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 --> 00:00:18,450 at ocw.mit.edu. 8 00:00:21,000 --> 00:00:24,090 JOHN ESSIGMANN: Let's take a look at storyboard number 10. 9 00:00:24,090 --> 00:00:26,040 Back in earlier sessions, we talked 10 00:00:26,040 --> 00:00:28,650 about the detail of glycolysis. 11 00:00:28,650 --> 00:00:30,270 One of the points that I emphasized 12 00:00:30,270 --> 00:00:32,520 is the fact that it's necessary to maintain 13 00:00:32,520 --> 00:00:37,020 redox neutrality in the cytoplasm of a mammalian cell. 14 00:00:37,020 --> 00:00:39,960 It's also necessary to maintain redox neutrality 15 00:00:39,960 --> 00:00:44,070 and prokaryotic cells, not just eukaryotic cells. 16 00:00:44,070 --> 00:00:47,400 At the glyceraldehyde 3-phosphate dehydrogenase step 17 00:00:47,400 --> 00:00:52,650 of glycolysis, NAD+ was consumed and converted to NADH. 18 00:00:52,650 --> 00:00:57,480 That means we have to find a way to convert NADH back to NAD+ 19 00:00:57,480 --> 00:01:01,140 in order to make glycolysis a continuous process. 20 00:01:01,140 --> 00:01:03,690 Back in lectures three and four, I said that there were three 21 00:01:03,690 --> 00:01:08,610 ways to convert the NADH back to NAD+ These were alcoholic 22 00:01:08,610 --> 00:01:11,460 fermentation in anaerobic cells. 23 00:01:11,460 --> 00:01:15,320 Homolactic fermentation, again, in an anaerobic environment. 24 00:01:15,320 --> 00:01:17,100 And the third is respiration, which occurs 25 00:01:17,100 --> 00:01:19,350 in the presence of oxygen. 26 00:01:19,350 --> 00:01:22,950 I'm going to loop back now and revisit this topic. 27 00:01:22,950 --> 00:01:25,770 I want to spotlight three general strategies 28 00:01:25,770 --> 00:01:29,820 that cells use to achieve redox neutrality in the cytoplasm. 29 00:01:29,820 --> 00:01:32,250 The first is lactate dehydrogenase. 30 00:01:32,250 --> 00:01:36,420 The second is called the glycerol-3-phosphate shuttle. 31 00:01:36,420 --> 00:01:39,930 And the third is called the malate-aspartate shuttle. 32 00:01:39,930 --> 00:01:44,130 Panel A of this figure shows the cytoplasm working in concert 33 00:01:44,130 --> 00:01:45,540 with the mitochondrion. 34 00:01:45,540 --> 00:01:47,640 You can see depicted on the left, 35 00:01:47,640 --> 00:01:49,620 the pathway of glycolysis. 36 00:01:49,620 --> 00:01:52,590 In the middle, pyruvate dehydrogenase. 37 00:01:52,590 --> 00:01:55,630 And to the right, the citric acid cycle or TCA cycle, 38 00:01:55,630 --> 00:01:57,870 which we just covered. 39 00:01:57,870 --> 00:02:00,540 There are two important boundary conditions for the discussion 40 00:02:00,540 --> 00:02:01,870 we're about to have. 41 00:02:01,870 --> 00:02:04,140 The first is that oxaloacetate is, 42 00:02:04,140 --> 00:02:07,260 as I mentioned earlier, present only in very small 43 00:02:07,260 --> 00:02:09,930 concentrations within the cell and especially 44 00:02:09,930 --> 00:02:11,850 in the mitochondrion. 45 00:02:11,850 --> 00:02:13,990 As a consequence, the mitochondrion 46 00:02:13,990 --> 00:02:17,490 does not have a transporter to allow it to escape. 47 00:02:17,490 --> 00:02:19,380 In other words, its concentration 48 00:02:19,380 --> 00:02:21,870 is preserved at about one micromolar 49 00:02:21,870 --> 00:02:24,420 inside the mitochondrion. 50 00:02:24,420 --> 00:02:27,840 The second boundary condition concerns the fact that NAD+ 51 00:02:27,840 --> 00:02:34,320 and NADH, as well as NADP+ and NADPH cannot go directly across 52 00:02:34,320 --> 00:02:37,120 the mitochondrial membrane. 