1 00:00:00,090 --> 00:00:02,490 The following content is provided under a Creative 2 00:00:02,490 --> 00:00:04,030 Commons license. 3 00:00:04,030 --> 00:00:06,330 Your support will help MIT OpenCourseWare 4 00:00:06,330 --> 00:00:10,720 continue to offer high quality educational resources for free. 5 00:00:10,720 --> 00:00:13,320 To make a donation or view additional materials 6 00:00:13,320 --> 00:00:17,280 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,280 --> 00:00:18,240 at ocw.mit.edu. 8 00:00:21,940 --> 00:00:24,180 PROFESSOR: Let's look at storyboard two. 9 00:00:24,180 --> 00:00:25,840 We're going to look in more detail 10 00:00:25,840 --> 00:00:28,624 at carbohydrate catabolism at this point. 11 00:00:28,624 --> 00:00:30,040 We're eventually going to be doing 12 00:00:30,040 --> 00:00:33,220 the pathway of glycolysis, but we have to get there first. 13 00:00:33,220 --> 00:00:34,870 And it depends on what precursors 14 00:00:34,870 --> 00:00:37,980 are available to enter the pathway of glycolysis. 15 00:00:37,980 --> 00:00:39,730 One option that we'll look at a little bit 16 00:00:39,730 --> 00:00:42,520 later is to take in glucose from the blood 17 00:00:42,520 --> 00:00:45,190 by way of a glucose carrier, and then 18 00:00:45,190 --> 00:00:48,560 phosphorylate the glucose using either hexokinase or 19 00:00:48,560 --> 00:00:49,960 glucokinase. 20 00:00:49,960 --> 00:00:53,530 The second option is to take glycogen, the polymeric storage 21 00:00:53,530 --> 00:00:57,370 form of glucose, and degrade it to glucose 1-phosphate, which 22 00:00:57,370 --> 00:01:00,010 will be then converted to glucose 6-phosphate which 23 00:01:00,010 --> 00:01:02,260 will enter the pathway of glycolysis. 24 00:01:02,260 --> 00:01:05,260 We're going to start with this pathway of glycogen breakdown, 25 00:01:05,260 --> 00:01:08,170 or glycogenolysis. 26 00:01:08,170 --> 00:01:10,760 Panel A shows the structure of glycogen, 27 00:01:10,760 --> 00:01:13,630 which consists of glucose monomeric units connected 28 00:01:13,630 --> 00:01:16,360 by a bond between the 1 and 4 carbons 29 00:01:16,360 --> 00:01:18,580 in the alpha configuration. 30 00:01:18,580 --> 00:01:20,140 Chemistry is going to be happening 31 00:01:20,140 --> 00:01:22,800 at the non-reducing end, which is to the left. 32 00:01:22,800 --> 00:01:25,030 The reducing end, which is to the right, 33 00:01:25,030 --> 00:01:28,660 is connected to a scaffolding protein called glycogenin. 34 00:01:28,660 --> 00:01:31,460 We'll come back to glycogenin later in the course. 35 00:01:31,460 --> 00:01:34,600 Let's look next at panel B. The reaction begins 36 00:01:34,600 --> 00:01:38,320 with the proteination of the red oxygen between the terminal two 37 00:01:38,320 --> 00:01:42,220 glucose moieties by a proton on the glycogen phosphorylase 38 00:01:42,220 --> 00:01:43,150 enzyme. 39 00:01:43,150 --> 00:01:46,180 The intermediate product is a resonant stabilized pair 40 00:01:46,180 --> 00:01:49,090 of positively charged species, or cations, 41 00:01:49,090 --> 00:01:52,300 an oxonium ion at the 1 prime oxygen, 42 00:01:52,300 --> 00:01:54,940 and a carbocation at the 1-carbon. 43 00:01:54,940 --> 00:01:57,220 The electrophilic carbocation is attacked 44 00:01:57,220 --> 00:02:00,160 by the negatively charged phosphate residue, which 45 00:02:00,160 --> 00:02:03,745 is non-covalently associated with glycogen phosphorylase 46 00:02:03,745 --> 00:02:07,070 to form glucose 1-phosphate. 47 00:02:07,070 --> 00:02:09,440 Note that the glucose 1-phosphate that forms 48 00:02:09,440 --> 00:02:12,460 has the covalently attached phosphate on the alpha, 49 00:02:12,460 --> 00:02:15,650 or bottom face, as it's drawn in the figure. 50 00:02:15,650 --> 00:02:18,020 The structure of glycogen phosphorylase 51 00:02:18,020 --> 00:02:21,170 allows attack of it's phosphate from the bottom, 52 00:02:21,170 --> 00:02:26,594 giving rise to alpha isomer only of glucose 1-phosphate. 53 00:02:26,594 --> 00:02:28,010 The structure of the phosphorylase 54 00:02:28,010 --> 00:02:30,290 precludes access of its phosphate 55 00:02:30,290 --> 00:02:32,360 to the top face of the sugar molecule, 56 00:02:32,360 --> 00:02:35,930 so you get only one stereoisomer of glucose 1-phosphate out 57 00:02:35,930 --> 00:02:37,320 of this reaction. 