:35.860 Pyruvate generated in the cytoplasm-- 00:18:35.860 --> 00:18:38.560 that's the compartment where glycolysis occurs-- 00:18:38.560 --> 00:18:41.650 goes through the porous outer membrane of the mitochondria. 00:18:41.650 --> 00:18:45.180 Then, the pyruvate encounters the membrane-bound pyruvate 00:18:45.180 --> 00:18:48.760 dehydrogenase complex, which is our next topic. 00:18:48.760 --> 00:18:52.210 In bacteria, which are in many ways like mitochondria, 00:18:52.210 --> 00:18:54.610 respiration happens in the cellular membrane 00:18:54.610 --> 00:18:56.980 and in the cell's cytoplasm. 00:18:56.980 --> 00:19:00.820 Let's take a look at panel A of storyboard seven. 00:19:00.820 --> 00:19:03.710 We're about to start our discussion of respiration, 00:19:03.710 --> 00:19:05.890 which is the oxidative metabolism 00:19:05.890 --> 00:19:10.090 of all metabolic fuels via the common intermediate acetyl CoA. 00:19:10.090 --> 00:19:12.040 In mammals, as I said earlier, these 00:19:12.040 --> 00:19:14.350 are mitochondrial reactions. 00:19:14.350 --> 00:19:16.360 At the outset, I also want to point out 00:19:16.360 --> 00:19:18.220 that we have seen that carbohydrates 00:19:18.220 --> 00:19:23.110 can be metabolized either anaerobically or aerobically. 00:19:23.110 --> 00:19:26.140 As we'll see when we use lipids as our metabolic fuels, 00:19:26.140 --> 00:19:29.510 they can only be metabolized aerobically. 00:19:29.510 --> 00:19:32.170 Lipids break down to acetyl CoA, which is then 00:19:32.170 --> 00:19:34.840 oxidized by the TCA cycle. 00:19:34.840 --> 00:19:36.670 Let's take a look at panel B. When 00:19:36.670 --> 00:19:39.460 we talked about alcoholic fermentation earlier, 00:19:39.460 --> 00:19:43.480 I said that yeast have a pyruvate decarboxylase complex, 00:19:43.480 --> 00:19:47.200 and I said that the reactions of the pyruvate decarboxylase 00:19:47.200 --> 00:19:51.310 complex are very similar to the reactions in the early part 00:19:51.310 --> 00:19:54.400 of the pyruvate dehydrogenase reaction, which 00:19:54.400 --> 00:19:56.450 is a little bit more complex. 00:19:56.450 --> 00:20:02.440 Pyruvate dehydrogenase has three activities, E1, E2, and E3. 00:20:02.440 --> 00:20:05.530 As with pyruvate decarboxylase, E1 00:20:05.530 --> 00:20:12.100 has a thiamine pyrophosphate unit, TPP, and ylide on the TPP 00:20:12.100 --> 00:20:14.350 attacks the middle carbon of the pyruvate. 00:20:14.350 --> 00:20:16.882 Specifically, it's ketone carbon. 00:20:16.882 --> 00:20:18.340 At this point, you're going to want 00:20:18.340 --> 00:20:20.800 to take a look at detailed notes that I've provided 00:20:20.800 --> 00:20:22.880 as supplemental material. 00:20:22.880 --> 00:20:24.670 This supplemental material will be 00:20:24.670 --> 00:20:29.000 referred to as slide one, slide two, and so on. 00:20:29.000 --> 00:20:32.210 Looking at slide one, you can see that decarboxylation 00:20:32.210 --> 00:20:34.220 happens exactly the same way that I 00:20:34.220 --> 00:20:38.200 described for the pyruvate decarboxylase system. 