53 00:02:37,120 --> 00:02:39,360 So in other words, there are two separate pools 54 00:02:39,360 --> 00:02:41,070 of this nucleotide co-factor. 55 00:02:41,070 --> 00:02:42,480 One in the cytoplasm. 56 00:02:42,480 --> 00:02:44,310 One in the mitochondrion. 57 00:02:44,310 --> 00:02:46,350 I'll come back to the importance of the two 58 00:02:46,350 --> 00:02:48,870 pools in just a few minutes. 59 00:02:48,870 --> 00:02:50,910 Let's look first, here in panel A, 60 00:02:50,910 --> 00:02:54,270 at the mechanism by which lactate dehydrogenase achieves 61 00:02:54,270 --> 00:02:57,120 redox neutrality in the cytoplasm. 62 00:02:57,120 --> 00:03:00,720 We've already covered this, so this is a bit of a review. 63 00:03:00,720 --> 00:03:03,810 Note that you see NAD+ getting converted to NADH 64 00:03:03,810 --> 00:03:05,040 in the cytoplasm. 65 00:03:05,040 --> 00:03:07,470 That's at that GAPDH or glyceraldehyde 66 00:03:07,470 --> 00:03:09,920 3-phosphate dehydrogenase step. 67 00:03:09,920 --> 00:03:11,820 The hatched lines that you see represent 68 00:03:11,820 --> 00:03:13,890 the flow of electrons. 69 00:03:13,890 --> 00:03:16,290 In other words, electrons flow from glucose 70 00:03:16,290 --> 00:03:18,900 and they end up in NADH. 71 00:03:18,900 --> 00:03:21,940 Then the lactate dehydrogenase enzyme 72 00:03:21,940 --> 00:03:26,580 transfers the electrons from NADH into lactate. 73 00:03:26,580 --> 00:03:29,040 So these electrons from glucose are 74 00:03:29,040 --> 00:03:31,890 involved in the reduction of the ketone functionality 75 00:03:31,890 --> 00:03:36,210 of pyruvate into the alcohol functionality of lactate. 76 00:03:36,210 --> 00:03:39,090 And that's where the electrons stay. 77 00:03:39,090 --> 00:03:41,310 The other product of this reaction, as you'll see, 78 00:03:41,310 --> 00:03:45,240 NAD+ which is now available to enable the oxidation 79 00:03:45,240 --> 00:03:47,610 of the next molecule of glucose. 80 00:03:47,610 --> 00:03:50,640 What happens to the lactate that's produced? 81 00:03:50,640 --> 00:03:53,700 In a working muscle cell, that lactate 82 00:03:53,700 --> 00:03:56,190 will escape from the cell, go into the blood, 83 00:03:56,190 --> 00:03:58,860 and then go to the liver or another organ that's 84 00:03:58,860 --> 00:04:02,550 capable of doing the pathway of gluconeogenesis. 85 00:04:02,550 --> 00:04:04,830 As I've mentioned in the past, gluconeogenesis 86 00:04:04,830 --> 00:04:07,080 is a pathway by which non carbohydrate 87 00:04:07,080 --> 00:04:11,820 precursors, such as lactate, are built back up into glucose. 88 00:04:11,820 --> 00:04:14,230 Keep that working muscle scenario in mind, 89 00:04:14,230 --> 00:04:16,110 because I'm going to come back to it later 90 00:04:16,110 --> 00:04:18,630 when I talk about physiological responses 91 00:04:18,630 --> 00:04:21,720 to stress, such as what I'll call the fight and flight 92 00:04:21,720 --> 00:04:23,550 scenario. 93 00:04:23,550 --> 00:04:26,490 That's all I'm going to say for now about the LDH shuttle. 94 00:04:26,490 --> 00:04:28,350 That is, lactate dehydrogenase shuttle 95 00:04:28,350 --> 00:04:31,410 in panel A, which is the first of the three pathways 96 00:04:31,410 --> 00:04:35,340 by which redox neutrality is maintained in the cytoplasm. 97 00:04:35,340 --> 00:04:38,040 The second pathway to retain redox 98 00:04:38,040 --> 00:04:41,040 neutrality is the glycerol-3-phosphate shuttle. 99 00:04:41,040 --> 00:04:43,590 This pathway is particularly active in the brain 100 00:04:43,590 --> 00:04:45,570 and in skeletal muscle. 101 00:04:45,570 --> 00:04:47,790 Once again, follow the hatched lines 102 00:04:47,790 --> 00:04:51,199 to follow the path of electrons as they go from glucose. 