1 00:00:00,000 --> 00:00:02,460 [SQUEAKING] 2 00:00:02,460 --> 00:00:03,936 [RUSTLING] 3 00:00:03,936 --> 00:00:06,396 [CLICKING] 4 00:00:10,840 --> 00:00:14,740 PROFESSOR: So last time, we discussed glycolysis 5 00:00:14,740 --> 00:00:15,850 as a pathway. 6 00:00:15,850 --> 00:00:17,950 And I just want to remind everybody 7 00:00:17,950 --> 00:00:22,010 that, like all pathways, glycolysis has to be favorable. 8 00:00:22,010 --> 00:00:25,570 That is, delta G across the entire pathway 9 00:00:25,570 --> 00:00:28,060 has to be less than 0. 10 00:00:28,060 --> 00:00:29,770 We spent a lot of time discussing 11 00:00:29,770 --> 00:00:31,510 the regulation of glycolysis. 12 00:00:31,510 --> 00:00:33,790 And I just want to revisit this quickly. 13 00:00:33,790 --> 00:00:36,850 And remember, we discussed that the steps that are regulated 14 00:00:36,850 --> 00:00:39,580 are those with the biggest, quote, unquote, 15 00:00:39,580 --> 00:00:40,960 bio-energetic cost. 16 00:00:40,960 --> 00:00:43,480 That is, the biggest drop in delta G 17 00:00:43,480 --> 00:00:47,630 is as molecules would proceed across the pathway. 18 00:00:47,630 --> 00:00:50,600 And those are shown here-- the hexokinase step, 19 00:00:50,600 --> 00:00:53,770 the phosphofructokinase step, and the pyruvate kinase 20 00:00:53,770 --> 00:00:56,580 step of the pathway. 21 00:00:56,580 --> 00:01:03,550 Now this is largely true of most pathways that we will consider, 22 00:01:03,550 --> 00:01:05,890 in that these regulated steps are often 23 00:01:05,890 --> 00:01:08,020 those with the biggest bio-energetic cost. 24 00:01:08,020 --> 00:01:11,770 And the reason for that is that it makes a lot of sense. 25 00:01:11,770 --> 00:01:16,960 Once you go through those steps, it's very difficult to go back. 26 00:01:16,960 --> 00:01:20,590 Also, you'll note that these are the steps that 27 00:01:20,590 --> 00:01:24,710 regulate entrance and exit from the pathway. 28 00:01:24,710 --> 00:01:27,850 And this also is something we'll commonly see and other pathways 29 00:01:27,850 --> 00:01:30,670 and also makes some sense. 30 00:01:30,670 --> 00:01:32,680 Just to remind everybody, I drew up 31 00:01:32,680 --> 00:01:36,640 here a skeletonized version of glycolysis 32 00:01:36,640 --> 00:01:42,160 and the most important regulatory features of it. 33 00:01:42,160 --> 00:01:44,470 Just to remind people that initially, 34 00:01:44,470 --> 00:01:47,350 hexokinase is regulated by glucose-6 phosphate. 35 00:01:47,350 --> 00:01:50,470 Glucose-6 phosphate negatively regulates hexokinase. 36 00:01:50,470 --> 00:01:53,740 And that's because if the glucose-6 phosphate levels 37 00:01:53,740 --> 00:01:56,620 in the cell go up such that it can't be shunted to glycogen-- 38 00:01:56,620 --> 00:01:58,390 something we'll talk about today-- 39 00:01:58,390 --> 00:02:00,460 or be sent through glycolysis, it 40 00:02:00,460 --> 00:02:03,100 makes sense not to trap more glucose. 41 00:02:03,100 --> 00:02:06,820 Once glucose-6 phosphate enters the rest 42 00:02:06,820 --> 00:02:09,370 of the glycolytic pathway, the key step of regulation 43 00:02:09,370 --> 00:02:11,110 is phosphofructokinase. 44 00:02:11,110 --> 00:02:15,740 It, of course, will have positive feedback, saying, 45 00:02:15,740 --> 00:02:17,710 let's put carbon into the pathway. 46 00:02:17,710 --> 00:02:22,510 If energy charge low, AMP is high, activates it. 47 00:02:22,510 --> 00:02:25,270 This molecule fructose 2,6-bisphosphate that we talked 48 00:02:25,270 --> 00:02:28,060 about earlier today-- we'll mention again today-- 49 00:02:28,060 --> 00:02:32,020 can be involved in some other signaling inputs 50 00:02:32,020 --> 00:02:34,240 to allosterically regulate the enzyme. 51 00:02:34,240 --> 00:02:37,420 FBP can feed forward to activate pyruvate kinase. 52 00:02:37,420 --> 00:02:39,380 You want to match input with output. 53 00:02:39,380 --> 00:02:42,640 And, of course, if PEP builds up, you want to slow it down. 54 00:02:42,640 --> 00:02:44,140 If a downstream product-- 55 00:02:44,140 --> 00:02:46,970 citrate-- builds up, you want to slow it down. 56 00:02:46,970 --> 00:02:50,570 And of course, a major output of glycolysis is ATP. 57 00:02:50,570 --> 00:02:56,110 And if ATP is high, no reason to continue oxidizing glucose 58 00:02:56,110 --> 00:02:57,670 carbons. 59 00:02:57,670 --> 00:03:03,370 Now this regulation we discussed is often 60 00:03:03,370 --> 00:03:09,340 done at the allosteric level by controlling enzyme rates. 61 00:03:09,340 --> 00:03:13,540 That can be via a small molecule binding, as I've shown up here. 62 00:03:13,540 --> 00:03:15,940 And it could also be via signaling. 63 00:03:15,940 --> 00:03:19,990 And that's really a place where fructose 2,6-bisphosphate 64 00:03:19,990 --> 00:03:24,700 as a combination of signaling, and allosteric small molecules 65 00:03:24,700 --> 00:03:25,540 comes into play. 66 00:03:25,540 --> 00:03:29,500 That is, you can have signaling that controls the enzymes that 67 00:03:29,500 --> 00:03:32,170 generate fructose 2,6-bisphosphate, 68 00:03:32,170 --> 00:03:35,440 thereby creating an additional molecule that can act 69 00:03:35,440 --> 00:03:41,230 as allosteric regulator of the enzyme PFK to generate FBP 70 00:03:41,230 --> 00:03:44,620 and commit carbon to glycolysis. 71 00:03:44,620 --> 00:03:48,160 Now we closed the discussion last time 72 00:03:48,160 --> 00:03:51,400 beginning to discuss how it is that organisms 73 00:03:51,400 --> 00:03:52,840 can make glucose. 74 00:03:52,840 --> 00:03:54,970 Obviously, this glucose carbon that 75 00:03:54,970 --> 00:03:59,020 is being oxidized to get ATP has to come from somewhere. 76 00:03:59,020 --> 00:04:02,140 We mentioned that plants make glucose during the day 77 00:04:02,140 --> 00:04:06,460 when they have plenty of excess energy from light from the sun 78 00:04:06,460 --> 00:04:10,090 but then use that glucose to do constant metabolism to maintain 79 00:04:10,090 --> 00:04:12,760 their ATP levels at night. 80 00:04:12,760 --> 00:04:16,329 And we as animals obviously take advantage of this 81 00:04:16,329 --> 00:04:18,700 by eating the glucose that ultimately 82 00:04:18,700 --> 00:04:24,760 was created by plants using the energy of the sun. 83 00:04:24,760 --> 00:04:31,840 We began to discuss an example of where us, as organisms, also 84 00:04:31,840 --> 00:04:33,620 make glucose. 85 00:04:33,620 --> 00:04:35,770 Of course, this is not net production of glucose. 86 00:04:35,770 --> 00:04:39,250 But for instance, if our muscle cells are working hard, 87 00:04:39,250 --> 00:04:40,630 lactate can build up. 88 00:04:40,630 --> 00:04:42,880 That lactate is excreted into the blood. 89 00:04:42,880 --> 00:04:44,800 That lactate can then be used by the liver 90 00:04:44,800 --> 00:04:46,090 to regenerate glucose. 91 00:04:46,090 --> 00:04:48,100 And one of the major jobs of the liver 92 00:04:48,100 --> 00:04:51,940 is to maintain a constant level of blood glucose 93 00:04:51,940 --> 00:04:57,530 to continue to supply fuel for various organs, 94 00:04:57,530 --> 00:05:01,600 including the muscle to be able to carry out the work 95 00:05:01,600 --> 00:05:03,770 that it needs to do. 96 00:05:03,770 --> 00:05:08,020 And so this whole process is called the Cori cycle. 97 00:05:08,020 --> 00:05:11,560 And a major job of the liver is thus 98 00:05:11,560 --> 00:05:14,650 generating glucose to maintain blood glucose levels. 99 00:05:14,650 --> 00:05:17,410 And the pathways used to do this is 100 00:05:17,410 --> 00:05:20,500 a pathway that we began to talk about last time 101 00:05:20,500 --> 00:05:29,380 called gluconeogenesis. 102 00:05:33,630 --> 00:05:37,260 And it's really a discussion of gluconeogenesis. 103 00:05:37,260 --> 00:05:41,670 That is, how can one build a pathway in plants, 104 00:05:41,670 --> 00:05:46,950 in the liver, wherever, as a way to generate glucose 105 00:05:46,950 --> 00:05:49,980 from something like lactate? 106 00:05:49,980 --> 00:05:52,110 Now, of course, we know from last time, 107 00:05:52,110 --> 00:05:54,540 lactate dehydrogenase can, obviously, it's 108 00:05:54,540 --> 00:05:57,450 a redox reaction to interconvert with pyruvate. 109 00:05:57,450 --> 00:05:59,820 And what gluconeogenesis really is 110 00:05:59,820 --> 00:06:03,480 it's a pathway that cells use to convert 111 00:06:03,480 --> 00:06:10,000 pyruvate or really anything else that can come into glycolysis-- 112 00:06:10,000 --> 00:06:13,310 and example being lactate. 113 00:06:13,310 --> 00:06:16,210 This, of course, can happen by the lactate dehydrogenase 114 00:06:16,210 --> 00:06:17,080 reaction. 115 00:06:17,080 --> 00:06:19,330 And then once it has that pyruvate, 116 00:06:19,330 --> 00:06:22,930 turn that pyruvate into glucose. 117 00:06:22,930 --> 00:06:25,720 That's really what we're talking about when we're 118 00:06:25,720 --> 00:06:29,070 discussing gluconeogenesis. 119 00:06:29,070 --> 00:06:33,360 Now how can we turn pyruvate into glucose? 120 00:06:33,360 --> 00:06:35,430 Well, we just discussed the whole pathway 121 00:06:35,430 --> 00:06:38,310 where we could turn glucose into pyruvate-- glycolysis. 122 00:06:38,310 --> 00:06:43,200 And so this is really the reverse of glycolysis. 123 00:06:43,200 --> 00:06:47,490 I said many times that all enzymes are reversible. 124 00:06:47,490 --> 00:06:49,890 So why can't we just run the pathway 125 00:06:49,890 --> 00:06:51,960 in the opposite direction? 126 00:06:51,960 --> 00:06:54,450 Well, hopefully, if you've been paying attention 127 00:06:54,450 --> 00:06:56,220 for the last several lectures, you 128 00:06:56,220 --> 00:06:59,380 realize there's two problems with doing that. 129 00:06:59,380 --> 00:07:03,720 The first is that, remember, why we're doing metabolism 130 00:07:03,720 --> 00:07:07,410 in the first place is to maintain a high ATP:ADP ratio 131 00:07:07,410 --> 00:07:11,220 under conditions so that the cell can use that ATP:ADP ratio 132 00:07:11,220 --> 00:07:14,430 to carry out otherwise unfavorable processes. 133 00:07:14,430 --> 00:07:16,200 And so thus, at a very high level, 134 00:07:16,200 --> 00:07:18,120 cells can't stop catabolism. 135 00:07:18,120 --> 00:07:21,960 Glycolysis or whatever has to continue to maintain 136 00:07:21,960 --> 00:07:23,967 ATP in the right range. 137 00:07:23,967 --> 00:07:26,550 And so now, if we're going to do something anabolic-- that is, 138 00:07:26,550 --> 00:07:29,790 build something, turn it into glucose, 139 00:07:29,790 --> 00:07:32,160 this has to happen in the background 140 00:07:32,160 --> 00:07:34,420 where catabolism has still happened. 141 00:07:34,420 --> 00:07:37,560 And so at least you need some kind of way 142 00:07:37,560 --> 00:07:39,690 to control both and prevent this from being 143 00:07:39,690 --> 00:07:42,540 some kind of futile cycle. 144 00:07:42,540 --> 00:07:48,150 Second, even if that ATP level is super high in the cell, 145 00:07:48,150 --> 00:07:52,470 it has extra energy, there is still a thermodynamic problem 146 00:07:52,470 --> 00:07:54,840 with just reversing glycolysis. 147 00:07:54,840 --> 00:07:58,140 And that is, remember, delta G has to be less than 0 148 00:07:58,140 --> 00:08:00,480 for any pathway to work. 149 00:08:00,480 --> 00:08:06,420 And so it then stands to reason that if glucose oxidation 150 00:08:06,420 --> 00:08:11,190 to pyruvate, if this reaction is favored, 151 00:08:11,190 --> 00:08:16,920 as it is in glycolysis, that delta G is less than 0. 152 00:08:16,920 --> 00:08:19,420 Well, under the conditions where that's occurring, 153 00:08:19,420 --> 00:08:21,750 if we want to then say, well, what about the reverse? 154 00:08:21,750 --> 00:08:23,680 Let's switch the signs-- 155 00:08:23,680 --> 00:08:26,280 pyruvate to glucose. 156 00:08:26,280 --> 00:08:28,770 We know that delta G is the same as it 157 00:08:28,770 --> 00:08:31,050 was in the opposite direction but, 158 00:08:31,050 --> 00:08:34,860 now, changing the signs from positive to negative 159 00:08:34,860 --> 00:08:36,179 or negative positive. 