58 00:02:37,320 --> 00:02:39,710 The other product shown at the bottom of panel C 59 00:02:39,710 --> 00:02:42,650 is the glycogen chain, which is truncated or shortened 60 00:02:42,650 --> 00:02:44,540 by one glucose unit. 61 00:02:44,540 --> 00:02:47,720 Now looking at the big picture, the epinephrine molecule 62 00:02:47,720 --> 00:02:49,850 produced as part of the stress response 63 00:02:49,850 --> 00:02:53,210 interacted with the cell membrane of the muscle cell. 64 00:02:53,210 --> 00:02:55,700 And that interaction ultimately activated glycogen 65 00:02:55,700 --> 00:02:59,480 phosphorylase to enable it to degrade glycogen, the storage 66 00:02:59,480 --> 00:03:03,200 form of glucose, and liberate glucose 1-phosphate, which 67 00:03:03,200 --> 00:03:06,290 is going to then find its way into glycolysis to generate 68 00:03:06,290 --> 00:03:08,270 fast energy to enable the student 69 00:03:08,270 --> 00:03:10,670 to be able to stand up in class and avoid 70 00:03:10,670 --> 00:03:12,410 the stressful situation. 71 00:03:12,410 --> 00:03:14,600 I'm not going to go through all of the details 72 00:03:14,600 --> 00:03:16,790 of the chemical reactions of glycolysis. 73 00:03:16,790 --> 00:03:18,320 You can find those in the book which 74 00:03:18,320 --> 00:03:21,750 does a good job of presenting those details. 75 00:03:21,750 --> 00:03:23,660 In the first step of glycolysis, we 76 00:03:23,660 --> 00:03:27,380 see the phosphorylated hexose, glucose 6-phosphate, 77 00:03:27,380 --> 00:03:31,040 be converted by phosphoglucoisomerase 78 00:03:31,040 --> 00:03:33,800 into the furanose fructose 6-phosphate. 79 00:03:33,800 --> 00:03:35,930 The next step involves the phosphorylation 80 00:03:35,930 --> 00:03:39,950 of the fructose 6-phosphate by phosphofructokinase 1. 81 00:03:39,950 --> 00:03:43,070 ATP is the phosphate donor, and you form fructose 82 00:03:43,070 --> 00:03:46,200 1,6-bisphosphate as the product. 83 00:03:46,200 --> 00:03:47,930 You'll notice that phosphofructokinase 84 00:03:47,930 --> 00:03:51,230 catalyzes one way, a thermodynamically irreversible 85 00:03:51,230 --> 00:03:52,560 reaction. 86 00:03:52,560 --> 00:03:55,280 This step, like hexokinase we talked about earlier, 87 00:03:55,280 --> 00:03:57,870 are sites of regulation of the pathway. 88 00:03:57,870 --> 00:04:01,820 The word glycolysis comes from the Greek word sugar splitting, 89 00:04:01,820 --> 00:04:03,740 and we're now at the step where the splitting 90 00:04:03,740 --> 00:04:06,560 of the sugar from one 6-carbon compound into two 91 00:04:06,560 --> 00:04:08,840 3-carbon compounds occurs. 92 00:04:08,840 --> 00:04:12,290 The enzyme that splits the sugar in half is called aldolase. 93 00:04:12,290 --> 00:04:13,940 And, again, I advise you to take a look 94 00:04:13,940 --> 00:04:17,300 at the book to see the detail of the chemical reaction. 95 00:04:17,300 --> 00:04:21,589 Briefly, the enzyme is going to form a proteinated Schiff base 96 00:04:21,589 --> 00:04:25,340 at the number two carbon of the fructose 1,6-bisphosphate. 97 00:04:25,340 --> 00:04:28,820 That proteinated Schiff base is going to draw electrons all 98 00:04:28,820 --> 00:04:32,660 the way over from the hydroxyl group on carbon 4 of fructose 99 00:04:32,660 --> 00:04:34,430 1,6-bisphosphate. 100 00:04:34,430 --> 00:04:36,110 That movement of electrons is going 101 00:04:36,110 --> 00:04:38,570 to result in cleavage of the molecule 102 00:04:38,570 --> 00:04:42,010 into two parts at the broken line shown in the figure. 103 00:04:42,010 --> 00:04:44,880 Carbons 1, 2, and 3 are going to form 104 00:04:44,880 --> 00:04:48,520 DHAP, or dihydroxyacetone phosphate, 105 00:04:48,520 --> 00:04:50,570 and carbons 4, 5, and 6 are going 106 00:04:50,570 --> 00:04:54,110 to form GAP, or glyceraldehyde 3-phosphate. 107 00:04:54,110 --> 00:04:58,720 GAP and DHAP are the products of the aldolase reaction. 108 00:04:58,720 --> 00:05:02,420 As an aside, this is also a branch point in the pathway. 109 00:05:02,420 --> 00:05:05,350 Dihydroxyacetone phosphate is an opportunity 110 00:05:05,350 --> 00:05:08,470 for the cell to make the glycerol backbones of lipids 111 00:05:08,470 --> 00:05:10,550 that we'll come to later. 112 00:05:10,550 --> 00:05:12,940 So we see here that glycolysis is indeed 113 00:05:12,940 --> 00:05:14,860 a resource that can be used for things 114 00:05:14,860 --> 00:05:17,810 other than energy generation. 115 00:05:17,810 --> 00:05:20,000 Now let's look at panel B. 116 00:05:20,000 --> 00:05:23,510 Imagine that a cell is committed to make as much energy 117 00:05:23,510 --> 00:05:24,500 as it can. 118 00:05:24,500 --> 00:05:26,210 For example, the stand up, sit down 119 00:05:26,210 --> 00:05:28,740 scenario we talked about earlier. 