103 00:04:51,199 --> 00:04:52,740 And ultimately, in this case, they're 104 00:04:52,740 --> 00:04:57,000 going to end up being deposited into oxygen to form water. 105 00:04:57,000 --> 00:05:01,520 Starting at the top, you see in NAD+ being reduced to NADH 106 00:05:01,520 --> 00:05:04,920 at the glyceraldehyde 3-phosphate dehydrogenase step, 107 00:05:04,920 --> 00:05:06,410 GAPDH. 108 00:05:06,410 --> 00:05:08,870 Next, we're going to temporarily borrow 109 00:05:08,870 --> 00:05:13,130 a molecule of dihydroxyacetone phosphate, DHAP. 110 00:05:13,130 --> 00:05:15,140 DHAP is a ketone. 111 00:05:15,140 --> 00:05:19,430 And what we're going to do is deposit the electrons from NADH 112 00:05:19,430 --> 00:05:22,610 into the ketone functionality to make the alcohol, 113 00:05:22,610 --> 00:05:24,710 glycerol-3-phosphate. 114 00:05:24,710 --> 00:05:27,350 The source of the electrons was NADH. 115 00:05:27,350 --> 00:05:29,730 And now you've accomplished your chemical goal, 116 00:05:29,730 --> 00:05:33,290 which was to restore the NAD+ pool -- the cytoplasm, 117 00:05:33,290 --> 00:05:37,880 but we borrowed a molecule of dihydroxyacetone phosphate. 118 00:05:37,880 --> 00:05:40,640 And we've somehow got to get that back. 119 00:05:40,640 --> 00:05:45,020 Let me point out, at this point, that the reduction of DHAP 120 00:05:45,020 --> 00:05:47,180 to glycerol-3-phosphate was accomplished 121 00:05:47,180 --> 00:05:51,410 by the cytoplasmic form of the enzyme glycerol-3-phosphate 122 00:05:51,410 --> 00:05:56,120 dehydrogenase, which catalyzed step 2 on the storyboarded. 123 00:05:56,120 --> 00:05:59,400 We're going to deal more with coenzyme q in the next lecture. 124 00:05:59,400 --> 00:06:01,505 But for now, it's a molecule, specifically 125 00:06:01,505 --> 00:06:05,270 a quinone, that's easily reduced to its hydroquinone form, 126 00:06:05,270 --> 00:06:07,130 called QH2. 127 00:06:07,130 --> 00:06:10,850 The structures of q, in QH2, are shown in the box. 128 00:06:10,850 --> 00:06:13,790 QH2 is in the mitochondrial membrane. 129 00:06:13,790 --> 00:06:16,130 In glycerol-3-phosphate dehydrogenase 130 00:06:16,130 --> 00:06:18,200 the mitochondrial version of it is 131 00:06:18,200 --> 00:06:22,640 present in the outer part of the mitochondrial inner membrane. 132 00:06:22,640 --> 00:06:27,230 Like NADH and FADH2, QH2, the hydroquinone, 133 00:06:27,230 --> 00:06:30,670 is what I've called a mobile electron carrier. 134 00:06:30,670 --> 00:06:33,750 QH2 is going to allow the electrons that started out 135 00:06:33,750 --> 00:06:37,280 in glucose or in any LDH of the gap DH step, 136 00:06:37,280 --> 00:06:40,070 to flow through the electron transport chain, which we'll 137 00:06:40,070 --> 00:06:41,570 come to in the next lecture. 138 00:06:41,570 --> 00:06:45,470 And flow into oxygen, which is reduced to form water. 139 00:06:45,470 --> 00:06:49,040 This terminal reduction is shown in step five. 140 00:06:49,040 --> 00:06:52,010 Effectively, in the glycerol-3-phosphate shuttle 141 00:06:52,010 --> 00:06:57,890 we're using oxygen in order to oxidize NADH back to NAD+ And 142 00:06:57,890 --> 00:07:01,130 once again, maintaining a constant supply of NAD+ is 143 00:07:01,130 --> 00:07:04,220 necessary in order to make glycolysis a continuous 144 00:07:04,220 --> 00:07:05,580 process. 145 00:07:05,580 --> 00:07:09,890 Panel C shows a third strategy for maintaining redox 146 00:07:09,890 --> 00:07:12,060 neutrality in the cytoplasm. 147 00:07:12,060 --> 00:07:14,990 This is called the malate-aspartate shuttle. 