160 00:08:36,179 --> 00:08:39,780 And so, thus, delta G, for the reverse reaction, 161 00:08:39,780 --> 00:08:42,179 must be greater than 0 and, therefore, must 162 00:08:42,179 --> 00:08:43,799 be spontaneous-- 163 00:08:43,799 --> 00:08:46,470 is not spontaneous. 164 00:08:46,470 --> 00:08:50,910 And therefore says that we need to have a different pathway 165 00:08:50,910 --> 00:08:52,950 simply for energetic reasons if we're 166 00:08:52,950 --> 00:08:55,440 going to go in the opposite direction. 167 00:08:55,440 --> 00:08:58,830 Now oxidizing glucose releases energy. 168 00:08:58,830 --> 00:09:01,760 That's how we keep the ATP:ADP ratio high. 169 00:09:01,760 --> 00:09:04,370 It stands to reason then that we are 170 00:09:04,370 --> 00:09:08,420 going to have to put energy in if we're going to run 171 00:09:08,420 --> 00:09:09,630 in the opposite direction. 172 00:09:09,630 --> 00:09:12,230 And so if we're going to build a pathway from pyruvate 173 00:09:12,230 --> 00:09:19,640 to glucose, you can guess, we're going to have to add couple 174 00:09:19,640 --> 00:09:23,180 that pathway to ATP-ADP hydrolysis-- 175 00:09:23,180 --> 00:09:27,440 that is, couple it to something favorable to now allow 176 00:09:27,440 --> 00:09:32,710 this otherwise unfavorable reaction to happen. 177 00:09:32,710 --> 00:09:37,480 Now this energy, of course, in plants can come from the sun. 178 00:09:37,480 --> 00:09:39,070 We'll discuss that in great detail 179 00:09:39,070 --> 00:09:40,990 when we get to photosynthesis. 180 00:09:40,990 --> 00:09:44,890 However, for the sake of you and I, I guess, 181 00:09:44,890 --> 00:09:47,800 what it says is, if you think about this, 182 00:09:47,800 --> 00:09:51,040 if you release energy from glycolysis, it makes sense 183 00:09:51,040 --> 00:09:52,750 then you have to add energy if you're 184 00:09:52,750 --> 00:09:55,030 going to do gluconeogenesis. 185 00:09:55,030 --> 00:09:58,330 But remember, to satisfy all the laws of thermodynamics, 186 00:09:58,330 --> 00:10:03,610 the net energy loss to the universe is going to be-- 187 00:10:03,610 --> 00:10:06,070 must exist, which means that it is 188 00:10:06,070 --> 00:10:09,550 going to cost more energy to make glucose 189 00:10:09,550 --> 00:10:13,040 than you're going to get back from breaking it down. 190 00:10:13,040 --> 00:10:16,780 That just, hopefully, fits with your intuition. 191 00:10:16,780 --> 00:10:18,500 And it actually, I guess, is good news, 192 00:10:18,500 --> 00:10:20,500 because when are we going to do anabolism? 193 00:10:20,500 --> 00:10:22,780 When are we going to store all these excess calories 194 00:10:22,780 --> 00:10:23,802 as glucose? 195 00:10:23,802 --> 00:10:25,510 Well, we're going to do it if we overeat. 196 00:10:25,510 --> 00:10:27,490 And if we overeat, and it costs us 197 00:10:27,490 --> 00:10:30,730 energy in the form of calories to actually store 198 00:10:30,730 --> 00:10:34,240 that energy for later, I guess, it means that when we overeat, 199 00:10:34,240 --> 00:10:38,320 we don't have things quite as bad as it could have been. 200 00:10:38,320 --> 00:10:39,460 All right. 201 00:10:39,460 --> 00:10:44,600 Now let's get back to the how one can actually do this. 202 00:10:44,600 --> 00:10:46,510 So if we want to think, what is a pathway 203 00:10:46,510 --> 00:10:50,020 that we can build to get from pyruvate back to glucose? 204 00:10:50,020 --> 00:10:54,320 Well, we know we're going to have to burn some ATP to do it. 205 00:10:54,320 --> 00:10:59,630 But what are the issues with simply reversing glycolysis? 206 00:10:59,630 --> 00:11:02,440 Well, we can come back here and say, well, 207 00:11:02,440 --> 00:11:05,290 what was driving glycolysis in the first place? 208 00:11:05,290 --> 00:11:08,300 And it was these big steps of free energy change 209 00:11:08,300 --> 00:11:10,780 acts of hexokinase, phosphofructokinase, 210 00:11:10,780 --> 00:11:12,160 and pyruvate kinase. 211 00:11:12,160 --> 00:11:14,320 Where as many of these other reactions, 212 00:11:14,320 --> 00:11:16,870 at least under these standard conditions that 213 00:11:16,870 --> 00:11:20,230 are estimated to exist in cells, are relatively 214 00:11:20,230 --> 00:11:21,320 close to equilibrium. 215 00:11:21,320 --> 00:11:24,280 So there's really no problem with reversing glycolysis 216 00:11:24,280 --> 00:11:26,950 across here or across there. 217 00:11:26,950 --> 00:11:29,860 The real issue comes from getting around the hexokinase 218 00:11:29,860 --> 00:11:33,580 to phosphofructokinase and the pyruvate kinase step. 219 00:11:33,580 --> 00:11:35,860 These are the places where the most energy 220 00:11:35,860 --> 00:11:37,450 is released, if you will, when you're 221 00:11:37,450 --> 00:11:39,110 going through glycolysis. 222 00:11:39,110 --> 00:11:40,600 So those are the steps we're going 223 00:11:40,600 --> 00:11:44,890 to have to add energy in order to reverse build a pathway that 224 00:11:44,890 --> 00:11:47,020 goes in the opposite direction and allows 225 00:11:47,020 --> 00:11:50,720 you to start with pyruvate and end up with glucose. 226 00:11:50,720 --> 00:12:04,070 And so what we will find is that gluconeogenesis and glycolysis 227 00:12:04,070 --> 00:12:13,690 use many of the same enzymes. 228 00:12:13,690 --> 00:12:17,380 However, not all of the enzymes are the same 229 00:12:17,380 --> 00:12:19,390 because we need different enzymes 230 00:12:19,390 --> 00:12:22,930 if we're going to get around this hexokinase, 231 00:12:22,930 --> 00:12:26,770 phosphofructokinase, and pyruvate kinase steps 232 00:12:26,770 --> 00:12:28,370 of the pathway. 233 00:12:28,370 --> 00:12:31,420 And so for gluconeogenesis, we essentially 234 00:12:31,420 --> 00:12:36,360 need four new enzymes in order to get past those places. 235 00:12:36,360 --> 00:12:38,110 So we're going to need a new enzyme called 236 00:12:38,110 --> 00:12:41,740 glucose 6-phosphatase. 237 00:12:41,740 --> 00:12:44,350 And glucose 6-phosphatase is going 238 00:12:44,350 --> 00:12:50,350 to allow us to get from glucose 6-phosphate 239 00:12:50,350 --> 00:12:53,250 and turn it back into glucose. 240 00:12:53,250 --> 00:12:54,270 All right. 241 00:12:54,270 --> 00:13:01,230 We're going to need an enzyme fructose 1,6-bisphosphatase, 242 00:13:01,230 --> 00:13:06,480 which is going to allow us to take FBP and turn it back 243 00:13:06,480 --> 00:13:14,190 into fructose 6-phosphate. 244 00:13:14,190 --> 00:13:17,250 And then we're going to need two enzymes to get around 245 00:13:17,250 --> 00:13:18,960 the pyruvate kinase step. 246 00:13:18,960 --> 00:13:25,290 These are two enzymes called PEPCK and PC. 247 00:13:25,290 --> 00:13:27,000 That stands for phosphoenolpyruvate 248 00:13:27,000 --> 00:13:29,440 carboxykinase and pyruvate carboxylase. 249 00:13:29,440 --> 00:13:32,430 I'll discuss these in great detail in a minute. 250 00:13:32,430 --> 00:13:44,800 And these are going to allow us to get from pyruvate back 251 00:13:44,800 --> 00:13:46,330 to PEP. 252 00:13:52,090 --> 00:13:56,560 Also, essentially, I'll add them up here to this diagram. 253 00:14:09,860 --> 00:14:14,140 So to get back from pyruvate to PEP, two enzymes-- 254 00:14:14,140 --> 00:14:17,860 pyruvate carboxylase and phosphoenolpyruvate 255 00:14:17,860 --> 00:14:23,480 carboxykinase to get from fructose 1,6-bisphosphate, 256 00:14:23,480 --> 00:14:28,390 fructose 6-phosphate, an enzyme called FBPase. 257 00:14:31,720 --> 00:14:40,410 And to get from glucose 6-phosphate back to glucose, 258 00:14:40,410 --> 00:14:44,130 an enzyme called glucose 6-phosphatase. 259 00:14:54,510 --> 00:14:55,560 OK. 260 00:14:55,560 --> 00:14:58,290 Now, of course, if you think back, 261 00:14:58,290 --> 00:15:00,930 we're also going to need to balance electrons 262 00:15:00,930 --> 00:15:02,010 in the pathway. 263 00:15:02,010 --> 00:15:04,980 Remember, glucose to pyruvate is an oxidation reaction. 264 00:15:04,980 --> 00:15:07,740 That is, we're generating NADH. 265 00:15:07,740 --> 00:15:10,210 That means to go in the reverse direction, 266 00:15:10,210 --> 00:15:15,720 we need a source of NADH to reverse that GAPDH reaction. 267 00:15:15,720 --> 00:15:18,240 I just want to point out that you're only 268 00:15:18,240 --> 00:15:22,860 doing gluconeogenesis if cells have plenty of energy. 269 00:15:22,860 --> 00:15:24,580 What does plenty of energy mean? 270 00:15:24,580 --> 00:15:28,230 Well, that means the ATP:ADP ratio is high 271 00:15:28,230 --> 00:15:30,600 because you're doing lots of metabolism. 272 00:15:30,600 --> 00:15:32,370 But if you're doing lots of metabolism, 273 00:15:32,370 --> 00:15:36,600 and the ATP:ADP ratio's high, that also means the NADH/NAD+ 274 00:15:36,600 --> 00:15:37,980 ratio is high. 275 00:15:37,980 --> 00:15:42,390 And so you can think of the NAD regeneration 276 00:15:42,390 --> 00:15:46,500 by gluconeogenesis as being, for all intents and purposes, 277 00:15:46,500 --> 00:15:49,690 an alternative to fermentation, if you will, 278 00:15:49,690 --> 00:15:51,600 as a way to keep catabolism going. 279 00:15:51,600 --> 00:15:53,640 That is, you're only doing this if you 280 00:15:53,640 --> 00:15:55,620 have plenty of NADH around. 281 00:15:55,620 --> 00:16:00,210 And so dealing with that electron balance 282 00:16:00,210 --> 00:16:02,970 is less of an issue. 283 00:16:02,970 --> 00:16:03,900 OK. 284 00:16:03,900 --> 00:16:06,480 Now let's discuss briefly how some of these enzymes work. 285 00:16:06,480 --> 00:16:10,950 So glucose 6-phosphate and fructose 1,6-bisphosphatase are 286 00:16:10,950 --> 00:16:13,110 very straightforward. 287 00:16:13,110 --> 00:16:17,070 Effectively, all they are is simply removing a phosphate. 288 00:16:17,070 --> 00:16:18,600 That's very favorable. 289 00:16:18,600 --> 00:16:20,250 Remember, when we did those reactions 290 00:16:20,250 --> 00:16:23,730 of hexokinase phosphofructokinase reactions 291 00:16:23,730 --> 00:16:27,750 and glycolysis, that, we had to couple otherwise 292 00:16:27,750 --> 00:16:29,040 unfavorable things-- 293 00:16:29,040 --> 00:16:32,560 adding a phosphate group to ATP hydrolysis. 294 00:16:32,560 --> 00:16:33,940 This is the reverse of that. 295 00:16:33,940 --> 00:16:35,490 And so the reverse of that, delta G 296 00:16:35,490 --> 00:16:37,200 is going to be less than 0. 297 00:16:37,200 --> 00:16:41,280 Delta G less than 0 means we just have a phosphatase there, 298 00:16:41,280 --> 00:16:43,710 just like you might have on a kinase enzyme. 299 00:16:43,710 --> 00:16:45,570 That's very straightforward. 300 00:16:45,570 --> 00:16:50,640 But there's a much bigger issue to get around this other step, 301 00:16:50,640 --> 00:16:52,440 this pyruvate kinase step. 302 00:16:52,440 --> 00:16:54,675 Remember, equilibrium strongly favored 303 00:16:54,675 --> 00:16:56,580 PEP to pyruvate conversion. 304 00:16:56,580 --> 00:16:58,830 That was one of the reasons why we could use that step 305 00:16:58,830 --> 00:17:03,300 to maintain a high ATP:ADP ratio in cells. 306 00:17:03,300 --> 00:17:05,280 Well, now, the issue becomes, how can we 307 00:17:05,280 --> 00:17:09,329 trap that phosphate back onto pyruvate 308 00:17:09,329 --> 00:17:13,619 while generating phosphoenolpyruvate? 309 00:17:13,619 --> 00:17:18,390 How one does that is an issue. 310 00:17:18,390 --> 00:17:21,060 Even if the ATP:ADP ratio is high, 311 00:17:21,060 --> 00:17:25,020 it's never high enough to reverse pyruvate kinase on its 312 00:17:25,020 --> 00:17:25,780 own. 313 00:17:25,780 --> 00:17:28,840 And so we need a new pathway with two steps in it. 314 00:17:28,840 --> 00:17:30,450 That's why there's two enzymes-- 315 00:17:30,450 --> 00:17:36,150 PC and PEPCK-- that are there to create a pathway where 316 00:17:36,150 --> 00:17:43,530 pyruvate to PEP now becomes favorable. 317 00:17:43,530 --> 00:17:47,490 That is, where delta G 0 prime is less than 0. 318 00:17:47,490 --> 00:17:50,640 That is, the equilibrium favors to the right. 319 00:17:50,640 --> 00:17:53,370 And under cellular conditions, this pathway 320 00:17:53,370 --> 00:17:58,810 can actually happen such that cells can net make glucose. 