1 00:00:00,500 --> 00:00:02,840 The following content is provided under a Creative 2 00:00:02,840 --> 00:00:04,380 Commons license. 3 00:00:04,380 --> 00:00:06,680 Your support will help MIT OpenCourseWare 4 00:00:06,680 --> 00:00:11,070 continue to offer high quality educational resources for free. 5 00:00:11,070 --> 00:00:13,670 To make a donation or view additional materials 6 00:00:13,670 --> 00:00:17,630 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,630 --> 00:00:24,840 at ocw.mit.edu 8 00:00:24,840 --> 00:00:26,940 BOGDAN FEDELES: Hi, welcome back. 9 00:00:26,940 --> 00:00:29,220 I am Dr. Bogdan Fedeles. 10 00:00:29,220 --> 00:00:31,660 Let's solve some more biochemistry problems. 11 00:00:31,660 --> 00:00:35,050 Today we're going to be talking about problem 3 of problem set 12 00:00:35,050 --> 00:00:36,000 10. 13 00:00:36,000 --> 00:00:40,300 Now this is a problem about the spontaneity of gluconeogenesis. 14 00:00:40,300 --> 00:00:42,360 As you guys have already learned, 15 00:00:42,360 --> 00:00:46,710 gluconeogenesis is the process by which non-sugar precursors 16 00:00:46,710 --> 00:00:49,650 are used to produce glucose. 17 00:00:49,650 --> 00:00:52,170 This problem should help you think 18 00:00:52,170 --> 00:00:54,750 of the following conundrum-- in a cell, 19 00:00:54,750 --> 00:00:59,490 when we have glucose we can make energy by doing glycolysis. 20 00:00:59,490 --> 00:01:01,080 But at the same time, the cell can 21 00:01:01,080 --> 00:01:04,650 take, for example, the endpoints of glycolysis such as pyruvate, 22 00:01:04,650 --> 00:01:08,190 or even further metabolites like amino acids, 23 00:01:08,190 --> 00:01:11,950 and use the pathway in reverse to make glucose. 24 00:01:11,950 --> 00:01:14,970 Now if one process is spontaneous 25 00:01:14,970 --> 00:01:17,820 in terms of thermodynamics, then how 26 00:01:17,820 --> 00:01:20,685 could we run the reaction in reverse? 27 00:01:20,685 --> 00:01:22,060 So here we're going to be looking 28 00:01:22,060 --> 00:01:25,230 at a couple of steps of gluconeogenesis that 29 00:01:25,230 --> 00:01:27,690 are the exact reverse of the reactions 30 00:01:27,690 --> 00:01:30,510 that you've seen already in glycolysis. 31 00:01:30,510 --> 00:01:32,730 And then in the end, we're going to be talking 32 00:01:32,730 --> 00:01:36,960 about the whole pathway level and make some comments 33 00:01:36,960 --> 00:01:40,770 on the spontaneity of both glycolysis and gluconeogenesis. 34 00:01:44,570 --> 00:01:50,040 Part A of the problem deals with the aldolase reaction, 35 00:01:50,040 --> 00:01:52,790 namely going from fructose 1,6 bisphosphate 36 00:01:52,790 --> 00:01:57,410 to glyceraldehyde 3-phosphate dihydroxyacetone phosphate. 37 00:01:57,410 --> 00:02:00,470 Now in gluconeogenesis, we will be going in reverse, starting 38 00:02:00,470 --> 00:02:03,050 with dihydroxyacetone phosphate and glyceraldehyde 39 00:02:03,050 --> 00:02:07,674 3-phosphate to regenerate fructose 1,6 bisphosphate. 40 00:02:07,674 --> 00:02:09,090 Let's take a look at the reaction. 41 00:02:14,450 --> 00:02:19,090 This is dihydroxyacetone phosphate. 42 00:02:19,090 --> 00:02:22,630 This is glyceraldehyde 3-phosphate or GAP. 43 00:02:22,630 --> 00:02:29,110 And these two come together in the aldolase reaction 44 00:02:29,110 --> 00:02:39,480 to form fructose 1,6 bisphosphate, or F 1,6 BP. 45 00:02:39,480 --> 00:02:41,890 Now the Part 1 of the question asked 46 00:02:41,890 --> 00:02:47,020 us to write down the detailed mechanism for this reaction, 47 00:02:47,020 --> 00:02:48,320 running in reverse. 48 00:02:48,320 --> 00:02:51,400 So as you guys know, aldolase, specifically the Class II 49 00:02:51,400 --> 00:02:55,540 aldolases, use an active site lysine residue 50 00:02:55,540 --> 00:02:59,320 to covalently bind the substrates 51 00:02:59,320 --> 00:03:01,250 during the course of the reaction. 