120 00:05:28,740 --> 00:05:33,470 In that case, the enzyme TIM, or triosephosphate isomerase, 121 00:05:33,470 --> 00:05:36,680 is going to interconvert very quickly dihydroxyacetone 122 00:05:36,680 --> 00:05:39,590 phosphate and glyceraldehyde 3-phosphate, 123 00:05:39,590 --> 00:05:41,230 and glyceraldehyde 2-phosphate is 124 00:05:41,230 --> 00:05:43,370 going to be the molecule that progresses 125 00:05:43,370 --> 00:05:46,270 along the glycolysis pathway. 126 00:05:46,270 --> 00:05:49,900 In the next step of glycolysis, glyceraldehyde 3-phosphate 127 00:05:49,900 --> 00:05:52,900 will be oxidized by glyceraldehyde 3-phosphate 128 00:05:52,900 --> 00:05:55,960 dehydrogenase, or GAPDH. 129 00:05:55,960 --> 00:05:58,330 Again, take a look at the mechanism in the book. 130 00:05:58,330 --> 00:06:03,380 In brief, it involves attack by a thiol residue of a GAPDH 131 00:06:03,380 --> 00:06:08,260 cysteine on the aldehydic carbon of glyceraldehyde 3-phosphate. 132 00:06:08,260 --> 00:06:12,270 The product is a thiohemiacetal which is then oxidized. 133 00:06:12,270 --> 00:06:16,950 Its electrons are transferred as hydride to NAD+ on the enzyme 134 00:06:16,950 --> 00:06:18,500 to form NADH. 135 00:06:18,500 --> 00:06:22,400 This is the only oxidation step in the pathway of glycolysis. 136 00:06:22,400 --> 00:06:25,780 The oxidation of the hemiacetal produces the thioester, 137 00:06:25,780 --> 00:06:28,810 and that thioester is a very high energy compound. 138 00:06:28,810 --> 00:06:32,080 The thioester is attacked by inorganic phosphate to form 139 00:06:32,080 --> 00:06:35,410 an acetylphosphate, another high energy compound, 140 00:06:35,410 --> 00:06:38,710 which is called 1,3-bisphosphoglycerate. 141 00:06:38,710 --> 00:06:42,170 The acetylphosphate of 1,3-bisphosphoglycerate then 142 00:06:42,170 --> 00:06:47,230 phosphorylates ADP to form ATP in the first ATP forming step 143 00:06:47,230 --> 00:06:48,610 of glycolysis. 144 00:06:48,610 --> 00:06:50,590 Keep in mind that two molecules of GAP 145 00:06:50,590 --> 00:06:53,646 have been formed in the upstream part of the pathway. 146 00:06:53,646 --> 00:06:55,270 So for each molecule of glucose, you're 147 00:06:55,270 --> 00:06:58,030 getting two molecules of ATP at this step. 148 00:06:58,030 --> 00:07:00,100 The enzyme that does this phosphorylation 149 00:07:00,100 --> 00:07:02,950 is phosphoglycerate kinase. 150 00:07:02,950 --> 00:07:06,670 After 1,3-bisphosphoglycerate has lost its terminal phosphate 151 00:07:06,670 --> 00:07:10,750 from the 1-carbon, it forms the acid 3-phosphoglycerate. 152 00:07:10,750 --> 00:07:13,060 As a reminder, mutases are enzymes 153 00:07:13,060 --> 00:07:15,280 that move a functional group from one atom 154 00:07:15,280 --> 00:07:18,230 to another on the same molecule. 155 00:07:18,230 --> 00:07:21,520 The next enzyme in the pathway is phosphoglycerate mutase, 156 00:07:21,520 --> 00:07:23,950 which in effect moves the phosphate from the 3 157 00:07:23,950 --> 00:07:27,220 to the 2-carbon, forming 2-phosphoglycerate, which 158 00:07:27,220 --> 00:07:30,850 is the next intermediate in the glycolysis pathway. 159 00:07:30,850 --> 00:07:33,040 Although it's easy to say that the phosphate is 160 00:07:33,040 --> 00:07:36,326 quote unquote "moved," that is somewhat inaccurate. 161 00:07:36,326 --> 00:07:38,450 If you look at the details of the step in the book, 162 00:07:38,450 --> 00:07:41,020 you'll see that the reaction starts with the transfer 163 00:07:41,020 --> 00:07:44,050 of a phosphate from the mutase protein to the substrate 164 00:07:44,050 --> 00:07:48,400 3-phosphoglycerate, forming an intermediate bis-phosphorylated 165 00:07:48,400 --> 00:07:51,850 product 2,3-bisphosphoglycerate. 166 00:07:51,850 --> 00:07:55,710 As an aside, this is the same powerful allosteric effector 167 00:07:55,710 --> 00:07:58,810 that JoAnne described when she taught us about how small 168 00:07:58,810 --> 00:08:01,840 molecules can dramatically reduce the affinity 169 00:08:01,840 --> 00:08:05,770 of hemoglobin for oxygen. In the case of the mutase, however, 170 00:08:05,770 --> 00:08:09,640 the enzyme will take the phosphate off of the 3-hydroxyl 171 00:08:09,640 --> 00:08:12,430 of 2,3-bisphosphoglycerate and the enzyme will 172 00:08:12,430 --> 00:08:14,900 re-phosphorylate itself. 173 00:08:14,900 --> 00:08:17,740 So the final product of the phosphoglycerate mutase 174 00:08:17,740 --> 00:08:21,150 reaction is 2-phosphoglycerate. 175 00:08:21,150 --> 00:08:23,390 Now let's turn to storyboard four. 176 00:08:23,390 --> 00:08:26,160 In panel A, we see the enzyme enolase, 177 00:08:26,160 --> 00:08:28,560 which removes the hydrogen from the 2-carbon 178 00:08:28,560 --> 00:08:30,210 of 2-phosphoglycerate. 