321 00:17:58,810 --> 00:18:03,660 Now these are two steps, both of which require ATP. 322 00:18:03,660 --> 00:18:06,540 And so you effectively get one ATP from PEP 323 00:18:06,540 --> 00:18:07,800 to pyruvate conversion. 324 00:18:07,800 --> 00:18:10,710 It costs you to ADPs or two ADP equivalents 325 00:18:10,710 --> 00:18:15,240 to go back from pyruvate to PEP, which makes sense. 326 00:18:15,240 --> 00:18:16,980 You should have to spend more energy 327 00:18:16,980 --> 00:18:21,000 to go upstream than you get back coming downstream. 328 00:18:21,000 --> 00:18:21,720 All right. 329 00:18:21,720 --> 00:18:23,490 Here's how this pathway works. 330 00:18:31,720 --> 00:18:38,970 So this is pyruvate, the first reaction 331 00:18:38,970 --> 00:18:50,470 catalyzed by PC, which stands for pyruvate carboxylase. 332 00:18:58,760 --> 00:19:10,110 Couples ATP hydrolysis to addition 333 00:19:10,110 --> 00:19:27,270 of a CO2 that generates this intermediate, which 334 00:19:27,270 --> 00:19:35,520 is called oxaloacetate, often abbreviated 335 00:19:35,520 --> 00:19:38,355 OAA for Oxaloacetic Acid. 336 00:19:41,160 --> 00:19:46,770 That oxaloacetate is then decarboxylated. 337 00:19:46,770 --> 00:19:54,790 That is, the CO2 that is lost is removed-- 338 00:19:54,790 --> 00:19:57,010 that was added is removed. 339 00:19:57,010 --> 00:20:03,250 This reaction is coupled to GTP and is catalyzed 340 00:20:03,250 --> 00:20:28,750 by this enzyme, PEPCK PEP carboxykinase 341 00:20:28,750 --> 00:20:33,070 and uses that phosphate from GTP to trap pyruvate in 342 00:20:33,070 --> 00:20:37,660 the enol form as PEP or phosphoenolpyruvate. 343 00:20:42,590 --> 00:20:43,550 great. 344 00:20:43,550 --> 00:20:49,990 So once you've regenerated PEP, now 345 00:20:49,990 --> 00:20:54,550 that PEP gets to this flat part, if we go over here 346 00:20:54,550 --> 00:20:57,230 to our delta G place. 347 00:20:57,230 --> 00:21:01,000 So now it can easily run back across this flat part 348 00:21:01,000 --> 00:21:02,050 of the curve. 349 00:21:02,050 --> 00:21:11,310 That is, go through enolase phosphoglycerate mutase, 350 00:21:11,310 --> 00:21:19,900 phosphoglycerate kinase, GAPDH, taking advantage of the high 351 00:21:19,900 --> 00:21:23,920 NADH/NAD+ ratio that would exist when you're doing 352 00:21:23,920 --> 00:21:27,580 gluconeogenesis-- triosephosphate isomerase, 353 00:21:27,580 --> 00:21:29,060 aldolase. 354 00:21:29,060 --> 00:21:33,190 This generates a DHAP and a glyceraldehyde 3-phosphate that 355 00:21:33,190 --> 00:21:39,340 you can then combine to give a fructose 1,6-bisphosphate. 356 00:21:39,340 --> 00:21:44,420 Now we get back here to now we have this steep part there. 357 00:21:44,420 --> 00:21:47,770 But rather than going through FBP, 358 00:21:47,770 --> 00:21:53,470 we can just have a phosphatase, fructose 1,6-bisphosphatase 359 00:21:53,470 --> 00:21:55,420 that releases the phosphate. 360 00:21:55,420 --> 00:22:01,390 Now, we have fructose 6-phosphate. 361 00:22:01,390 --> 00:22:03,640 That fructose 6-phosphate can then 362 00:22:03,640 --> 00:22:07,720 go through glucose 6-phosphate isomerase 363 00:22:07,720 --> 00:22:12,490 and generate glucose 6-phosphate. 364 00:22:12,490 --> 00:22:15,770 That now gets back to this other steep hill. 365 00:22:15,770 --> 00:22:17,200 So this is relatively flat. 366 00:22:17,200 --> 00:22:19,720 Now, you have this steep hill around hexokinase. 367 00:22:19,720 --> 00:22:25,885 But rather than coupling it to adding to ATP-ADP hydrolysis, 368 00:22:25,885 --> 00:22:31,480 you just remove the phosphate with glucose 6-phosphatase. 369 00:22:31,480 --> 00:22:35,560 And in the end, now we have a pathway 370 00:22:35,560 --> 00:22:45,380 using these four extra enzymes that give us 371 00:22:45,380 --> 00:22:48,980 a way to start at pyruvate and run the opposite direction 372 00:22:48,980 --> 00:22:52,190 and generate glucose. 373 00:22:52,190 --> 00:23:00,120 And you can do so in a way that is thermodynamically favorable. 374 00:23:00,120 --> 00:23:11,340 In other words, we can start with 2 pyruvate and use 4 ATP-- 375 00:23:17,730 --> 00:23:22,770 so and ATP to turn pyruvate twice into oxaloacetate. 376 00:23:22,770 --> 00:23:24,060 So that's two. 377 00:23:24,060 --> 00:23:30,390 And then another ATP at phosphoglycerate kinase 378 00:23:30,390 --> 00:23:33,910 to go backwards there to go from-- 379 00:23:37,090 --> 00:23:42,960 to run that reaction backwards, plus 2 GTP. 380 00:23:42,960 --> 00:23:48,610 Those 2 GTP are effectively the equivalent of an ATP. 381 00:23:48,610 --> 00:23:52,530 So this would be 6 ATP equivalents. 382 00:23:52,530 --> 00:23:55,590 Why are the GTPs equivalent of ATP? 383 00:23:55,590 --> 00:23:59,910 I just want to point out that cells have enzymes 384 00:23:59,910 --> 00:24:05,580 that basically can interconvert ATP and GDP or, really, 385 00:24:05,580 --> 00:24:13,800 any nucleoside triphosphate and diphosphate to GTP plus ADP. 386 00:24:13,800 --> 00:24:18,960 This has a delta G 0 prime that is effectively 0. 387 00:24:18,960 --> 00:24:21,855 And so cells basically match their ATP 388 00:24:21,855 --> 00:24:25,560 to ADP ratios with any other ratios 389 00:24:25,560 --> 00:24:30,330 of nucleoside triphosphates and diphosphates because 390 00:24:30,330 --> 00:24:31,240 of this reaction. 391 00:24:31,240 --> 00:24:35,385 And so that's why the two GTP are roughly an ATP equivalent. 392 00:24:35,385 --> 00:24:41,910 So we'll just call it 6 ATP plus 2 NADH 393 00:24:41,910 --> 00:24:45,550 to reverse the GAPDH step. 394 00:24:45,550 --> 00:25:00,080 That gives us 6 ADP plus 2 NAD+ plus 6 inorganic phosphate plus 395 00:25:00,080 --> 00:25:03,000 a glucose. 396 00:25:03,000 --> 00:25:10,020 The delta G 0 prime for this entire coupled pathway 397 00:25:10,020 --> 00:25:15,150 is on the order of negative 9 kcals per mol, 398 00:25:15,150 --> 00:25:19,920 with the delta G less than 0, equilibrium lies to the right. 399 00:25:19,920 --> 00:25:25,240 Net synthesis of glucose is favorable. 400 00:25:25,240 --> 00:25:29,610 And so if we just think about this in ATP terms, 401 00:25:29,610 --> 00:25:32,010 remember, when we discussed glycolysis, 402 00:25:32,010 --> 00:25:37,470 you harvest net two ATP from conversion of glucose 403 00:25:37,470 --> 00:25:38,910 into two pyruvate. 404 00:25:38,910 --> 00:25:43,860 If we reverse the reaction doing gluconeogenesis, in ATP terms, 405 00:25:43,860 --> 00:25:48,090 we have to invest six ATP to turn that pyruvate back 406 00:25:48,090 --> 00:25:49,530 into glucose. 407 00:25:49,530 --> 00:25:50,320 Makes sense. 408 00:25:50,320 --> 00:25:52,320 We didn't make a perpetual motion machine. 409 00:25:52,320 --> 00:25:54,210 Both pathways are favorable. 410 00:25:54,210 --> 00:25:58,350 But it costs more energy to make a glucose than we can get out 411 00:25:58,350 --> 00:26:00,915 by burning the glucose. 412 00:26:00,915 --> 00:26:03,940 I should say, the energy isn't just the ATP. 413 00:26:03,940 --> 00:26:06,720 There's also this NADH. 414 00:26:06,720 --> 00:26:12,180 That NADH that's produced from glycolysis also is storing 415 00:26:12,180 --> 00:26:15,960 energy, if you will, from oxidation and an NADH/NAD+ 416 00:26:15,960 --> 00:26:19,560 ratio, and you're consuming that going in the opposite 417 00:26:19,560 --> 00:26:21,690 direction. 418 00:26:21,690 --> 00:26:26,130 You only do gluconeogenesis if you have a high ATP:ADP ratio, 419 00:26:26,130 --> 00:26:30,090 of course, and a NADH/NAH+ plus ratio-- 420 00:26:30,090 --> 00:26:32,640 that is, if you have it under the right conditions 421 00:26:32,640 --> 00:26:35,310 to actually run this pathway and have 422 00:26:35,310 --> 00:26:37,960 it make sense for the cell. 423 00:26:37,960 --> 00:26:39,250 All right. 424 00:26:39,250 --> 00:26:41,950 Now I want to discuss, briefly, how 425 00:26:41,950 --> 00:26:45,160 these enzymes in gluconeogenesis work. 426 00:26:45,160 --> 00:26:48,910 I'm not going to spend time on fructose 1,6-bisphosphatase 427 00:26:48,910 --> 00:26:50,530 or glucose 6-phosphatase. 428 00:26:50,530 --> 00:26:54,580 As I said before, this is just hydrolysis 429 00:26:54,580 --> 00:26:57,400 of that phosphate-alcohol bond. 430 00:26:57,400 --> 00:27:00,650 That's relatively straightforward. 431 00:27:00,650 --> 00:27:04,360 But I do want to spend time on pyruvate carboxylase 432 00:27:04,360 --> 00:27:08,500 and PEP carboxykinase because this introduces 433 00:27:08,500 --> 00:27:10,900 a new reaction, carboxylation. 434 00:27:10,900 --> 00:27:16,750 But it also introduces a new concept in metabolism 435 00:27:16,750 --> 00:27:21,760 that is actually quite important in eukaryotic cells. 436 00:27:21,760 --> 00:27:36,350 And that is this issue of so-called compartmentalized 437 00:27:36,350 --> 00:27:37,670 metabolism. 438 00:27:37,670 --> 00:27:41,210 And, of course, you've learned in introductory biology 439 00:27:41,210 --> 00:27:43,790 that a big difference between eukaryotes and prokaryotes 440 00:27:43,790 --> 00:27:46,580 is eukaryotes have all of these organelles-- that 441 00:27:46,580 --> 00:27:50,030 is, these membrane-bound structures within cells that 442 00:27:50,030 --> 00:27:52,440 carry out various functions. 443 00:27:52,440 --> 00:27:55,370 So one reason that it's useful to have 444 00:27:55,370 --> 00:27:58,280 these different membrane-bound organelles 445 00:27:58,280 --> 00:28:03,050 is it creates different compartments within the cell. 446 00:28:03,050 --> 00:28:04,080 What does that mean? 447 00:28:04,080 --> 00:28:11,420 Well, if you have different compartments, 448 00:28:11,420 --> 00:28:18,610 that means you can create different conditions 449 00:28:18,610 --> 00:28:21,160 within each compartment. 450 00:28:21,160 --> 00:28:23,403 And that's very important for metabolism. 451 00:28:23,403 --> 00:28:25,195 Well, why is that important for metabolism? 452 00:28:25,195 --> 00:28:30,070 Well, because, remember, whether or not reactions are favorable 453 00:28:30,070 --> 00:28:33,190 depend on delta G, which, of course, is 454 00:28:33,190 --> 00:28:35,680 dependent on the equilibrium constant, which 455 00:28:35,680 --> 00:28:39,130 is related to delta G 0 prime but also 456 00:28:39,130 --> 00:28:43,420 to the ratio of the products and reactants 457 00:28:43,420 --> 00:28:46,330 within that compartment. 458 00:28:46,330 --> 00:28:48,160 And so by having different compartments, 459 00:28:48,160 --> 00:28:53,500 you can have different ratios of metabolites-- say, 460 00:28:53,500 --> 00:29:00,580 a different ratio of ATP to ATP or a different ratio of NADH 461 00:29:00,580 --> 00:29:03,190 to NAD+. 462 00:29:03,190 --> 00:29:08,320 Having those different ratios means that because delta G is 463 00:29:08,320 --> 00:29:16,575 proportional to the ratio of the reactants over the products-- 464 00:29:16,575 --> 00:29:22,330 I'm sorry-- products over reactants, 465 00:29:22,330 --> 00:29:24,880 by having different ratios of things 466 00:29:24,880 --> 00:29:29,920 like ATP and ADP, NAD, NADH, you can make different reactions 467 00:29:29,920 --> 00:29:33,370 more or less favorable depending on which 468 00:29:33,370 --> 00:29:37,270 compartment in the cell that they're located. 469 00:29:37,270 --> 00:29:41,710 Now it turns out that glycolysis and most of gluconeogenesis 470 00:29:41,710 --> 00:29:45,310 takes place in the cytosol of eukaryotic cells. 471 00:29:45,310 --> 00:29:47,890 The cytosol is basically the space 472 00:29:47,890 --> 00:29:52,450 inside the cell that's not inside another organelle. 473 00:29:52,450 --> 00:29:58,000 However, pyruvate carboxylase is a reaction that takes place 474 00:29:58,000 --> 00:30:00,820 in mitochondria because mitochondria have 475 00:30:00,820 --> 00:30:04,790 a particularly high ATP:ADP ratio. 476 00:30:04,790 --> 00:30:08,260 And it turns out, this helps favor the pyruvate carboxylase 477 00:30:08,260 --> 00:30:09,560 reaction. 478 00:30:09,560 --> 00:30:12,460 And so we'll talk a lot about reactions in the site cytosol 479 00:30:12,460 --> 00:30:14,140 versus the mitochondria. 