00:20:38.200 --> 00:20:41.020 Now take a look at slides two through six. 00:20:41.020 --> 00:20:44.080 In the case of PDH, pyruvate dehydrogenase, 00:20:44.080 --> 00:20:48.610 unlike the situation with PDC, pyruvate decarboxylase, 00:20:48.610 --> 00:20:51.190 restructuring of the carboxyethyl group 00:20:51.190 --> 00:20:54.310 is going to result in the formation of our carbanion 00:20:54.310 --> 00:20:58.610 that's going to attack the disulfide of lipoic acid. 00:20:58.610 --> 00:21:01.040 Looking at slide seven, you'll see the conversion 00:21:01.040 --> 00:21:04.520 of the hydroxyethyl to a keto functionality 00:21:04.520 --> 00:21:08.410 jettisons the TPP, resulting in a thioester in which there 00:21:08.410 --> 00:21:12.110 is an acyl group connected to lipoic acid. 00:21:12.110 --> 00:21:15.110 At this point, the thiol of coenzyme A 00:21:15.110 --> 00:21:18.140 attacks the keto oxygen of the thioester, 00:21:18.140 --> 00:21:20.630 producing acetyl CoA, which is going 00:21:20.630 --> 00:21:23.750 to become a very important molecule as we move ahead. 00:21:23.750 --> 00:21:26.900 The second product is reduced lipoic acid. 00:21:26.900 --> 00:21:30.320 Technically, the formation of reduced lipoic acid 00:21:30.320 --> 00:21:34.070 is the oxidation step of the pyruvate dehydrogenase 00:21:34.070 --> 00:21:35.120 reaction. 00:21:35.120 --> 00:21:37.490 The decarboxylation step that happened 00:21:37.490 --> 00:21:40.880 a few steps earlier is basically the production of CO2 00:21:40.880 --> 00:21:44.720 that we eventually will breathe out when we exhale. 00:21:44.720 --> 00:21:46.940 Now let's look at slide eight. 00:21:46.940 --> 00:21:49.940 The reducing equivalents on the E2 subunits 00:21:49.940 --> 00:21:52.280 present as reduced lipoic acid will 00:21:52.280 --> 00:21:56.840 move across the E2 subunit toward the E3 subunit. 00:21:56.840 --> 00:22:00.860 The E3 subunit has an oxidized disulfide bond on it, 00:22:00.860 --> 00:22:03.070 which was created by the connection of two 00:22:03.070 --> 00:22:05.060 cysteines on the protein. 00:22:05.060 --> 00:22:07.550 That oxidized disulfide is then reduced 00:22:07.550 --> 00:22:09.650 by transfer of the reducing equivalents 00:22:09.650 --> 00:22:13.100 from the reduced lipoic acid to the disulfide. 00:22:13.100 --> 00:22:15.050 And then finally, the reducing equivalents 00:22:15.050 --> 00:22:20.890 are passed from the reduced disulfide to FAD to form FADH2. 00:22:20.890 --> 00:22:25.270 That FADH2 passes along its reducing equivalents to NAD+ 00:22:25.270 --> 00:22:26.860 forming NADH. 00:22:26.860 --> 00:22:31.670 This NADH is soluble and will move to its next location. 00:22:31.670 --> 00:22:34.910 Specifically, this NADH will return to the mitochondrial 00:22:34.910 --> 00:22:37.820 membrane-- actually to another place in the mitochondrial 00:22:37.820 --> 00:22:38.840 membrane-- 00:22:38.840 --> 00:22:42.230 an enzyme called complex one, and be oxidized in the electron 00:22:42.230 --> 00:22:44.000 transport chain. 00:22:44.000 --> 00:22:47.810 Overall, one pyruvate enters the PDH complex. 00:22:47.810 --> 00:22:50.750 We lose its carboxylate as CO2. 00:22:50.750 --> 00:22:56.390 We generate from pyruvate's residue an acetyl coenzyme A. 00:22:56.390 --> 00:22:58.700 And at the very end, we get an NADH, 00:22:58.700 --> 00:23:01.310 which will then go on to the electron transport complex 00:23:01.310 --> 00:23:02.870 to be oxidized. 00:23:02.870 --> 00:23:05.570 The formation of NAD+, as I mentioned above, 00:23:05.570 --> 00:23:08.840 is critical to allow further oxidation of reduce. 00:23:08.840 --> 00:23:12.490 That is, energy rich molecule such as glucose.