148 00:07:14,990 --> 00:07:19,310 and this pathway is operative in heart, liver, and kidney. 149 00:07:19,310 --> 00:07:21,950 To the left we see the production of NADH, 150 00:07:21,950 --> 00:07:25,730 just as we did in the previous two small pathways. 151 00:07:25,730 --> 00:07:27,740 At step one, let's assume that there's 152 00:07:27,740 --> 00:07:31,100 a molecule of oxaloacetate present as part 153 00:07:31,100 --> 00:07:35,120 of the cytoplasmic pool of organic acids. 154 00:07:35,120 --> 00:07:38,180 Oxaloacetate or OA is ketone. 155 00:07:38,180 --> 00:07:41,630 And the cytoplasmic form of the enzyme malate dehydrogenase, 156 00:07:41,630 --> 00:07:44,090 working in the reverse direction from the one 157 00:07:44,090 --> 00:07:46,550 that we see operative in the TCA cycle 158 00:07:46,550 --> 00:07:49,910 is able to reduce the oxaloacetate to malate. 159 00:07:49,910 --> 00:07:54,500 We just reduce to ketone OA to an alcohol malate. 160 00:07:54,500 --> 00:07:57,020 In step three, the accumulating malate 161 00:07:57,020 --> 00:07:59,120 is transported by a malate transporter 162 00:07:59,120 --> 00:08:02,130 into the mitochondrial matrix, which is, of course, 163 00:08:02,130 --> 00:08:04,700 the location of the TCA cycle. 164 00:08:04,700 --> 00:08:06,530 At this point, we're going to be using one 165 00:08:06,530 --> 00:08:09,320 of the steps of the TCA cycle. 166 00:08:09,320 --> 00:08:12,560 Specifically, we're going to use malate dehydrogenase, 167 00:08:12,560 --> 00:08:14,600 the mitochondrial version of the enzyme 168 00:08:14,600 --> 00:08:17,930 this time, to convert malate to oxaloacetate. 169 00:08:17,930 --> 00:08:20,120 That reaction is an oxidation. 170 00:08:20,120 --> 00:08:23,540 We use the mitochondrial pool of NAD+ to carry out that 171 00:08:23,540 --> 00:08:24,740 oxidation. 172 00:08:24,740 --> 00:08:27,440 In effect, we're using the electrons that came in from 173 00:08:27,440 --> 00:08:32,309 malate to reduce NAD+ to end NADH in the mitochondrion. 174 00:08:32,309 --> 00:08:35,240 Now, take a careful look at step four. 175 00:08:35,240 --> 00:08:38,390 Looking to the left you see the hatched lines go all the way 176 00:08:38,390 --> 00:08:42,140 back to glucose, which was the source of the electrons. 177 00:08:42,140 --> 00:08:46,280 To the right, the hatched lines by step five, 178 00:08:46,280 --> 00:08:50,060 go to the electron transport chain all the way to oxygen. 179 00:08:50,060 --> 00:08:53,390 We haven't done the electron transport chain as yet. 180 00:08:53,390 --> 00:08:56,660 So you're just going to have to trust me for a little while. 181 00:08:56,660 --> 00:09:01,160 There's an enzyme, NADH dehydrogenase, 182 00:09:01,160 --> 00:09:03,620 in the mitochondrial inner membrane that will take 183 00:09:03,620 --> 00:09:08,120 the electrons from NADH and eventually regenerate the NAD+ 184 00:09:08,120 --> 00:09:10,840 in the mitochondrial matrix. 185 00:09:10,840 --> 00:09:13,110 In step five, we're taking the electrons 186 00:09:13,110 --> 00:09:16,690 from the NADH produced by malate dehydrogenase 187 00:09:16,690 --> 00:09:19,770 and entering those electrons into the electron transport 188 00:09:19,770 --> 00:09:20,670 chain. 189 00:09:20,670 --> 00:09:22,800 Then, in a manner that's quite similar to what 190 00:09:22,800 --> 00:09:25,650 we did in the previous shuttle, the glycerol-3-phosphate 191 00:09:25,650 --> 00:09:27,690 shuttle, those electrons are going 192 00:09:27,690 --> 00:09:30,870 to be transferred to oxygen to make water. 193 00:09:30,870 --> 00:09:33,450 Before I go on, let's review a little bit. 194 00:09:33,450 --> 00:09:36,580 Between step one and step two in the cytoplasm, 195 00:09:36,580 --> 00:09:40,870 we deposited electrons into oxaloacetate to make malate. 