480 00:30:14,140 --> 00:30:18,820 But basically, here's glucose to PEP. 481 00:30:18,820 --> 00:30:21,790 Whether you do that in one direction by glycolysis 482 00:30:21,790 --> 00:30:24,160 or the other direction by gluconeogenesis, 483 00:30:24,160 --> 00:30:26,710 that happens in the cytosol as does 484 00:30:26,710 --> 00:30:29,530 turning that PEP into pyruvate. 485 00:30:29,530 --> 00:30:32,590 But if you're going to reverse the reaction, 486 00:30:32,590 --> 00:30:37,300 eukaryotes take advantage of a different compartment, 487 00:30:37,300 --> 00:30:44,950 the mitochondria, with a high ATP to ADP ratio. 488 00:30:44,950 --> 00:30:50,860 And basically, in the mitochondria, 489 00:30:50,860 --> 00:30:54,820 turn that pyruvate into oxaloacetic acid-- 490 00:30:54,820 --> 00:30:58,690 that is, carry out the pyruvate carboxylase reaction. 491 00:30:58,690 --> 00:31:07,480 And then that pyruvate carboxylase reaction 492 00:31:07,480 --> 00:31:11,470 generates oxaloacetic acid, and then PEPCK 493 00:31:11,470 --> 00:31:14,680 can, either in the mitochondria or in the cytosol, 494 00:31:14,680 --> 00:31:17,290 generate PEP. 495 00:31:17,290 --> 00:31:22,765 And so here's a place where one can run one reaction-- 496 00:31:22,765 --> 00:31:25,870 PEP to pyruvate of t make ATP in the cytosol-- 497 00:31:25,870 --> 00:31:27,570 but a different reaction-- 498 00:31:27,570 --> 00:31:29,200 pyruvate carboxylase to turn pyruvate 499 00:31:29,200 --> 00:31:31,630 into oxaloacetic acid that would then 500 00:31:31,630 --> 00:31:36,310 be used for PEPCK to turn that back into PEP 501 00:31:36,310 --> 00:31:40,300 and have that take place in the mitochondria. 502 00:31:40,300 --> 00:31:45,100 I want to point out, just so for MCAT exams and things like 503 00:31:45,100 --> 00:31:49,300 that, PEPCK is classically defined 504 00:31:49,300 --> 00:31:52,030 as a cytosolic activity, at least 505 00:31:52,030 --> 00:31:53,920 in terms of gluconeogenesis. 506 00:31:53,920 --> 00:31:56,740 Although, there is a mitochondrial isoform of PEPCK. 507 00:31:56,740 --> 00:31:59,788 And it's at least somewhat debated. 508 00:31:59,788 --> 00:32:01,330 There's some evidence that, at least, 509 00:32:01,330 --> 00:32:06,070 in some tissues, that might be important for gluconeogenesis 510 00:32:06,070 --> 00:32:06,620 as well. 511 00:32:06,620 --> 00:32:10,345 And so that's why I draw it as PEPCK using oxaloacetate 512 00:32:10,345 --> 00:32:14,330 to PEP either in the mitochondria or in the cytosol 513 00:32:14,330 --> 00:32:19,780 because that's an area of active investigation right now. 514 00:32:19,780 --> 00:32:21,490 All right. 515 00:32:21,490 --> 00:32:26,200 Let's start by talking about how does pyruvate carboxylase work? 516 00:32:26,200 --> 00:32:29,890 This is an example of a carboxylase reaction, that is, 517 00:32:29,890 --> 00:32:34,480 adding a CO2 group to a molecule to make a carbon-carbon bond. 518 00:32:34,480 --> 00:32:39,010 That purple CO2 is added to the CH3 of pyruvate of 519 00:32:39,010 --> 00:32:42,100 to make oxaloacetate. 520 00:32:42,100 --> 00:32:48,790 And how this happens, in many cases, uses a cofactor. 521 00:32:48,790 --> 00:32:53,110 And that cofactor is called biotin. 522 00:32:53,110 --> 00:32:58,240 Biotin, like many cofactors, is a vitamin. 523 00:32:58,240 --> 00:33:01,480 And this provides a useful functional group. 524 00:33:01,480 --> 00:33:06,430 In this case, enzymes that use biotin 525 00:33:06,430 --> 00:33:11,560 use ATP to basically drive CO2 addition to biotin. 526 00:33:11,560 --> 00:33:14,740 And then that CO2 on the biotin is then 527 00:33:14,740 --> 00:33:18,790 activated to be transferred to another molecule 528 00:33:18,790 --> 00:33:21,410 in a carboxylation reaction. 529 00:33:21,410 --> 00:33:21,910 All right. 530 00:33:21,910 --> 00:33:24,940 So what does biotin look like? 531 00:33:24,940 --> 00:33:25,990 I will draw it for you. 532 00:33:53,880 --> 00:33:56,780 So this, here, would be biotin. 533 00:33:56,780 --> 00:34:02,390 I've drawn it in the enolate form of biotin. 534 00:34:02,390 --> 00:34:05,180 I'll draw it in a different way in a second. 535 00:34:05,180 --> 00:34:13,429 Biotin is typically bound to a lysine molecule 536 00:34:13,429 --> 00:34:16,520 in the active site of an enzyme that uses this. 537 00:34:16,520 --> 00:34:19,580 And so this, here, is basically a peptide bond 538 00:34:19,580 --> 00:34:26,179 between the terminal amino group, the epsilon amino group 539 00:34:26,179 --> 00:34:30,440 of the side chain of a lysine in a residue 540 00:34:30,440 --> 00:34:32,270 that basically makes a peptide bond 541 00:34:32,270 --> 00:34:38,690 to link biotin into the active site of the enzyme that's 542 00:34:38,690 --> 00:34:40,580 using it. 543 00:34:40,580 --> 00:34:45,320 Biotin is often drawn in the keto form. 544 00:34:45,320 --> 00:34:48,050 And the active part of biotin is basically 545 00:34:48,050 --> 00:34:50,429 this top part of the molecule. 546 00:34:50,429 --> 00:34:55,429 And so to draw it to see differences between the enolate 547 00:34:55,429 --> 00:34:58,190 and keto form, I just want to show that quickly. 548 00:34:58,190 --> 00:35:04,160 I drew it in the enolate form because it's easier 549 00:35:04,160 --> 00:35:06,320 to see, I think, how it-- 550 00:35:14,310 --> 00:35:19,700 so this, here, would be the keto form where 551 00:35:19,700 --> 00:35:23,340 biotin is drawn most commonly. 552 00:35:23,340 --> 00:35:26,300 And basically, the reaction, the way biotin 553 00:35:26,300 --> 00:35:29,070 picks up a CO2 is as follows. 554 00:35:29,070 --> 00:35:37,630 And that is CO2 can exist particularly 555 00:35:37,630 --> 00:35:46,610 under basic conditions as CO2 or as-- 556 00:35:51,440 --> 00:35:53,040 this is bicarbonate. 557 00:35:56,750 --> 00:35:57,500 OK. 558 00:35:57,500 --> 00:36:03,740 So bicarbonate-- turns out, some enzymes use CO2 directly. 559 00:36:03,740 --> 00:36:06,610 Some enzymes use bicarbonate. 560 00:36:06,610 --> 00:36:11,600 It turns out that pyruvate carboxylase uses bicarbonate. 561 00:36:11,600 --> 00:36:15,710 So biotin bound in the active site of pyruvate carboxylase 562 00:36:15,710 --> 00:36:20,610 will utilize bicarbonate in the following way. 563 00:36:20,610 --> 00:36:23,810 And I'll just point out that the pH of the mitochondria 564 00:36:23,810 --> 00:36:26,420 is also more basic, which also helps 565 00:36:26,420 --> 00:36:29,750 this pyruvate carboxylation reaction. 566 00:36:29,750 --> 00:36:30,290 All right. 567 00:36:30,290 --> 00:36:33,590 So what happens is this bicarbonate 568 00:36:33,590 --> 00:36:37,355 is phosphorylated by ATP. 569 00:36:45,980 --> 00:36:55,400 This phospho intermediate can then 570 00:36:55,400 --> 00:37:07,060 react with this active site here of biotin 571 00:37:07,060 --> 00:37:11,540 to release the phosphate. 572 00:37:11,540 --> 00:37:29,580 And now, you now have this activated CO2 group 573 00:37:29,580 --> 00:37:33,640 that's attached to the biotin. 574 00:37:43,960 --> 00:37:48,940 Here's pyruvate drawn in the enol form. 575 00:37:48,940 --> 00:37:52,540 You can look back in your notes about how to interconvert it 576 00:37:52,540 --> 00:37:53,980 between keto and the enol form. 577 00:37:53,980 --> 00:37:56,240 I've shown that several times. 578 00:37:56,240 --> 00:38:09,640 And then this, then, can end up adding this CO2 579 00:38:09,640 --> 00:38:23,980 to the end of pyruvate to generate 580 00:38:23,980 --> 00:38:30,710 oxaloacetic acid plus regenerate the biotin 581 00:38:30,710 --> 00:38:34,830 cofactor in the right form. 582 00:38:34,830 --> 00:38:37,040 It'll be the keto form, which can then 583 00:38:37,040 --> 00:38:38,660 get back to the enol form. 584 00:38:38,660 --> 00:38:42,990 And it's ready to do another catalytic cycle. 585 00:38:42,990 --> 00:38:46,310 And so, basically, pyruvate carboxylase 586 00:38:46,310 --> 00:38:51,140 uses ATP to produce this carboxylate of biotin. 587 00:38:51,140 --> 00:38:53,735 And then uses that to add the CO2 588 00:38:53,735 --> 00:38:58,850 to pyruvate to generate oxaloacetic acid. 589 00:38:58,850 --> 00:39:03,760 Now I want to point out that oxaloacetic acid is 590 00:39:03,760 --> 00:39:09,400 both an alpha keto acid like pyruvate was. 591 00:39:09,400 --> 00:39:11,750 And it's a beta keto acid. 592 00:39:11,750 --> 00:39:20,380 And so this ketone is alpha to that carboxylic acid. 593 00:39:20,380 --> 00:39:23,930 And it's beta to that carboxylic acid. 594 00:39:23,930 --> 00:39:29,170 So it's both an alpha and a beta keto acid. 595 00:39:29,170 --> 00:39:31,970 And it turns out that the way PEPCK 596 00:39:31,970 --> 00:39:38,350 works is it takes advantage of the favorable decarbonization 597 00:39:38,350 --> 00:39:40,720 of a beta keto acid, which we talked about 598 00:39:40,720 --> 00:39:48,280 last time, along with GTP to phosphorylate pyruvate and trap 599 00:39:48,280 --> 00:39:50,720 it in the enol form. 600 00:39:50,720 --> 00:39:52,980 And so this is how PEPCK works. 601 00:39:52,980 --> 00:39:59,350 So I'm going to draw GTP in this very stylized way-- 602 00:39:59,350 --> 00:40:02,095 so guanine with three phosphate groups. 603 00:40:13,990 --> 00:40:17,010 There's oxaloacetate. 604 00:40:30,750 --> 00:40:36,180 That decarboxylates-- takes advantage 605 00:40:36,180 --> 00:40:44,330 of this being a beta keto acid to decarboxylate and add 606 00:40:44,330 --> 00:40:45,170 the phosphate. 607 00:40:54,100 --> 00:40:58,400 And that's how we can generate PEP. 608 00:40:58,400 --> 00:40:59,040 OK. 609 00:40:59,040 --> 00:40:59,540 Great. 610 00:40:59,540 --> 00:41:03,560 And so that's how you can build a different pathway that 611 00:41:03,560 --> 00:41:07,490 uses ATP and GTP for energy input 612 00:41:07,490 --> 00:41:12,850 in order to run glycolysis in the opposite direction-- 613 00:41:12,850 --> 00:41:14,750 gluconeogenesis. 614 00:41:14,750 --> 00:41:15,470 All right. 615 00:41:15,470 --> 00:41:20,180 So how is gluconeogenesis regulated? 616 00:41:20,180 --> 00:41:23,840 Well, just like the principles we talked about in glycolysis, 617 00:41:23,840 --> 00:41:26,100 it works in a way that makes sense. 618 00:41:26,100 --> 00:41:27,770 And so you can guess that the steps that 619 00:41:27,770 --> 00:41:30,200 are going to be regulated are exactly 620 00:41:30,200 --> 00:41:33,710 the ones that you might guess. 621 00:41:33,710 --> 00:41:37,330 They were the same steps that were 622 00:41:37,330 --> 00:41:39,520 regulated running glycolysis. 623 00:41:39,520 --> 00:41:42,730 It's going to be these big changes where 624 00:41:42,730 --> 00:41:48,310 energy changes occur across the pathway, which are also 625 00:41:48,310 --> 00:41:50,620 the entrance and exit of the pathway. 626 00:41:50,620 --> 00:41:55,640 And basically, regulation has to be reciprocal. 627 00:41:55,640 --> 00:41:59,830 So in glycolysis, you want to increase glycolysis 628 00:41:59,830 --> 00:42:05,500 under conditions where you need to produce ATP. 629 00:42:05,500 --> 00:42:08,500 And you want to decrease glycolysis 630 00:42:08,500 --> 00:42:16,960 if you have enough ATP or enough of another downstream product, 631 00:42:16,960 --> 00:42:18,700 such as citrate. 632 00:42:18,700 --> 00:42:19,870 All right. 633 00:42:19,870 --> 00:42:28,160 Well, gluconeogenesis is going to be exactly the opposite. 634 00:42:28,160 --> 00:42:32,810 You certainly don't want to do gluconeogenesis 635 00:42:32,810 --> 00:42:34,490 if you need energy release. 636 00:42:34,490 --> 00:42:38,060 If you need ATP, you don't want to run gluconeogenesis. 637 00:42:38,060 --> 00:42:42,530 However, you want to increase gluconeogenesis, 638 00:42:42,530 --> 00:42:46,800 if the cell has ATP excess, you also 639 00:42:46,800 --> 00:42:52,080 want to do it if you have excess of other products 640 00:42:52,080 --> 00:42:59,450 like citrate because why go through the trouble of sending 641 00:42:59,450 --> 00:43:02,150 things down glycolysis if you have nowhere to put it. 642 00:43:02,150 --> 00:43:06,020 You might as well, instead, make glucose or shunt that glucose 643 00:43:06,020 --> 00:43:09,630 off to produce glycogen. 