179 00:08:30,210 --> 00:08:31,950 This is not an easy task. 180 00:08:31,950 --> 00:08:35,130 The PK of that hydrogen is about 30. 181 00:08:35,130 --> 00:08:38,820 Nevertheless, the reaction does occur and liberates water. 182 00:08:38,820 --> 00:08:41,520 The product is phosphoenolpyruvate, 183 00:08:41,520 --> 00:08:46,770 usually abbreviated PEP, or PEP, a very high energy compound. 184 00:08:46,770 --> 00:08:50,160 We're now almost at the end of the pathway of glycolysis. 185 00:08:50,160 --> 00:08:52,170 And as I mentioned earlier, one usually 186 00:08:52,170 --> 00:08:55,650 looks for highly exergonic steps near the beginnings 187 00:08:55,650 --> 00:08:59,460 or ends of pathways to see where the pathway is regulated. 188 00:08:59,460 --> 00:09:03,270 Pyruvate kinase, or PK, the last step in the pathway 189 00:09:03,270 --> 00:09:05,980 is such a regulation point. 190 00:09:05,980 --> 00:09:09,280 The pyruvate kinase reaction occurs in two steps. 191 00:09:09,280 --> 00:09:11,470 In the first step, phosphoenolpyruvate 192 00:09:11,470 --> 00:09:14,560 phosphorylates ADP to form ATP. 193 00:09:14,560 --> 00:09:18,430 The enol product then undergoes enol-keto tautomerization, 194 00:09:18,430 --> 00:09:20,740 yielding the ketone pyruvate. 195 00:09:20,740 --> 00:09:24,040 And pyruvate is the end of the pathway. 196 00:09:24,040 --> 00:09:26,450 Let's turn now to story board five, 197 00:09:26,450 --> 00:09:29,130 panel A. Let's take a look at this pathway 198 00:09:29,130 --> 00:09:31,110 from a higher altitude. 199 00:09:31,110 --> 00:09:32,760 First, as I just mentioned, the pathway 200 00:09:32,760 --> 00:09:34,920 is regulated at the top and bottom 201 00:09:34,920 --> 00:09:37,290 specifically at the hexokinase step, 202 00:09:37,290 --> 00:09:41,100 the glycogen phosphorylase step, and the pyruvate kinase step. 203 00:09:41,100 --> 00:09:43,620 It's also regulated in the middle, specifically 204 00:09:43,620 --> 00:09:47,010 at the phosphofructokinase one step. 205 00:09:47,010 --> 00:09:49,320 Regulation can be allosteric, which we 206 00:09:49,320 --> 00:09:51,960 shall see as the case with PFK. 207 00:09:51,960 --> 00:09:53,115 It also can be covalent. 208 00:09:53,115 --> 00:09:55,740 We saw that this is the case with glycogen phosphorylase, 209 00:09:55,740 --> 00:09:57,342 or GP. 210 00:09:57,342 --> 00:09:58,800 The last lecture, we'll take a look 211 00:09:58,800 --> 00:10:01,680 at regulation of these enzymes in great detail. 212 00:10:01,680 --> 00:10:04,500 As a second issue, let's now look at the pathway 213 00:10:04,500 --> 00:10:08,490 as drawn in summary form in panel B of this storyboard. 214 00:10:08,490 --> 00:10:12,600 We start with a single molecule of the 6-carbon sugar glucose. 215 00:10:12,600 --> 00:10:15,600 Hexokinase or glucokinase will utilize 216 00:10:15,600 --> 00:10:19,140 one ATP to form a phosphorylated intermediate. 217 00:10:19,140 --> 00:10:23,040 Phosphofructokinase-1 will use a second ATP to form a doubly 218 00:10:23,040 --> 00:10:26,670 phosphorylated hexose, fructose 1,6-bisphosphate. 219 00:10:26,670 --> 00:10:29,490 Bis-phosphorylated hexose will split 220 00:10:29,490 --> 00:10:33,210 into two trioses, glyceraldehyde 3-phosphate and 221 00:10:33,210 --> 00:10:35,310 dihydroxyacetone phosphate. 222 00:10:35,310 --> 00:10:37,320 These are inter-convertible. 223 00:10:37,320 --> 00:10:40,590 The chemical species glyceraldehyde 3-phosphate 224 00:10:40,590 --> 00:10:42,630 is subjected to oxidation. 225 00:10:42,630 --> 00:10:46,200 Because we get two molecules of GAP per molecule of glucose, 226 00:10:46,200 --> 00:10:51,030 GAP oxidation will produce two molecules of NADH. 227 00:10:51,030 --> 00:10:52,680 In the next step, we're going to make 228 00:10:52,680 --> 00:10:56,730 two ATP's using the enzyme phosphoglycerate kinase. 229 00:10:56,730 --> 00:10:59,000 At this point, we're ATP neutral. 230 00:10:59,000 --> 00:11:03,210 We've consumed two ATP's and we've generated two ATP's. 231 00:11:03,210 --> 00:11:07,050 And lastly, the enzyme pyruvate kinase is going to generate 232 00:11:07,050 --> 00:11:09,210 an additional two ATP's. 233 00:11:09,210 --> 00:11:13,140 I put those in a box, because these are the two net ATP's 234 00:11:13,140 --> 00:11:14,410 for the whole pathway. 235 00:11:14,410 --> 00:11:17,101 So that's the pathway of glycolysis. 236 00:11:17,101 --> 00:11:19,350 I want to give you a little bit of a preview of coming 237 00:11:19,350 --> 00:11:21,180 attractions at this point. 