196 00:09:40,870 --> 00:09:44,830 That step restored in NAD+ levels in the cytoplasm, 197 00:09:44,830 --> 00:09:46,830 which is what we wanted to accomplish. 198 00:09:46,830 --> 00:09:50,670 However, we've consumed a molecule of oxaloacetate. 199 00:09:50,670 --> 00:09:52,650 And as I've mentioned before, the cell 200 00:09:52,650 --> 00:09:55,470 has to try to preserve the concentration of this very 201 00:09:55,470 --> 00:09:57,010 precious molecule. 202 00:09:57,010 --> 00:10:00,630 We now have to find a way to restore oxaloacetate 203 00:10:00,630 --> 00:10:03,550 that we borrowed in step one. 204 00:10:03,550 --> 00:10:06,300 Now let's look back at step four, 205 00:10:06,300 --> 00:10:08,770 where malate was converted to oxaloacetate 206 00:10:08,770 --> 00:10:11,080 in the mitochondrial matrix. 207 00:10:11,080 --> 00:10:14,410 Because the molecule of malate came from the cytoplasm, 208 00:10:14,410 --> 00:10:17,140 this is a net increase in the mitochondrial matrix 209 00:10:17,140 --> 00:10:19,590 of one unit of malate and, ultimately, 210 00:10:19,590 --> 00:10:22,060 one unit of oxaloacetate. 211 00:10:22,060 --> 00:10:26,050 We need to find a way to get that molecule of oxaloacetate 212 00:10:26,050 --> 00:10:28,660 back out into the cytoplasm, in order to make 213 00:10:28,660 --> 00:10:31,290 the shuttle a continuous one. 214 00:10:31,290 --> 00:10:33,960 In the co-factor section of 5.07, 215 00:10:33,960 --> 00:10:35,760 JoAnne taught us about the ways that 216 00:10:35,760 --> 00:10:39,450 pyridoxal phosphate and pyridoxamine work, in order 217 00:10:39,450 --> 00:10:43,920 to put amino groups into organic acids, such as oxaloacetate. 218 00:10:43,920 --> 00:10:47,170 And that's what's going to happen in this case. 219 00:10:47,170 --> 00:10:51,230 Oxaloacetate is converted into its amino acid homolog, 220 00:10:51,230 --> 00:10:52,970 aspartic acid. 221 00:10:52,970 --> 00:10:56,480 Why did we do this emanation reaction? 222 00:10:56,480 --> 00:10:59,960 Well, there's no way to get oxaloacetate directly out 223 00:10:59,960 --> 00:11:03,540 of the mitochondria because there's no transporter for it. 224 00:11:03,540 --> 00:11:06,710 But there is a good transporter, the apartheid transporter, 225 00:11:06,710 --> 00:11:10,460 that will take aspartic acid out into the cytoplasm. 226 00:11:10,460 --> 00:11:13,880 So the oxaloacetate is converted, temporarily, 227 00:11:13,880 --> 00:11:16,850 into aspartic acid in the mitochondrion. 228 00:11:16,850 --> 00:11:19,310 And that aspartic acid then slips out 229 00:11:19,310 --> 00:11:23,500 through its transporter to the cytoplasm. 230 00:11:23,500 --> 00:11:26,830 Once in the cytoplasm, there's a similar pyridoxal mediated 231 00:11:26,830 --> 00:11:29,860 mechanism to deaminate the aspartate 232 00:11:29,860 --> 00:11:33,430 to regenerate the cytoplasmic molecule of acetate 233 00:11:33,430 --> 00:11:36,660 that we borrowed at step one a few minutes ago. 234 00:11:36,660 --> 00:11:39,070 In panel D I summarize. 235 00:11:39,070 --> 00:11:42,580 That we've looked at three different small pathways that 236 00:11:42,580 --> 00:11:46,180 enable the cytoplasm of the cell to always have enough NAD+ 237 00:11:46,180 --> 00:11:49,320 to oxidize glucose to pyruvate. 238 00:11:49,320 --> 00:11:52,720 These pathways are first, the lactate dehydrogenase system. 239 00:11:52,720 --> 00:11:55,260 Second, the glycerol-3-phosphate shuttle. 240 00:11:55,260 --> 00:11:57,970 And third, the malate departed shuttle.