644 00:43:09,630 --> 00:43:20,780 And so let me just add that regulation here. 645 00:43:20,780 --> 00:43:26,510 And so major regulators of gluconeogenesis are as follows. 646 00:43:26,510 --> 00:43:31,920 And so high levels of citrate is going to stimulate fructose 647 00:43:31,920 --> 00:43:34,320 1,6-bisphosphatase. 648 00:43:34,320 --> 00:43:37,560 So high levels of citrate will inhibit things coming through 649 00:43:37,560 --> 00:43:42,360 glycolysis and activate gluconeogenesis by acting 650 00:43:42,360 --> 00:43:49,200 at fructose 1,6-bisphosphatase to match the energy 651 00:43:49,200 --> 00:43:51,590 considerations. 652 00:43:51,590 --> 00:43:56,015 If you have high levels of AMP, energy charge is low. 653 00:43:56,015 --> 00:44:01,070 You want to stimulate glycolysis at phosphofructokinase. 654 00:44:01,070 --> 00:44:05,990 Similarly, that's going to inhibit gluconeogenesis 655 00:44:05,990 --> 00:44:07,700 at FBPase. 656 00:44:07,700 --> 00:44:11,780 And it turns out, high levels of ADP-- 657 00:44:11,780 --> 00:44:15,240 I'm sorry-- yeah, high levels of ADP, low energy charge, 658 00:44:15,240 --> 00:44:18,530 also inhibits PEPCK because you also 659 00:44:18,530 --> 00:44:23,890 don't want to try to generate PEP under those conditions. 660 00:44:29,990 --> 00:44:31,770 Great. 661 00:44:31,770 --> 00:44:32,280 All right. 662 00:44:32,280 --> 00:44:37,860 Now in animals, we also want to regulate glucose catabolism 663 00:44:37,860 --> 00:44:41,340 and production under signaling control. 664 00:44:41,340 --> 00:44:45,280 And this gets back to this other topic we talked about, 665 00:44:45,280 --> 00:44:48,690 such as the liver's job being to maintain a constant level 666 00:44:48,690 --> 00:44:50,580 of blood sugars. 667 00:44:50,580 --> 00:44:54,480 Most of you are very familiar with blood sugar control 668 00:44:54,480 --> 00:44:55,830 through diabetes. 669 00:44:55,830 --> 00:44:58,560 You've probably heard that this is under hormonal control, 670 00:44:58,560 --> 00:45:01,650 that the job of insulin is to lower blood glucose. 671 00:45:01,650 --> 00:45:04,950 And you also have hormones like epinephrine or glucagon 672 00:45:04,950 --> 00:45:07,650 whose job is to raise blood glucose. 673 00:45:07,650 --> 00:45:09,330 Why do you want to raise blood glucose? 674 00:45:09,330 --> 00:45:12,840 Well, if you have some kind of fight-or-flight response-- 675 00:45:12,840 --> 00:45:15,090 you see that lion, and you need to run away, 676 00:45:15,090 --> 00:45:17,130 your body has this adrenaline rush. 677 00:45:17,130 --> 00:45:18,600 That's what epinephrine is. 678 00:45:18,600 --> 00:45:21,450 That basically wants to supply more energy to your muscles, 679 00:45:21,450 --> 00:45:24,930 so that you can run away effectively. 680 00:45:24,930 --> 00:45:27,870 So it turns out, both insulin and glucose act 681 00:45:27,870 --> 00:45:29,370 on lots of tissues in the body. 682 00:45:29,370 --> 00:45:32,340 I'm going to focus on what they do in the liver. 683 00:45:32,340 --> 00:45:34,320 And that's because in the liver, this 684 00:45:34,320 --> 00:45:39,460 is a major organ that regulates blood glucose levels. 685 00:45:39,460 --> 00:45:44,130 So if you have excess glucose around, what do you want to do? 686 00:45:44,130 --> 00:45:46,950 You want to stimulate glucose uptake 687 00:45:46,950 --> 00:45:49,860 into cells, its metabolism, and its storage. 688 00:45:49,860 --> 00:45:52,200 And so insulin with high blood sugar 689 00:45:52,200 --> 00:45:56,760 basically wants to stimulate, taking glucose into the liver 690 00:45:56,760 --> 00:46:00,460 and storing it as glycogen. 691 00:46:00,460 --> 00:46:03,580 If you want to run away from the lion, 692 00:46:03,580 --> 00:46:05,470 well, now you have epinephrine around. 693 00:46:05,470 --> 00:46:06,970 You want to make sure that as you're 694 00:46:06,970 --> 00:46:08,770 consuming that glucose in your blood 695 00:46:08,770 --> 00:46:10,660 that you're continually making more. 696 00:46:10,660 --> 00:46:14,320 And so you want the epinephrine to stimulate release of glucose 697 00:46:14,320 --> 00:46:18,160 either from gluconeogenesis or from the breakdown 698 00:46:18,160 --> 00:46:23,650 of glycogen. And both of these hormones act-- 699 00:46:23,650 --> 00:46:27,190 insulin and epinephrine-- act at a level 700 00:46:27,190 --> 00:46:29,530 where they can regulate enzymes of glycolysis 701 00:46:29,530 --> 00:46:33,430 and gluconeogenesis as well as entry and exit of glucose 702 00:46:33,430 --> 00:46:36,380 monomers in and out of glycogen. 703 00:46:36,380 --> 00:46:44,350 And so this is an example of how fructose 2,6-bisphosphate 704 00:46:44,350 --> 00:46:44,930 works. 705 00:46:44,930 --> 00:46:49,150 And so remember you learned from Professor Yaffe 706 00:46:49,150 --> 00:46:57,590 that epinephrine acts via cyclic AMP signaling, which 707 00:46:57,590 --> 00:46:59,690 turns on a kinase-- 708 00:46:59,690 --> 00:47:03,740 protein kinase A. And that protein kinase A can regulate 709 00:47:03,740 --> 00:47:09,680 enzymes that produce or break down, basically, 710 00:47:09,680 --> 00:47:12,560 these enzymes that produce or break down fructose 711 00:47:12,560 --> 00:47:15,590 2,6-bisphosphatase which, in turn, 712 00:47:15,590 --> 00:47:20,990 regulates PFK activities such that you can match your need 713 00:47:20,990 --> 00:47:25,640 to burn that glucose versus doing gluconeogenesis 714 00:47:25,640 --> 00:47:28,790 in the case of the liver, so you can do enough gluconeogenesis 715 00:47:28,790 --> 00:47:36,830 in order to have glucose around for the body to use in that 716 00:47:36,830 --> 00:47:40,530 fight-or-flight response being driven by epinephrine. 717 00:47:40,530 --> 00:47:46,270 Now a major effective insulin and epinephrine signaling, 718 00:47:46,270 --> 00:47:53,020 though, is actually on release or storage of glucose molecules 719 00:47:53,020 --> 00:47:56,140 in glycogen. And I alluded last time in talking 720 00:47:56,140 --> 00:48:00,520 about the regulation hexokinase that glucose 6-phosphate is 721 00:48:00,520 --> 00:48:04,420 basically the entry point to get glucose units in 722 00:48:04,420 --> 00:48:08,260 and out of glycogen. And I want to discuss 723 00:48:08,260 --> 00:48:12,550 how you add and subtract those glucose 724 00:48:12,550 --> 00:48:14,680 units into storage polymers. 725 00:48:14,680 --> 00:48:17,230 Now I'm going to discuss it in the context of glycogen 726 00:48:17,230 --> 00:48:19,010 storage in humans. 727 00:48:19,010 --> 00:48:22,690 But remember, plants actually store things also 728 00:48:22,690 --> 00:48:24,730 as glucose polymers. 729 00:48:24,730 --> 00:48:26,260 The chemistry is very similar. 730 00:48:26,260 --> 00:48:30,760 But, obviously, the regulation is very different. 731 00:48:30,760 --> 00:48:34,960 Now you'll hopefully recall from past 732 00:48:34,960 --> 00:48:39,430 lectures that these storage polymers of glucose-- 733 00:48:39,430 --> 00:48:43,450 glycogen in humans, starch in plants-- 734 00:48:43,450 --> 00:48:48,670 are basically these alpha 1,4 linkages of glucose molecules. 735 00:48:48,670 --> 00:48:50,410 Remember, there was a non-reducing end 736 00:48:50,410 --> 00:48:52,580 and a reducing end of the molecule. 737 00:48:52,580 --> 00:48:55,420 So starch was a straight-chain polymer. 738 00:48:55,420 --> 00:48:59,950 Glycogen had these alpha 1,6 branch points 739 00:48:59,950 --> 00:49:02,200 that made this branch polymer, where 740 00:49:02,200 --> 00:49:07,870 there's lots of non-reducing ends and a single reducing end. 741 00:49:07,870 --> 00:49:10,300 And each of these non-reducing ends 742 00:49:10,300 --> 00:49:13,810 is a polymer of glucose molecules with this alpha 1,4 743 00:49:13,810 --> 00:49:17,590 linkage that, at the branch point, has this alpha 1,6 744 00:49:17,590 --> 00:49:23,200 linkage to make this long-chain branched polymer. 745 00:49:23,200 --> 00:49:25,630 Now when we discussed this at the time, 746 00:49:25,630 --> 00:49:27,730 I mentioned that this is useful because you 747 00:49:27,730 --> 00:49:30,430 have all these non-reducing ends that 748 00:49:30,430 --> 00:49:34,270 can be used to add or subtract glucose monomers. 749 00:49:34,270 --> 00:49:35,200 And that's great. 750 00:49:35,200 --> 00:49:38,140 This is a nice compact form of storage with lots of places 751 00:49:38,140 --> 00:49:40,240 to put or remove glucose from. 752 00:49:40,240 --> 00:49:42,490 You either store it quickly, insulin 753 00:49:42,490 --> 00:49:44,020 driving glucose storage-- 754 00:49:44,020 --> 00:49:47,710 or remove it quickly, epinephrine driving release 755 00:49:47,710 --> 00:49:52,040 of glucose from the glycogen. 756 00:49:52,040 --> 00:49:56,020 And so if we're going to have a way to add glucose polymers 757 00:49:56,020 --> 00:49:59,110 or break them down, those are two separate pathways, just 758 00:49:59,110 --> 00:50:01,660 like glycolysis and gluconeogenesis-- 759 00:50:01,660 --> 00:50:04,240 reciprocal activities, but we need two pathways 760 00:50:04,240 --> 00:50:08,650 to do it because delta G has to be less than 0 for each pathway 761 00:50:08,650 --> 00:50:09,640 to work. 762 00:50:09,640 --> 00:50:12,370 Also, we have to be able to regulate these separately 763 00:50:12,370 --> 00:50:16,150 because we don't want to have a futile cycle. 764 00:50:16,150 --> 00:50:23,590 Now the way you add and subtract glucose polymers to glycogen 765 00:50:23,590 --> 00:50:32,190 acts through glucose 6-phosphate being first converted 766 00:50:32,190 --> 00:50:34,170 to glucose 1-phosphate. 767 00:50:38,000 --> 00:50:44,950 So this, here, is alpha glucose 6-phosphate. 768 00:50:44,950 --> 00:50:47,450 Remember, it's alpha because I drew that hydroxyl at the one 769 00:50:47,450 --> 00:50:49,650 position pointing down. 770 00:50:49,650 --> 00:50:56,140 This, obviously, can enter glycolysis and come 771 00:50:56,140 --> 00:51:01,330 in from glucose via hexokinase. 772 00:51:01,330 --> 00:51:01,830 All right. 773 00:51:04,380 --> 00:51:10,380 This is first converted via a mutase reaction 774 00:51:10,380 --> 00:51:14,865 to move the phosphate from the 6 to the 1 position of glucose. 775 00:51:26,870 --> 00:51:32,870 So this, here, is glucose 1-phosphate. 776 00:51:32,870 --> 00:51:34,580 The way the mutase reaction works 777 00:51:34,580 --> 00:51:37,220 is exactly the same analogous mechanism 778 00:51:37,220 --> 00:51:39,840 that I explained for phosphoglycerate mutase 779 00:51:39,840 --> 00:51:41,390 in glycolysis-- 780 00:51:41,390 --> 00:51:44,840 moves the phosphate in this case from the 6 to the 1 position 781 00:51:44,840 --> 00:51:46,760 of glucose. 782 00:51:46,760 --> 00:51:50,150 And then once you have that glucose 1-phosphate, that 783 00:51:50,150 --> 00:51:58,310 can be added to a non-reducing end of a glycogen polymer 784 00:51:58,310 --> 00:52:09,560 using an enzyme called Glycogen Synthase, GS. 785 00:52:09,560 --> 00:52:16,590 So that'll give me a glycogen molecule 786 00:52:16,590 --> 00:52:20,730 with one additional monomer added to the non-reducing end 787 00:52:20,730 --> 00:52:22,770 of the polymer. 788 00:52:22,770 --> 00:52:26,340 And you release that glucose back off 789 00:52:26,340 --> 00:52:29,400 via a different pathway, a different enzyme 790 00:52:29,400 --> 00:52:31,560 called phosphorylase. 791 00:52:35,180 --> 00:52:37,250 All right. 792 00:52:37,250 --> 00:52:44,400 Now the activity of these enzymes-- 793 00:52:44,400 --> 00:52:46,850 glycogen synthase and phosphorylase-- 794 00:52:46,850 --> 00:52:51,090 that is, the pathway to add glucose 1-phosphate glucose 795 00:52:51,090 --> 00:52:55,590 monomer to the polymer versus remove it from the polymer 796 00:52:55,590 --> 00:53:02,760 is under hormonal signaling control in animals. 797 00:53:02,760 --> 00:53:07,905 And basically, that hormonal signaling works as follows. 