238 00:11:21,180 --> 00:11:23,940 What you'll notice is that the pathway involves an oxidation 239 00:11:23,940 --> 00:11:28,800 step in which we consumed two NAD+ molecules and generated 240 00:11:28,800 --> 00:11:30,630 two NADH's. 241 00:11:30,630 --> 00:11:33,000 NAD+ is derived from a vitamin. 242 00:11:33,000 --> 00:11:35,400 We'll only have limited amounts of it. 243 00:11:35,400 --> 00:11:38,490 We need to find a way to regenerate the NAD+ in order 244 00:11:38,490 --> 00:11:40,950 to process the next molecule of glucose, 245 00:11:40,950 --> 00:11:43,440 and we'll see that nature has several ways to solve that 246 00:11:43,440 --> 00:11:44,940 problem. 247 00:11:44,940 --> 00:11:48,960 Nature actually has three ways to regenerate NAD+. 248 00:11:48,960 --> 00:11:52,140 The first we'll call alcoholic fermentation. 249 00:11:52,140 --> 00:11:54,780 The second is homolactic fermentation, 250 00:11:54,780 --> 00:11:56,940 and the third is respiration. 251 00:11:56,940 --> 00:12:00,060 Alcoholic fermentation and homolactic fermentation 252 00:12:00,060 --> 00:12:03,390 occur in the absence of oxygen. That is, anaerobically. 253 00:12:03,390 --> 00:12:07,050 Respiration by definition is an aerobic process. 254 00:12:07,050 --> 00:12:10,470 Now we'll look at each of these mechanisms of regeneration 255 00:12:10,470 --> 00:12:15,090 of NAD+ in some detail, but also, in addition to NAD+, 256 00:12:15,090 --> 00:12:17,910 you're going to have to generate a number of other products that 257 00:12:17,910 --> 00:12:19,980 can be useful to the cell. 258 00:12:19,980 --> 00:12:21,450 We'll see those later. 259 00:12:21,450 --> 00:12:25,110 Let's look at panel C. Under anaerobic conditions, 260 00:12:25,110 --> 00:12:27,840 yeast will take pyruvate and convert it initially 261 00:12:27,840 --> 00:12:32,610 to acetaldehyde, and then reduce the acetaldehyde to ethanol. 262 00:12:32,610 --> 00:12:35,610 These are the reactions of alcoholic fermentation. 263 00:12:35,610 --> 00:12:39,240 Yeast uses an enzyme called pyruvate decarboxylase 264 00:12:39,240 --> 00:12:40,850 to process the pyruvate. 265 00:12:40,850 --> 00:12:44,730 Pyruvate decarboxylase, or PDC, has on it 266 00:12:44,730 --> 00:12:48,240 a covalently attached thiamin pyrophosphate. 267 00:12:48,240 --> 00:12:51,750 Thiamin is derived from vitamin B1. 268 00:12:51,750 --> 00:12:55,650 As we go through the pyruvate decarboxylase reactions, 269 00:12:55,650 --> 00:12:57,540 at the outset I want you to keep in mind 270 00:12:57,540 --> 00:13:00,120 that PDC, pyruvate decarboxylase, 271 00:13:00,120 --> 00:13:03,090 is very similar to the front end of the chemical reaction 272 00:13:03,090 --> 00:13:05,910 series that's conducted by an enzyme present in mammals 273 00:13:05,910 --> 00:13:06,840 like us. 274 00:13:06,840 --> 00:13:10,200 That enzyme complex has pyruvate dehydrogenase, 275 00:13:10,200 --> 00:13:12,120 which we'll come to a little later when we 276 00:13:12,120 --> 00:13:14,100 talk about respiration. 277 00:13:14,100 --> 00:13:17,490 The thiazole ring in TPP forms an ylide. 278 00:13:17,490 --> 00:13:21,750 That means that despite the fact that the PKA of the thiamin 279 00:13:21,750 --> 00:13:23,880 pyrophosphate is about 19, you are 280 00:13:23,880 --> 00:13:27,970 able to form a carbanion at the carbon of the thiazolium ring 281 00:13:27,970 --> 00:13:28,470 system. 282 00:13:28,470 --> 00:13:31,470 That carbanion attacks the middle carbon of pyruvate, 283 00:13:31,470 --> 00:13:35,140 converting it from a ketone to an alcohol. 284 00:13:35,140 --> 00:13:38,710 Now you have the thiazolium ring system with the positive charge 285 00:13:38,710 --> 00:13:41,590 beta to the carboxylate of pyruvate. 286 00:13:41,590 --> 00:13:43,660 That system readily decarboxylates 287 00:13:43,660 --> 00:13:47,080 as shown, liberating thiamin pyrophosphate and the product, 288 00:13:47,080 --> 00:13:48,820 acetaldehyde. 289 00:13:48,820 --> 00:13:50,800 The next enzyme in this small pathway 290 00:13:50,800 --> 00:13:53,770 is alcohol dehydrogenase which utilizes 291 00:13:53,770 --> 00:13:57,550 NADH, which came in, in principle, from the GAPDH 292 00:13:57,550 --> 00:13:59,740 step of glycolysis. 293 00:13:59,740 --> 00:14:04,690 Alcohol dehydrogenase uses the glycolysis-derived NADH 294 00:14:04,690 --> 00:14:08,650 to reduce the aldehyde functionality of acetaldehyde 295 00:14:08,650 --> 00:14:12,130 to form the product of this pathway, ethanol. 296 00:14:12,130 --> 00:14:17,240 Ethanol is an alcohol, hence the name alcoholic fermentation. 