798 00:53:32,780 --> 00:53:46,090 So phosphorylase can be phosphorylated 799 00:53:46,090 --> 00:53:55,640 or dephosphorylated via a signaling enzyme, an enzyme 800 00:53:55,640 --> 00:54:07,330 like phosphorylase kinase, which is 801 00:54:07,330 --> 00:54:15,310 downstream of PKA, which is downstream of epinephrine 802 00:54:15,310 --> 00:54:16,420 signaling. 803 00:54:16,420 --> 00:54:17,410 OK. 804 00:54:17,410 --> 00:54:22,750 And so when phosphorylase is phosphorylated by phosphorylase 805 00:54:22,750 --> 00:54:25,660 kinase-- that has, has a phosphate group added is 806 00:54:25,660 --> 00:54:27,430 a signaling cascade-- 807 00:54:27,430 --> 00:54:29,320 it is in the active state. 808 00:54:29,320 --> 00:54:36,290 And when it's dephosphorylated, it's in the inactive state. 809 00:54:36,290 --> 00:54:40,820 Well, glycogen synthase also is subject to regulation 810 00:54:40,820 --> 00:54:44,990 by protein phosphorylation on the enzyme by a kinase. 811 00:54:44,990 --> 00:54:48,750 Except this has the opposite relationship. 812 00:54:48,750 --> 00:54:56,460 So when it is in the phosphorylated state, 813 00:54:56,460 --> 00:54:59,580 it is inactive. 814 00:54:59,580 --> 00:55:03,180 But it is in the non-phosphorylated state, 815 00:55:03,180 --> 00:55:05,720 it is active. 816 00:55:05,720 --> 00:55:14,310 And so protein kinase A, which is downstream of epinephrine, 817 00:55:14,310 --> 00:55:18,720 can both activate phosphorylation 818 00:55:18,720 --> 00:55:21,840 of phosphorylase and glycogen synthase. 819 00:55:21,840 --> 00:55:23,610 And that makes sense. 820 00:55:23,610 --> 00:55:27,090 You want to release glycogen glucose monomers 821 00:55:27,090 --> 00:55:30,930 from glycogen. You turn on, by phosphorylation, the pathway 822 00:55:30,930 --> 00:55:32,100 to release them. 823 00:55:32,100 --> 00:55:36,940 And you turn off the pathway to store them. 824 00:55:36,940 --> 00:55:39,475 Another kinase that phosphorylates 825 00:55:39,475 --> 00:55:43,900 to glycogen synthase is a kinase called GSK or Glycogen Synthase 826 00:55:43,900 --> 00:55:44,950 Kinase. 827 00:55:44,950 --> 00:55:50,200 This kinase is inhibited by insulin signaling. 828 00:55:50,200 --> 00:55:55,300 And so this is a negative of a negative. 829 00:55:55,300 --> 00:55:58,480 So effectively, a negative of a negative 830 00:55:58,480 --> 00:56:03,610 will keep it in the inactive state. 831 00:56:03,610 --> 00:56:08,410 And so insulin, by inhibiting the ability of GSK 832 00:56:08,410 --> 00:56:11,650 to put glycogen synthase in the inactive state, 833 00:56:11,650 --> 00:56:13,900 will make glycogen synthase active. 834 00:56:13,900 --> 00:56:16,000 And when you have high insulin around, 835 00:56:16,000 --> 00:56:18,850 that actives glycogen synthesis, and you 836 00:56:18,850 --> 00:56:21,820 store glucose monomers as glycogen. 837 00:56:21,820 --> 00:56:24,020 Get them out of the blood. 838 00:56:24,020 --> 00:56:26,230 OK. 839 00:56:26,230 --> 00:56:40,820 Now let's discuss the chemistry that 840 00:56:40,820 --> 00:56:45,950 allows us to do this glycogen synthase and phosphorylates 841 00:56:45,950 --> 00:56:46,850 reactions. 842 00:57:14,490 --> 00:57:16,740 Now as we go through this, what you will see 843 00:57:16,740 --> 00:57:22,420 is that when we break down the polymer, energy is released. 844 00:57:22,420 --> 00:57:24,440 So that's going to be breaking down a polymer. 845 00:57:24,440 --> 00:57:26,510 That's the right direction of entropy. 846 00:57:26,510 --> 00:57:28,370 And so that's going to be released. 847 00:57:28,370 --> 00:57:30,350 And storing it is going to require energy. 848 00:57:30,350 --> 00:57:32,392 That is, you're going to have to have some energy 849 00:57:32,392 --> 00:57:33,830 input to build a polymer. 850 00:57:33,830 --> 00:57:36,740 But nature actually does this in a way that 851 00:57:36,740 --> 00:57:41,560 is actually quite efficient. 852 00:57:41,560 --> 00:57:50,430 First, let's talk about how you store glucose 1-phosphate units 853 00:57:50,430 --> 00:57:54,870 by adding them to glycogen. So this uses energy input 854 00:57:54,870 --> 00:57:59,280 from UTP, which is an ATP equivalent for exactly 855 00:57:59,280 --> 00:58:00,780 the reason I described earlier. 856 00:58:00,780 --> 00:58:06,000 That is, because ATP plus UDP, like any other interconversion 857 00:58:06,000 --> 00:58:08,730 of nucleoside triphosphates and diphosphates 858 00:58:08,730 --> 00:58:10,410 is very close to equilibrium. 859 00:58:10,410 --> 00:58:13,830 So the UTP to UDP ratio should be similar to the ATP the ADP 860 00:58:13,830 --> 00:58:14,670 ratio. 861 00:58:14,670 --> 00:58:17,220 And so UTP is really an ATP equivalent. 862 00:58:17,220 --> 00:58:20,820 But nature, for whatever reason, decided to use UTP here. 863 00:58:20,820 --> 00:58:27,180 And it generates a nucleoside sugar called UDP glucose. 864 00:58:27,180 --> 00:58:30,510 And so let me show you what that is. 865 00:58:38,210 --> 00:58:41,455 So here is glucose 1-phosphate. 866 00:58:52,300 --> 00:58:59,770 I will draw a stylized version of UTP here. 867 00:59:24,080 --> 00:59:29,990 And so this molecule with the UDP added to the glucose 868 00:59:29,990 --> 00:59:36,320 is UDP glucose. 869 00:59:36,320 --> 00:59:41,600 This will also generate a pyrophosphate. 870 00:59:41,600 --> 00:59:46,220 And that pyrophosphate can be hydrolyzed 871 00:59:46,220 --> 00:59:49,550 to generate two inorganic phosphates, effectively, 872 00:59:49,550 --> 00:59:54,020 like building any other polymer, by doing this downstream-- 873 00:59:54,020 --> 00:59:57,290 keeping that product of the first reaction low 874 00:59:57,290 --> 01:00:02,360 helps pull that reaction forward for polymer synthesis. 875 01:00:02,360 --> 01:00:11,810 This UDP glucose can now react with the non-reducing end. 876 01:00:21,370 --> 01:00:25,570 So this, here, will be the non-reducing end 877 01:00:25,570 --> 01:00:28,070 of the polymer. 878 01:00:28,070 --> 01:00:31,580 That's the OH group at the 4 position, 879 01:00:31,580 --> 01:00:33,440 at the non-producing end. 880 01:00:33,440 --> 01:00:46,360 And so that then generates that alpha 1,4 881 01:00:46,360 --> 01:00:50,480 bond to add to the polymer. 882 01:00:50,480 --> 01:00:57,340 So here, we have glycogen n plus 1 plus releases UDP. 883 01:00:57,340 --> 01:01:02,740 And so the net to add a glucose 1-phosphate 884 01:01:02,740 --> 01:01:06,100 to the non-reducing end of the growing glycogen polymer 885 01:01:06,100 --> 01:01:10,720 is net conversion of a UTP to a UDP. 886 01:01:10,720 --> 01:01:17,380 So that is one ATP equivalent to add glucose 1-phosphate 887 01:01:17,380 --> 01:01:23,000 to the end of the polymer. 888 01:01:23,000 --> 01:01:25,180 Now, of course, you had it to also 889 01:01:25,180 --> 01:01:30,690 have an ATP from hexokinase to capture that glucose 890 01:01:30,690 --> 01:01:32,550 6-phosphate to begin with. 891 01:01:32,550 --> 01:01:37,890 But effectively, this is two ADP molecules 892 01:01:37,890 --> 01:01:49,320 to add a glucose to glycogen. All right. 893 01:01:49,320 --> 01:01:52,590 Now if we're generating starch, that's all we have to do, 894 01:01:52,590 --> 01:01:53,940 just build a long polymer. 895 01:01:53,940 --> 01:01:55,650 But if we're making glycogen, remember, 896 01:01:55,650 --> 01:02:00,510 glycogen has all of these branch points on them as well. 897 01:02:00,510 --> 01:02:02,550 And to do the branch points, you basically 898 01:02:02,550 --> 01:02:05,640 have to make this alpha 1,6 bond, which then gives you 899 01:02:05,640 --> 01:02:11,540 two new non-reducing ends that you can add more subunits to. 900 01:02:11,540 --> 01:02:15,030 The way nature does this is as follows. 901 01:02:15,030 --> 01:02:22,110 And so if I just draw this here in a stylized way, 902 01:02:22,110 --> 01:02:28,020 it turns out, once you get to about seven units 903 01:02:28,020 --> 01:02:29,960 here in the growing polymer-- 904 01:02:29,960 --> 01:02:34,500 so this is the reducing end, and this is the non-reducing end 905 01:02:34,500 --> 01:02:35,680 of the polymer. 906 01:02:35,680 --> 01:02:39,750 And so the polymer is growing by adding things to that end. 907 01:02:39,750 --> 01:02:41,970 Once you get about seven, there's 908 01:02:41,970 --> 01:02:45,750 an enzyme that will basically cleave this, and move it 909 01:02:45,750 --> 01:02:50,050 and make a new alpha 1,6 bond. 910 01:02:50,050 --> 01:02:51,420 OK. 911 01:02:51,420 --> 01:02:52,300 Here we go. 912 01:02:52,300 --> 01:02:59,060 We have our reducing and our non-reducing end 913 01:02:59,060 --> 01:03:06,090 and basically transfers these 7 units over. 914 01:03:06,090 --> 01:03:09,630 And now, we have two non-reducing ends 915 01:03:09,630 --> 01:03:16,950 that we can continue to grow the polymer from. 916 01:03:16,950 --> 01:03:20,820 You can think of this like the airplane analogy. 917 01:03:20,820 --> 01:03:24,150 When you load an airplane, at least the way that's 918 01:03:24,150 --> 01:03:25,980 most efficient, the people who are 919 01:03:25,980 --> 01:03:28,590 sitting in the back of the plane, they get on first. 920 01:03:28,590 --> 01:03:30,990 But then, they're the last people off 921 01:03:30,990 --> 01:03:34,110 the plane because you add basically 922 01:03:34,110 --> 01:03:36,420 to these non-reducing ends, and then 923 01:03:36,420 --> 01:03:38,820 you subtract from those non-reducing ends. 924 01:03:38,820 --> 01:03:42,900 And so the first glucose added, the one at the reducing end, 925 01:03:42,900 --> 01:03:47,420 is the last one that is going to be removed. 926 01:03:47,420 --> 01:03:48,980 All right. 927 01:03:48,980 --> 01:03:51,650 Now phosphorylase is the enzyme that 928 01:03:51,650 --> 01:03:53,750 allows you to do the opposite-- that is, 929 01:03:53,750 --> 01:03:58,310 to break that alpha 1,4 bond and release monomers. 930 01:03:58,310 --> 01:04:00,740 And it's called phosphorylase because it 931 01:04:00,740 --> 01:04:05,330 uses phosphate to break that alpha 1,4 bond, 932 01:04:05,330 --> 01:04:08,150 taking advantage of the fact that breaking down the polymer 933 01:04:08,150 --> 01:04:12,080 is favorable and actually releasing the glucose from it 934 01:04:12,080 --> 01:04:15,030 again is a glucose 1-phosphate. 935 01:04:15,030 --> 01:04:18,410 And so at the non-reducing end of the molecule-- 936 01:04:33,160 --> 01:04:39,960 so this, here, would be the non-reducing end 937 01:04:39,960 --> 01:04:42,270 of the glycogen polymer. 938 01:04:42,270 --> 01:04:55,920 You basically have a phosphate molecule that takes that off, 939 01:04:55,920 --> 01:05:21,940 so you have a glycogen n minus 1 plus a glucose 1 phosphate, 940 01:05:21,940 --> 01:05:23,910 which can now undergo a mutase reaction back 941 01:05:23,910 --> 01:05:26,340 to glucose 6-phosphate that could then be released 942 01:05:26,340 --> 01:05:28,200 from the cell if it's a liver, or it 943 01:05:28,200 --> 01:05:30,870 could be burned in glycolysis. 944 01:05:30,870 --> 01:05:33,450 Now, obviously, if you're doing breakdown, that is, 945 01:05:33,450 --> 01:05:36,240 your chewing back from non-reducing ends, 946 01:05:36,240 --> 01:05:42,990 eventually, you're going to hit a situation where 947 01:05:42,990 --> 01:05:47,220 you come to where there's just a nub here with an alpha 1,6 bond 948 01:05:47,220 --> 01:05:48,030 there. 949 01:05:48,030 --> 01:05:51,930 That is, chew it back till you hit this single monomer 950 01:05:51,930 --> 01:05:56,040 with a non-reducing end here to this alpha 1,6 break point. 951 01:05:56,040 --> 01:06:00,760 Turns out, you just cleave off that nub, if you will. 952 01:06:00,760 --> 01:06:04,920 And so that nub is then just released as free glucose. 953 01:06:04,920 --> 01:06:08,340 So other than release of that nub, what it means 954 01:06:08,340 --> 01:06:14,280 is that you actually basically get 955 01:06:14,280 --> 01:06:19,330 no ATP to actually break down in the phosphorylase reaction. 956 01:06:19,330 --> 01:06:24,630 So the net cost to store a glucose 1-phosphate molecule 957 01:06:24,630 --> 01:06:31,440 is one ATP via the UTP to UDP conversion in the glycogen 958 01:06:31,440 --> 01:06:32,880 synthase reaction. 