297 00:14:17,240 --> 00:14:21,070 So looking at this small pathway in total, what you see 298 00:14:21,070 --> 00:14:24,820 is that you form CO2 as a first product, which 299 00:14:24,820 --> 00:14:27,040 could be the bubbles in a carbonated beverage 300 00:14:27,040 --> 00:14:30,250 or what makes bread rise, and form ethanol 301 00:14:30,250 --> 00:14:32,200 as the other major product. 302 00:14:32,200 --> 00:14:34,630 And, of course, you get your NAD+ back, 303 00:14:34,630 --> 00:14:37,030 which you can then return to glycolysis, 304 00:14:37,030 --> 00:14:41,200 specifically the GAPDH step of glycolysis, 305 00:14:41,200 --> 00:14:43,780 to enable metabolic processing of the next molecule 306 00:14:43,780 --> 00:14:45,320 of glucose. 307 00:14:45,320 --> 00:14:47,050 So this is the pathway that yeast 308 00:14:47,050 --> 00:14:49,450 and other alcohol-forming organisms 309 00:14:49,450 --> 00:14:53,300 use to maintain redox neutrality within the cell. 310 00:14:53,300 --> 00:14:57,412 I'm on storyboard six, and we're going to start with panel D. 311 00:14:57,412 --> 00:14:59,620 The second general mechanism that we're going to look 312 00:14:59,620 --> 00:15:02,170 at that concerns the regeneration of NAD+ 313 00:15:02,170 --> 00:15:05,950 for glycolysis is called homolactic fermentation. 314 00:15:05,950 --> 00:15:08,800 This occurs in mammals and in lactic acid bacteria. 315 00:15:08,800 --> 00:15:11,170 And like alcoholic fermentation, it 316 00:15:11,170 --> 00:15:13,640 is also a process that occurs anaerobically. 317 00:15:13,640 --> 00:15:16,300 That is, in the absence of oxygen. 318 00:15:16,300 --> 00:15:19,570 As you can see, pyruvate is a keto acid, 319 00:15:19,570 --> 00:15:23,470 and the ketone at the number 2 carbon can be easily reduced. 320 00:15:23,470 --> 00:15:27,310 In this case, NADH will transfer hydride to the ketone 321 00:15:27,310 --> 00:15:31,000 in order to reduce it to the alcohol lactate. 322 00:15:31,000 --> 00:15:34,600 The net reaction here involves consumption of one NADH 323 00:15:34,600 --> 00:15:37,110 and the production of one NAD+. 324 00:15:37,110 --> 00:15:40,570 And this NAD+ of course, can go back and be utilized to enable 325 00:15:40,570 --> 00:15:43,390 oxidation of the next molecule of glucose passing through 326 00:15:43,390 --> 00:15:45,130 the glycolytic pathway. 327 00:15:45,130 --> 00:15:46,870 When a mammal is running hard, this 328 00:15:46,870 --> 00:15:49,120 is the pathway by which we achieve redox 329 00:15:49,120 --> 00:15:51,280 neutrality and glycolysis. 330 00:15:51,280 --> 00:15:54,100 When we exercise intensely, lactate 331 00:15:54,100 --> 00:15:58,040 is produced in excess to keep the glycolytic pathway active. 332 00:15:58,040 --> 00:16:01,820 The lactate causes the blood pH to go down. 333 00:16:01,820 --> 00:16:05,110 That is, the blood becomes more acidic because lactic acid has 334 00:16:05,110 --> 00:16:07,030 a low PKA. 335 00:16:07,030 --> 00:16:10,750 I also want to point out that this anaerobic pathway is also 336 00:16:10,750 --> 00:16:14,050 the basis for production of lactate by lactic acid 337 00:16:14,050 --> 00:16:18,580 bacteria, which is critical to the manufacturing of yogurt. 338 00:16:18,580 --> 00:16:22,690 Let's look now at panel E. The third pathway to regenerate 339 00:16:22,690 --> 00:16:25,660 NAD+ for glycolysis is respiration. 340 00:16:25,660 --> 00:16:28,540 We're going to be going through respiration in some detail 341 00:16:28,540 --> 00:16:30,670 later, but right now I'm going to give you 342 00:16:30,670 --> 00:16:33,160 a very high level view of it. 343 00:16:33,160 --> 00:16:36,280 In the way of an introduction, the mitochondrial intermembrane 344 00:16:36,280 --> 00:16:40,030 is very well equipped to be able to transport electrons. 345 00:16:40,030 --> 00:16:41,770 Those electrons will travel along 346 00:16:41,770 --> 00:16:45,700 in an electron transport chain to oxygen, reducing the oxygen 347 00:16:45,700 --> 00:16:47,710 we breathe into water. 348 00:16:47,710 --> 00:16:50,660 This is a highly energy generating process, 349 00:16:50,660 --> 00:16:53,110 and the energy that's generated is part of the driving 350 00:16:53,110 --> 00:16:55,670 force for the synthesis of ATP. 351 00:16:55,670 --> 00:16:58,780 The details of how a respiring organism generates ATP 352 00:16:58,780 --> 00:16:59,930 is covered later. 353 00:16:59,930 --> 00:17:03,130 For right now, however, let's just say that the mitochondrial 354 00:17:03,130 --> 00:17:07,660 membrane oxidizes NADH to regenerate the NAD+ needed 355 00:17:07,660 --> 00:17:09,130 to sustain glycolysis. 356 00:17:09,130 --> 00:17:10,810 And, again, we'll see the details 357 00:17:10,810 --> 00:17:12,510 of how this happens later. 358 00:17:12,510 --> 00:17:16,089 Later, I'll also cover the ways that redox neutrality is 359 00:17:16,089 --> 00:17:18,190 maintained in a mammalian cell. 360 00:17:18,190 --> 00:17:23,500 In brief, NAD+ is generated from NADH in aerobes by a series 361 00:17:23,500 --> 00:17:26,560 of reactions that I call quote unquote "the shuttles," 362 00:17:26,560 --> 00:17:28,870 which will be covered in section 12. 363 00:17:28,870 --> 00:17:32,525 Before we go on, let me give you a little recap of where we are. 364 00:17:32,525 --> 00:17:34,900 We've seen that there are a couple of optional beginnings 365 00:17:34,900 --> 00:17:36,650 for glycolysis. 366 00:17:36,650 --> 00:17:39,250 It can begin with intake of glucose from the blood, 367 00:17:39,250 --> 00:17:41,560 or it can begin with the breakdown of glycogen 368 00:17:41,560 --> 00:17:42,340 by glycogenolysis. 369 00:17:42,340 --> 00:17:47,950 The formal pathway takes glucose as glucose 6-phosphate down 370 00:17:47,950 --> 00:17:49,390 to pyruvate. 371 00:17:49,390 --> 00:17:52,300 We get a total of two ATP's in that process, 372 00:17:52,300 --> 00:17:55,720 and we produce two NADH's. 373 00:17:55,720 --> 00:17:59,530 Now, however, we've got to have a way to be able to regenerate 374 00:17:59,530 --> 00:18:03,970 our NAD+ from those NADH's in order to be able to make 375 00:18:03,970 --> 00:18:07,870 the pathway ready to process the next molecule of glucose. 376 00:18:07,870 --> 00:18:11,980 Accordingly, nature developed three endings to the pathway 377 00:18:11,980 --> 00:18:15,370 that result in the regeneration of NAD+. 378 00:18:15,370 --> 00:18:18,160 These endings are alcoholic fermentation, 379 00:18:18,160 --> 00:18:21,280 homolactic fermentation, and respiration. 380 00:18:21,280 --> 00:18:24,670 Looking at that picture in panel E once again, in us, 381 00:18:24,670 --> 00:18:27,020 respiration happens in the mitochondria 382 00:18:27,020 --> 00:18:29,740 and primarily in the mitochondrial intermembrane 383 00:18:29,740 --> 00:18:33,550 and in the jelly-like mitochondrial matrix. 384 00:18:33,550 --> 00:18:35,860 Pyruvate generated in the cytoplasm-- 385 00:18:35,860 --> 00:18:38,560 that's the compartment where glycolysis occurs-- 386 00:18:38,560 --> 00:18:41,650 goes through the porous outer membrane of the mitochondria. 387 00:18:41,650 --> 00:18:45,180 Then, the pyruvate encounters the membrane-bound pyruvate 388 00:18:45,180 --> 00:18:48,760 dehydrogenase complex, which is our next topic. 389 00:18:48,760 --> 00:18:52,210 In bacteria, which are in many ways like mitochondria, 390 00:18:52,210 --> 00:18:54,610 respiration happens in the cellular membrane 391 00:18:54,610 --> 00:18:56,980 and in the cell's cytoplasm. 392 00:18:56,980 --> 00:19:00,820 Let's take a look at panel A of storyboard seven. 393 00:19:00,820 --> 00:19:03,710 We're about to start our discussion of respiration, 394 00:19:03,710 --> 00:19:05,890 which is the oxidative metabolism 395 00:19:05,890 --> 00:19:10,090 of all metabolic fuels via the common intermediate acetyl CoA. 396 00:19:10,090 --> 00:19:12,040 In mammals, as I said earlier, these 397 00:19:12,040 --> 00:19:14,350 are mitochondrial reactions. 398 00:19:14,350 --> 00:19:16,360 At the outset, I also want to point out 399 00:19:16,360 --> 00:19:18,220 that we have seen that carbohydrates 400 00:19:18,220 --> 00:19:23,110 can be metabolized either anaerobically or aerobically. 401 00:19:23,110 --> 00:19:26,140 As we'll see when we use lipids as our metabolic fuels, 402 00:19:26,140 --> 00:19:29,510 they can only be metabolized aerobically. 403 00:19:29,510 --> 00:19:32,170 Lipids break down to acetyl CoA, which is then 404 00:19:32,170 --> 00:19:34,840 oxidized by the TCA cycle. 405 00:19:34,840 --> 00:19:36,670 Let's take a look at panel B. When 406 00:19:36,670 --> 00:19:39,460 we talked about alcoholic fermentation earlier, 407 00:19:39,460 --> 00:19:43,480 I said that yeast have a pyruvate decarboxylase complex, 408 00:19:43,480 --> 00:19:47,200 and I said that the reactions of the pyruvate decarboxylase 409 00:19:47,200 --> 00:19:51,310 complex are very similar to the reactions in the early part 410 00:19:51,310 --> 00:19:54,400 of the pyruvate dehydrogenase reaction, which 411 00:19:54,400 --> 00:19:56,450 is a little bit more complex. 412 00:19:56,450 --> 00:20:02,440 Pyruvate dehydrogenase has three activities, E1, E2, and E3. 413 00:20:02,440 --> 00:20:05,530 As with pyruvate decarboxylase, E1 414 00:20:05,530 --> 00:20:12,100 has a thiamine pyrophosphate unit, TPP, and ylide on the TPP 415 00:20:12,100 --> 00:20:14,350 attacks the middle carbon of the pyruvate. 