959 01:06:32,880 --> 01:06:36,600 And you get that one glucose 1-phosphate back. 960 01:06:36,600 --> 01:06:40,470 And so two ATPs to put a glucose tin glycogen. 961 01:06:40,470 --> 01:06:42,030 And then you get it directly back 962 01:06:42,030 --> 01:06:45,630 out as a glucose 1-phosphate. 963 01:06:45,630 --> 01:06:48,960 That's a phosphorylated glucose that can then enter glycolysis. 964 01:06:48,960 --> 01:06:52,890 So that's one less phosphate you need to spend on glycolysis. 965 01:06:52,890 --> 01:06:57,640 And so it's incredibly efficient to store glucose in that way. 966 01:06:57,640 --> 01:07:00,240 And so it's actually slightly greater than one ATP 967 01:07:00,240 --> 01:07:04,185 to store it as glycogen because you lose in ATP with that nub 968 01:07:04,185 --> 01:07:06,300 removal at the branch polymer. 969 01:07:06,300 --> 01:07:07,710 But for all intents and purposes, 970 01:07:07,710 --> 01:07:12,850 it costs one ATP to store your glucose as glycogen, 971 01:07:12,850 --> 01:07:15,210 which is pretty amazing. 972 01:07:15,210 --> 01:07:16,140 All right. 973 01:07:16,140 --> 01:07:19,710 Now before I leave the topic of sugar storage, 974 01:07:19,710 --> 01:07:22,890 I want to make some comments on some sugars that are stored 975 01:07:22,890 --> 01:07:24,930 in forms other than glucose. 976 01:07:24,930 --> 01:07:27,090 And want to talk about these because these sugars 977 01:07:27,090 --> 01:07:29,280 are, of course, important parts of our diet. 978 01:07:29,280 --> 01:07:35,270 And so as we've mentioned in prior lectures, 979 01:07:35,270 --> 01:07:42,890 two of these sugars are sucrose and lactose. 980 01:07:42,890 --> 01:07:45,440 So remember, sucrose is a disaccharide 981 01:07:45,440 --> 01:07:50,450 of glucose plus fructose. 982 01:07:50,450 --> 01:07:58,390 And lactose is a disaccharide of glucose plus galactose. 983 01:07:58,390 --> 01:08:01,300 So those are storage forms of sugar-- sucrose 984 01:08:01,300 --> 01:08:03,430 in the case of plants, lactose in the case 985 01:08:03,430 --> 01:08:06,430 of milk made by mammals. 986 01:08:06,430 --> 01:08:11,080 And so to break those sugars down, 987 01:08:11,080 --> 01:08:13,897 basically, you break the disaccharide. 988 01:08:13,897 --> 01:08:15,355 Now, you have the glucose molecules 989 01:08:15,355 --> 01:08:16,479 you know how to handle. 990 01:08:16,479 --> 01:08:17,783 That's glycolysis. 991 01:08:17,783 --> 01:08:19,450 But I want to spend a little bit of time 992 01:08:19,450 --> 01:08:22,540 how you deal with the fructose and the galactose 993 01:08:22,540 --> 01:08:25,420 that you get out from these sugars 994 01:08:25,420 --> 01:08:28,430 because this actually relates to some things 995 01:08:28,430 --> 01:08:31,569 that I'm sure you've all read about in the news. 996 01:08:31,569 --> 01:08:36,340 For instance, fructose as a sugar in our diet 997 01:08:36,340 --> 01:08:39,430 is actually quite controversial. 998 01:08:39,430 --> 01:08:42,040 Lots of stuff out there that says fructose 999 01:08:42,040 --> 01:08:46,540 was cooked up in the devil's kitchen and is this evil thing. 1000 01:08:46,540 --> 01:08:48,250 There's actually others who argue 1001 01:08:48,250 --> 01:08:50,410 that, oh, fructose is the same as glucose, 1002 01:08:50,410 --> 01:08:52,490 the same number of calories. 1003 01:08:52,490 --> 01:08:53,920 There's both sides of the debate. 1004 01:08:53,920 --> 01:08:56,470 Both sides are actually making factual claims. 1005 01:08:56,470 --> 01:08:58,930 And I want you to understand the biochemistry, 1006 01:08:58,930 --> 01:09:01,930 so you can actually judge for yourself 1007 01:09:01,930 --> 01:09:04,729 who's right in those claims. 1008 01:09:04,729 --> 01:09:05,229 All right. 1009 01:09:05,229 --> 01:09:06,771 So let's start with talking about how 1010 01:09:06,771 --> 01:09:09,010 you metabolize fructose. 1011 01:09:09,010 --> 01:09:18,490 And so fructose is first captured by phosphorylation 1012 01:09:18,490 --> 01:09:25,390 to generate fructose 1-phosphate. 1013 01:09:25,390 --> 01:09:31,510 This is carried out by an enzyme called ketohexokinase. 1014 01:09:31,510 --> 01:09:35,229 Ketohexokinase is an enzyme this present 1015 01:09:35,229 --> 01:09:36,640 in your gut and your liver. 1016 01:09:36,640 --> 01:09:39,729 And so fructose in the diet, from breaking down sucrose 1017 01:09:39,729 --> 01:09:41,859 or from high-fructose corn syrup, whatever, 1018 01:09:41,859 --> 01:09:45,880 is captured with ketohexokinase to make fructose 1-phosphate. 1019 01:09:45,880 --> 01:09:48,479 Turns out that phosphofructokinase, 1020 01:09:48,479 --> 01:09:51,069 our old friend from glycolysis, is less efficient 1021 01:09:51,069 --> 01:09:54,170 than ketohexokinase but can also carry out this reaction. 1022 01:09:54,170 --> 01:09:56,410 And remember, what is it do in glycolysis? 1023 01:09:56,410 --> 01:09:58,395 It adds a phosphate to the 1 position 1024 01:09:58,395 --> 01:09:59,395 of fructose 6-phosphate. 1025 01:09:59,395 --> 01:10:01,240 Well, it can also add a phosphate 1026 01:10:01,240 --> 01:10:06,040 to the 1 position of fructose give you fructose 1-phosphate. 1027 01:10:06,040 --> 01:10:06,880 OK. 1028 01:10:06,880 --> 01:10:10,030 Once you have this fructose 1-phosphate, which, remember, 1029 01:10:10,030 --> 01:10:14,620 is not in glycolysis, it can be a substrate 1030 01:10:14,620 --> 01:10:20,590 for the glycolytic enzyme aldolase. 1031 01:10:20,590 --> 01:10:22,810 What does aldolase do in glycolysis? 1032 01:10:22,810 --> 01:10:26,080 It splits FBP into dihydroxyacetone phosphate 1033 01:10:26,080 --> 01:10:28,300 and glyceraldehyde 3-phosphate. 1034 01:10:28,300 --> 01:10:31,580 Well, here, you only have a phosphate on the 1 position. 1035 01:10:31,580 --> 01:10:34,600 And so if you look back at how aldolase works, what you'll see 1036 01:10:34,600 --> 01:10:39,190 is that if aldolase acts on fructose 1-phosphate, 1037 01:10:39,190 --> 01:10:41,710 you'll generate a dihydroxyacetone phosphate. 1038 01:10:41,710 --> 01:10:45,070 That phosphate will go to the dihydroxyacetone half, which, 1039 01:10:45,070 --> 01:10:50,500 of course, is perfectly good substrate for glycolysis. 1040 01:10:50,500 --> 01:10:55,060 The other product of aldolase acting on fructose 1-phosphate 1041 01:10:55,060 --> 01:11:01,690 is just glyceraldehyde without the phosphate on it. 1042 01:11:08,680 --> 01:11:10,710 And then there's another enzyme that 1043 01:11:10,710 --> 01:11:16,670 will phosphorylate glyceraldehyde to generate 1044 01:11:16,670 --> 01:11:21,230 glyceraldehyde 3-phosphate, which can then also, of course, 1045 01:11:21,230 --> 01:11:23,210 enter glycolysis. 1046 01:11:23,210 --> 01:11:27,170 And so fructose is a hexose just like glucose. 1047 01:11:27,170 --> 01:11:29,090 You generate two trioses-- 1048 01:11:29,090 --> 01:11:31,670 DHAP and glyceraldehyde 3-phosphate. 1049 01:11:31,670 --> 01:11:35,600 That costs you two ATP to do it, exactly the same cost 1050 01:11:35,600 --> 01:11:41,010 is what you spent to get glucose to make DHAP and glyceraldehyde 1051 01:11:41,010 --> 01:11:42,270 3-phosphate. 1052 01:11:42,270 --> 01:11:44,510 And so people are absolutely right when 1053 01:11:44,510 --> 01:11:47,360 they say metabolizing fructose has 1054 01:11:47,360 --> 01:11:53,180 exactly the same caloric cost as metabolizing glucose-- 1055 01:11:53,180 --> 01:11:55,700 two ATPs to get it into the trioses. 1056 01:11:55,700 --> 01:11:57,200 And then whatever you get out of it, 1057 01:11:57,200 --> 01:12:00,710 you get out of it two ATPs, four ATPs-- 1058 01:12:00,710 --> 01:12:05,120 so net two ATPs if you just do fermentation, more 1059 01:12:05,120 --> 01:12:07,310 if you do complete oxidation. 1060 01:12:07,310 --> 01:12:10,670 But the bottom line is that from that standpoint, 1061 01:12:10,670 --> 01:12:13,400 you get exactly the same number of calories 1062 01:12:13,400 --> 01:12:16,880 from burning glucose versus fructose. 1063 01:12:16,880 --> 01:12:20,480 However, I want to point out that the regulation will not 1064 01:12:20,480 --> 01:12:21,690 be the same. 1065 01:12:21,690 --> 01:12:26,210 And that's because in this fructose metabolism, 1066 01:12:26,210 --> 01:12:30,680 you will notice that there was no glucose 6-phosphate 1067 01:12:30,680 --> 01:12:35,360 and no FBP generated. 1068 01:12:35,360 --> 01:12:40,030 That means you are avoiding all of the feedbacks 1069 01:12:40,030 --> 01:12:44,182 that control how much glucose you send to glycolysis-- 1070 01:12:44,182 --> 01:12:45,640 first to glycogen. Remember, that's 1071 01:12:45,640 --> 01:12:49,390 what glucose 6-phosphate does, acting on trapping sugar. 1072 01:12:49,390 --> 01:12:52,330 And more importantly, you're actually 1073 01:12:52,330 --> 01:12:56,830 not using phosphofructokinase in a way 1074 01:12:56,830 --> 01:13:02,470 to fit with the rest of the feedback regulation. 1075 01:13:02,470 --> 01:13:05,680 I also show that here on this slide. 1076 01:13:05,680 --> 01:13:11,740 It's basically a better version of what I drew up here. 1077 01:13:11,740 --> 01:13:17,440 And I now want to point out this storage of citrate 1078 01:13:17,440 --> 01:13:21,400 and this feedback regulation of citrate. 1079 01:13:21,400 --> 01:13:23,650 So we'll talk about the specifics 1080 01:13:23,650 --> 01:13:26,170 of this more in other lectures. 1081 01:13:26,170 --> 01:13:29,620 But it turns out, citrate is the precursor 1082 01:13:29,620 --> 01:13:33,100 for how one stores carbon as fat. 1083 01:13:33,100 --> 01:13:36,190 And so if you're putting fructose glucose 1084 01:13:36,190 --> 01:13:39,640 into the system and citrate is too high, 1085 01:13:39,640 --> 01:13:41,740 it'll shut off phosphofructokinase 1086 01:13:41,740 --> 01:13:44,260 and stop carbon coming down the system, 1087 01:13:44,260 --> 01:13:46,930 shunting it instead to glycogen or just saying don't give me 1088 01:13:46,930 --> 01:13:48,970 any more glucose at all. 1089 01:13:48,970 --> 01:13:51,340 If I'm doing the same thing with fructose, 1090 01:13:51,340 --> 01:13:53,050 those feedbacks don't exist. 1091 01:13:53,050 --> 01:13:57,880 Citrate doesn't feed back on any of the enzymes that 1092 01:13:57,880 --> 01:14:00,670 are regulated for fructose metabolism. 1093 01:14:00,670 --> 01:14:02,920 And so the idea is carbon keeps getting 1094 01:14:02,920 --> 01:14:06,920 dumped into citrate, which ultimately ends up as fat. 1095 01:14:06,920 --> 01:14:10,690 And this really underlies, at least on a theoretical basis, 1096 01:14:10,690 --> 01:14:14,950 why there may be a connection between fructose and obesity or 1097 01:14:14,950 --> 01:14:16,660 metabolic syndrome. 1098 01:14:16,660 --> 01:14:21,160 I've posted articles on Stellar, one from The Popular Press, 1099 01:14:21,160 --> 01:14:24,400 one from a scientific journal that discusses the controversy, 1100 01:14:24,400 --> 01:14:25,290 discusses this. 1101 01:14:25,290 --> 01:14:27,040 But now, at least, you have the background 1102 01:14:27,040 --> 01:14:30,410 to draw some of your own conclusions. 1103 01:14:30,410 --> 01:14:31,400 OK. 1104 01:14:31,400 --> 01:14:33,590 That's fructose metabolism. 1105 01:14:33,590 --> 01:14:37,820 What about galactose metabolism? 1106 01:14:37,820 --> 01:14:45,710 Well, galactose metabolism interfaces with, effectively, 1107 01:14:45,710 --> 01:14:50,270 glycogen metabolism or, at least, glucose 1-phosphate-- 1108 01:14:50,270 --> 01:14:55,460 or sorry, UDP glucose and does so in a way that 1109 01:14:55,460 --> 01:15:00,530 is a little bit non-intuitive. 1110 01:15:00,530 --> 01:15:03,590 But it's actually very important to understand 1111 01:15:03,590 --> 01:15:07,010 galactose metabolism, at least for those of you 1112 01:15:07,010 --> 01:15:10,985 who are looking to go to medical school. 1113 01:15:10,985 --> 01:15:15,110 It turns out that galactose metabolism and issues 1114 01:15:15,110 --> 01:15:17,570 with metabolizing galactose are what 1115 01:15:17,570 --> 01:15:20,960 underlie a set of rare genetic diseases leading 1116 01:15:20,960 --> 01:15:25,243 to a set of conditions called galactosemias. 