416 00:20:14,350 --> 00:20:16,882 Specifically, it's ketone carbon. 417 00:20:16,882 --> 00:20:18,340 At this point, you're going to want 418 00:20:18,340 --> 00:20:20,800 to take a look at detailed notes that I've provided 419 00:20:20,800 --> 00:20:22,880 as supplemental material. 420 00:20:22,880 --> 00:20:24,670 This supplemental material will be 421 00:20:24,670 --> 00:20:29,000 referred to as slide one, slide two, and so on. 422 00:20:29,000 --> 00:20:32,210 Looking at slide one, you can see that decarboxylation 423 00:20:32,210 --> 00:20:34,220 happens exactly the same way that I 424 00:20:34,220 --> 00:20:38,200 described for the pyruvate decarboxylase system. 425 00:20:38,200 --> 00:20:41,020 Now take a look at slides two through six. 426 00:20:41,020 --> 00:20:44,080 In the case of PDH, pyruvate dehydrogenase, 427 00:20:44,080 --> 00:20:48,610 unlike the situation with PDC, pyruvate decarboxylase, 428 00:20:48,610 --> 00:20:51,190 restructuring of the carboxyethyl group 429 00:20:51,190 --> 00:20:54,310 is going to result in the formation of our carbanion 430 00:20:54,310 --> 00:20:58,610 that's going to attack the disulfide of lipoic acid. 431 00:20:58,610 --> 00:21:01,040 Looking at slide seven, you'll see the conversion 432 00:21:01,040 --> 00:21:04,520 of the hydroxyethyl to a keto functionality 433 00:21:04,520 --> 00:21:08,410 jettisons the TPP, resulting in a thioester in which there 434 00:21:08,410 --> 00:21:12,110 is an acyl group connected to lipoic acid. 435 00:21:12,110 --> 00:21:15,110 At this point, the thiol of coenzyme A 436 00:21:15,110 --> 00:21:18,140 attacks the keto oxygen of the thioester, 437 00:21:18,140 --> 00:21:20,630 producing acetyl CoA, which is going 438 00:21:20,630 --> 00:21:23,750 to become a very important molecule as we move ahead. 439 00:21:23,750 --> 00:21:26,900 The second product is reduced lipoic acid. 440 00:21:26,900 --> 00:21:30,320 Technically, the formation of reduced lipoic acid 441 00:21:30,320 --> 00:21:34,070 is the oxidation step of the pyruvate dehydrogenase 442 00:21:34,070 --> 00:21:35,120 reaction. 443 00:21:35,120 --> 00:21:37,490 The decarboxylation step that happened 444 00:21:37,490 --> 00:21:40,880 a few steps earlier is basically the production of CO2 445 00:21:40,880 --> 00:21:44,720 that we eventually will breathe out when we exhale. 446 00:21:44,720 --> 00:21:46,940 Now let's look at slide eight. 447 00:21:46,940 --> 00:21:49,940 The reducing equivalents on the E2 subunits 448 00:21:49,940 --> 00:21:52,280 present as reduced lipoic acid will 449 00:21:52,280 --> 00:21:56,840 move across the E2 subunit toward the E3 subunit. 450 00:21:56,840 --> 00:22:00,860 The E3 subunit has an oxidized disulfide bond on it, 451 00:22:00,860 --> 00:22:03,070 which was created by the connection of two 452 00:22:03,070 --> 00:22:05,060 cysteines on the protein. 453 00:22:05,060 --> 00:22:07,550 That oxidized disulfide is then reduced 454 00:22:07,550 --> 00:22:09,650 by transfer of the reducing equivalents 455 00:22:09,650 --> 00:22:13,100 from the reduced lipoic acid to the disulfide. 456 00:22:13,100 --> 00:22:15,050 And then finally, the reducing equivalents 457 00:22:15,050 --> 00:22:20,890 are passed from the reduced disulfide to FAD to form FADH2. 458 00:22:20,890 --> 00:22:25,270 That FADH2 passes along its reducing equivalents to NAD+ 459 00:22:25,270 --> 00:22:26,860 forming NADH. 460 00:22:26,860 --> 00:22:31,670 This NADH is soluble and will move to its next location. 461 00:22:31,670 --> 00:22:34,910 Specifically, this NADH will return to the mitochondrial 462 00:22:34,910 --> 00:22:37,820 membrane-- actually to another place in the mitochondrial 463 00:22:37,820 --> 00:22:38,840 membrane-- 464 00:22:38,840 --> 00:22:42,230 an enzyme called complex one, and be oxidized in the electron 465 00:22:42,230 --> 00:22:44,000 transport chain. 466 00:22:44,000 --> 00:22:47,810 Overall, one pyruvate enters the PDH complex. 467 00:22:47,810 --> 00:22:50,750 We lose its carboxylate as CO2. 468 00:22:50,750 --> 00:22:56,390 We generate from pyruvate's residue an acetyl coenzyme A. 469 00:22:56,390 --> 00:22:58,700 And at the very end, we get an NADH, 470 00:22:58,700 --> 00:23:01,310 which will then go on to the electron transport complex 471 00:23:01,310 --> 00:23:02,870 to be oxidized. 472 00:23:02,870 --> 00:23:05,570 The formation of NAD+, as I mentioned above, 473 00:23:05,570 --> 00:23:08,840 is critical to allow further oxidation of reduce. 474 00:23:08,840 --> 00:23:12,490 That is, energy rich molecule such as glucose.