1117 01:15:25,243 --> 01:15:26,660 I guess, if you're a pediatrician, 1118 01:15:26,660 --> 01:15:27,830 you may encounter these. 1119 01:15:27,830 --> 01:15:29,600 If you're not, these are quite rare. 1120 01:15:29,600 --> 01:15:32,810 Although med schools love to ask about these, 1121 01:15:32,810 --> 01:15:34,700 and certainly, they're very popular on things 1122 01:15:34,700 --> 01:15:36,170 like board exams. 1123 01:15:36,170 --> 01:15:38,540 So it's something you should know about. 1124 01:15:38,540 --> 01:15:43,400 And so I just want to briefly introduce galactose metabolism 1125 01:15:43,400 --> 01:15:45,110 to you. 1126 01:15:45,110 --> 01:15:47,870 So this is galactose. 1127 01:15:47,870 --> 01:15:52,400 Like all sugars, galactose is initially 1128 01:15:52,400 --> 01:15:55,620 trapped by phosphorylation. 1129 01:15:55,620 --> 01:16:00,450 It's phosphorylated on the 1 position to trap it, 1130 01:16:00,450 --> 01:16:01,815 just like fructose. 1131 01:16:07,920 --> 01:16:13,710 And so this is galactose 1-phosphate. 1132 01:16:13,710 --> 01:16:17,860 Remember, galactose is an epimer of glucose. 1133 01:16:17,860 --> 01:16:21,270 So it differs from glucose here by the stereochemistry 1134 01:16:21,270 --> 01:16:25,810 of the hydroxyl group at this 4 carbon. 1135 01:16:25,810 --> 01:16:28,350 And so the way galactose, once it's 1136 01:16:28,350 --> 01:16:31,680 trapped as galactose 1-phosphate is metabolized 1137 01:16:31,680 --> 01:16:37,740 is it will react with UDP glucose. 1138 01:16:52,780 --> 01:16:59,560 So here is UDP glucose from glycogen metabolism 1139 01:16:59,560 --> 01:17:01,960 drawn in a very stylized way. 1140 01:17:08,880 --> 01:17:26,890 That will generate a UDP galactose 1141 01:17:26,890 --> 01:17:37,560 and a glucose 1-phosphate, which, 1142 01:17:37,560 --> 01:17:46,530 of course, can enter glycolysis, can be 1143 01:17:46,530 --> 01:17:50,530 stored as glycogen, et cetera. 1144 01:17:50,530 --> 01:17:57,250 This UDP galactose can then be turned into UDP glucose 1145 01:17:57,250 --> 01:18:00,950 via an epimerase reaction. 1146 01:18:00,950 --> 01:18:03,280 And so remember, the epimerase reaction 1147 01:18:03,280 --> 01:18:05,290 needs to change the stereochemistry 1148 01:18:05,290 --> 01:18:08,020 of the hydroxyl group at this 4 position, 1149 01:18:08,020 --> 01:18:10,870 such that it's pointing up in galactose 1150 01:18:10,870 --> 01:18:15,020 or pointing down in glucose. 1151 01:18:15,020 --> 01:18:19,850 The way epimerases work is that they require a cofactor. 1152 01:18:19,850 --> 01:18:21,740 That cofactor is NAD. 1153 01:18:21,740 --> 01:18:24,150 and they work in the following way. 1154 01:18:24,150 --> 01:18:34,220 And so if I just draw, here's the 4 position of galactose, 1155 01:18:34,220 --> 01:18:47,640 the 4 carbon of galactose, with the hydroxyl group pointing up, 1156 01:18:47,640 --> 01:18:55,740 an epimerase can basically remove 1157 01:18:55,740 --> 01:18:58,800 a hydride from the bottom face of the molecule, 1158 01:18:58,800 --> 01:19:03,660 transfer those two electrons to NAD to generate NADH. 1159 01:19:03,660 --> 01:19:14,010 Now, you get this lactone intermediate at the 4 carbon. 1160 01:19:14,010 --> 01:19:29,430 And then you can have two electrons from an NADH can be 1161 01:19:29,430 --> 01:19:35,160 added to the opposite face of the molecule that regenerates 1162 01:19:35,160 --> 01:19:36,750 an NAD+. 1163 01:19:36,750 --> 01:19:45,450 And now, we've effectively changed the stereochemistry 1164 01:19:45,450 --> 01:19:48,810 at the 4 carbon to now be pointing 1165 01:19:48,810 --> 01:19:52,060 such the hydroxyl points down, and that's glucose. 1166 01:19:52,060 --> 01:19:55,680 And so the way epimerases work is they basically 1167 01:19:55,680 --> 01:19:59,640 remove and add electrons from different faces 1168 01:19:59,640 --> 01:20:01,500 of the pyranose ring. 1169 01:20:01,500 --> 01:20:04,740 And that changes the stereochemistry 1170 01:20:04,740 --> 01:20:10,180 of which direction the hydroxyl group is pointing. 1171 01:20:10,180 --> 01:20:14,440 And so effectively, this is how we metabolize 1172 01:20:14,440 --> 01:20:18,780 all kinds of different sugars, either 1173 01:20:18,780 --> 01:20:22,050 putting them in glycogen or other starch, 1174 01:20:22,050 --> 01:20:24,120 some other storage carbohydrate that we 1175 01:20:24,120 --> 01:20:27,450 can get them later, and then use them 1176 01:20:27,450 --> 01:20:32,310 in glycolysis as a way to drive glucose oxidation as a way 1177 01:20:32,310 --> 01:20:38,230 to release energy and keep ATP high in the cell. 1178 01:20:38,230 --> 01:20:38,730 All right. 1179 01:20:38,730 --> 01:20:40,830 Now I want to shift topics and begin 1180 01:20:40,830 --> 01:20:43,740 to discuss how we can oxidize pyruvate 1181 01:20:43,740 --> 01:20:49,760 from glycolysis to make CO2 as an alternative 1182 01:20:49,760 --> 01:20:54,410 to how we can release energy from carbohydrate oxidation 1183 01:20:54,410 --> 01:20:57,910 rather than just use fermentation. 1184 01:20:57,910 --> 01:21:01,950 Now you'll see that the enzymes and ways that we do this 1185 01:21:01,950 --> 01:21:05,280 is going to also be how we make citrate. 1186 01:21:05,280 --> 01:21:08,730 And so this isn't just about releasing energy. 1187 01:21:08,730 --> 01:21:11,100 It's also about storing energy because you'll see, 1188 01:21:11,100 --> 01:21:14,190 citrate is a precursor for storage as fat. 1189 01:21:14,190 --> 01:21:17,730 The reactions downstream of pyruvate oxidation 1190 01:21:17,730 --> 01:21:20,370 also make lots of other useful intermediates 1191 01:21:20,370 --> 01:21:23,400 that can be used to synthesize other things cells need-- 1192 01:21:23,400 --> 01:21:25,630 amino acids, nucleic acids. 1193 01:21:25,630 --> 01:21:29,040 And so this pathways we're going to discuss 1194 01:21:29,040 --> 01:21:31,470 are going to come up over and over again 1195 01:21:31,470 --> 01:21:34,000 throughout the course. 1196 01:21:34,000 --> 01:21:37,490 Now to facilitate this discussion, 1197 01:21:37,490 --> 01:21:42,870 I'm really going to at least initially focus on catabolism. 1198 01:21:42,870 --> 01:21:48,450 And so just to remind you, if we do glycolysis 1199 01:21:48,450 --> 01:21:56,350 by turning glucose pyruvate, this involves oxidation. 1200 01:21:56,350 --> 01:22:02,180 So we need to dispose of those electrons somewhere. 1201 01:22:02,180 --> 01:22:07,490 In other words, that NADH has to be regenerated NAD. 1202 01:22:07,490 --> 01:22:11,540 Fermentation of pyruvate into something like lactate 1203 01:22:11,540 --> 01:22:14,900 is a way to do that and allows net ATP production 1204 01:22:14,900 --> 01:22:20,040 from glycolysis without the need for any oxygen. 1205 01:22:20,040 --> 01:22:23,760 However, if we're going to completely oxidize 1206 01:22:23,760 --> 01:22:29,810 that pyruvate CO2 we're going to generate many more electrons 1207 01:22:29,810 --> 01:22:33,630 as waste that we have to put somewhere. 1208 01:22:33,630 --> 01:22:38,500 Oxygen is a particularly good electron acceptor. 1209 01:22:38,500 --> 01:22:41,170 And so we can reduce oxygen to water 1210 01:22:41,170 --> 01:22:43,540 as a way to deal with that electron waste, 1211 01:22:43,540 --> 01:22:46,600 and that allows us to, just burning wood, 1212 01:22:46,600 --> 01:22:49,150 release lots of energy from glucose 1213 01:22:49,150 --> 01:22:52,490 by transferring those electrons ultimately to oxygen. 1214 01:22:52,490 --> 01:22:54,760 And of course, that same process-- 1215 01:22:54,760 --> 01:22:58,150 oxygen to water-- if we're using the pyruvate to fully oxidize 1216 01:22:58,150 --> 01:23:01,150 it, also is necessary to regenerate 1217 01:23:01,150 --> 01:23:04,820 the NAD to run glycolysis. 1218 01:23:04,820 --> 01:23:08,330 Now ultimately, the series of reactions 1219 01:23:08,330 --> 01:23:12,830 that allow chemically conversion of pyruvate to CO2 1220 01:23:12,830 --> 01:23:26,660 is called the TCA cycle for try Tricarboxylic Acid Cycle 1221 01:23:26,660 --> 01:23:33,380 or sometimes referred to as the Krebs cycle 1222 01:23:33,380 --> 01:23:41,800 and also is referred to as the citric acid cycle-- 1223 01:23:41,800 --> 01:23:47,240 so TCA, Tricarboxylic Acid Cycle, Krebs cycle, 1224 01:23:47,240 --> 01:23:48,790 citric acid cycle-- 1225 01:23:48,790 --> 01:23:52,490 all synonyms for the same thing. 1226 01:23:52,490 --> 01:24:00,350 What these cycles are is basically 1227 01:24:00,350 --> 01:24:14,185 a way to take two carbon units. 1228 01:24:20,570 --> 01:24:23,050 And those two carbon units can come 1229 01:24:23,050 --> 01:24:29,290 from the oxidation of pyruvate or from the oxidation 1230 01:24:29,290 --> 01:24:31,390 of lots of other fuels. 1231 01:24:31,390 --> 01:24:35,890 And those two carbon units are combined 1232 01:24:35,890 --> 01:24:37,315 with four carbon units. 1233 01:24:41,670 --> 01:24:45,380 You'll see oxaloacetate is the four-carbon unit-- 1234 01:24:45,380 --> 01:24:47,370 oxaloacetate gluconeogenesis. 1235 01:24:47,370 --> 01:24:50,580 That oxaloacetate could come from the pyruvate-carboxylase 1236 01:24:50,580 --> 01:24:51,570 reaction. 1237 01:24:51,570 --> 01:24:55,530 Or it could come, as you'll see, from the TCA cycle itself. 1238 01:24:55,530 --> 01:25:03,150 This generates a six-carbon molecule, which is citrate. 1239 01:25:03,150 --> 01:25:07,740 Citrate is a tricarboxylic acid, hence the citric acid cycle 1240 01:25:07,740 --> 01:25:10,350 or the tricarboxylic acid cycle. 1241 01:25:10,350 --> 01:25:13,050 And then those six carbon units are 1242 01:25:13,050 --> 01:25:17,010 reoxidized, releasing two CO2 molecules, 1243 01:25:17,010 --> 01:25:19,380 back to four-carbon oxaloacetate. 1244 01:25:19,380 --> 01:25:21,960 And hence, this is a cycle. 1245 01:25:21,960 --> 01:25:24,090 Now this cycle was initially described 1246 01:25:24,090 --> 01:25:27,150 by Hans Krebs, who predicted the cycle largely 1247 01:25:27,150 --> 01:25:29,310 based on chemistry-- one of the most amazing feats 1248 01:25:29,310 --> 01:25:31,350 in the discovery of metabolism. 1249 01:25:31,350 --> 01:25:34,530 Turned out, he was right. 1250 01:25:34,530 --> 01:25:40,000 And thus, it's also referred to as his name, the Krebs cycle. 1251 01:25:40,000 --> 01:25:43,890 Now lots of oxidation is happening in this cycle. 1252 01:25:43,890 --> 01:25:47,400 You're oxidizing the molecule, releasing CO2. 1253 01:25:47,400 --> 01:25:49,530 That means lots of energy is released. 1254 01:25:49,530 --> 01:25:52,590 Those electrons ultimately will be transferred oxygen. And so 1255 01:25:52,590 --> 01:25:58,810 we can use this to make lots of ATP and do lots of other work. 1256 01:25:58,810 --> 01:26:05,150 Where are these reactions all occur is in the mitochondria, 1257 01:26:05,150 --> 01:26:10,700 specifically in the matrix of the mitochondria. 1258 01:26:10,700 --> 01:26:15,380 Remember, the mitochondria, from cell biology, 1259 01:26:15,380 --> 01:26:17,780 is a double-membrane organelle. 1260 01:26:17,780 --> 01:26:20,360 The innermost part of the mitochondria 1261 01:26:20,360 --> 01:26:22,370 is referred to as the matrix. 1262 01:26:22,370 --> 01:26:27,170 And these have conditions that favor pyruvate carboxylase 1263 01:26:27,170 --> 01:26:29,900 reaction, as I talked about, but also favor 1264 01:26:29,900 --> 01:26:32,840 other oxidation reactions, such as those 1265 01:26:32,840 --> 01:26:36,550 that occur in the TCA cycle. 1266 01:26:36,550 --> 01:26:40,050 And so next time, what I will start with 1267 01:26:40,050 --> 01:26:44,490 is by being more explicit about what chemistry is happening 1268 01:26:44,490 --> 01:26:47,700 to allow these two carbon units to combine 1269 01:26:47,700 --> 01:26:52,020 with oxaloacetate 4-carbon to generate citrate and go over 1270 01:26:52,020 --> 01:26:55,020 the reactions that then allow you to run a cycle 1271 01:26:55,020 --> 01:27:02,040 to regenerate oxaloacetate, that is, run the TCA cycle as a way 1272 01:27:02,040 --> 01:27:05,630 to oxidize carbon. 1273 01:27:05,630 --> 01:27:07,180 Thanks.