52 00:03:01,250 --> 00:03:04,960 So this reaction will proceed by dihydroxyacetone phosphate 53 00:03:04,960 --> 00:03:09,150 binding to the active site lysine of the aldolase. 54 00:03:09,150 --> 00:03:11,500 Here we have the dihydroxyacetone phosphate 55 00:03:11,500 --> 00:03:13,810 and the lysine in the active site. 56 00:03:13,810 --> 00:03:15,730 So as a first step to the mechanism, 57 00:03:15,730 --> 00:03:18,250 the lysine is going to react as the carbonyl group 58 00:03:18,250 --> 00:03:19,720 in the dihydroxyacetone phosphate 59 00:03:19,720 --> 00:03:23,932 to form a Schiff's base, or an imine. 60 00:03:23,932 --> 00:03:25,390 As you can imagine, the reaction is 61 00:03:25,390 --> 00:03:28,570 going to be catalyzed by a general base, which 62 00:03:28,570 --> 00:03:32,140 is going to remove one of the protons 63 00:03:32,140 --> 00:03:34,810 from the lysine, which then becomes a good nucleophile 64 00:03:34,810 --> 00:03:36,940 and can attack the carbonyl. 65 00:03:36,940 --> 00:03:40,870 Which can then will be protonated by a general acid. 66 00:03:40,870 --> 00:03:47,240 So this way you obtain an intermediate as shown here, 67 00:03:47,240 --> 00:03:51,070 which can then lose the water molecule 68 00:03:51,070 --> 00:03:52,750 to form the Schiff base. 69 00:03:52,750 --> 00:03:58,770 Once again, we need a base that will take this proton 70 00:03:58,770 --> 00:04:05,510 and then the water will need another acid molecule to leave. 71 00:04:05,510 --> 00:04:07,880 So what we have formed here, this 72 00:04:07,880 --> 00:04:13,640 is the protonated Schiff base, or what we call an iminium ion. 73 00:04:13,640 --> 00:04:18,110 And if you look at it from the point of view 74 00:04:18,110 --> 00:04:20,540 of where we started from, which was a carbonyl group, 75 00:04:20,540 --> 00:04:22,775 this is a version of an activated carbonyl. 76 00:04:22,775 --> 00:04:27,620 A carbonyl that would it's now poised to do chemistry. 77 00:04:27,620 --> 00:04:33,490 Once again, this is a iminium ion. 78 00:04:33,490 --> 00:04:35,320 Now as we talked in the carbonyl video, 79 00:04:35,320 --> 00:04:37,890 iminium ions are activated carbonyls 80 00:04:37,890 --> 00:04:41,050 that can now undergo some of the carbonyl reactions 81 00:04:41,050 --> 00:04:42,730 with more ease. 82 00:04:42,730 --> 00:04:45,910 For example, this one is going to do an enolization. 83 00:04:45,910 --> 00:04:49,150 So we have highlighted here the alpha hydrogen 84 00:04:49,150 --> 00:04:51,080 next to the carbonyl group. 85 00:04:51,080 --> 00:04:54,910 So we're going to form the enol by removing 86 00:04:54,910 --> 00:04:58,710 this hydrogen, and the carbonyl with its positive charge 87 00:04:58,710 --> 00:05:02,110 on the nitrogen, so it's at a very good electron sync. 88 00:05:02,110 --> 00:05:06,100 So what we're forming here is an imine 89 00:05:06,100 --> 00:05:08,080 that's bound to a double bond. 90 00:05:08,080 --> 00:05:10,170 So this we're going to call an enamine. 91 00:05:13,174 --> 00:05:17,900 So now this is the reactive version 92 00:05:17,900 --> 00:05:19,430 of the dihydroxyacetone phosphate. 93 00:05:19,430 --> 00:05:22,070 Now it's poised to react with the other partner 94 00:05:22,070 --> 00:05:27,170 in the reaction, GAP- glyceraldehyde 3-phosphate, 95 00:05:27,170 --> 00:05:28,410 which we've shown here. 96 00:05:28,410 --> 00:05:34,620 Now GAP is going to be the carbonyl component of this aldo 97 00:05:34,620 --> 00:05:38,760 reaction, while the enamine from the dihydroxyacetone phosphate 98 00:05:38,760 --> 00:05:43,810 is going to be the enolic component. 99 00:05:43,810 --> 00:05:48,940 So the enol is going to be a very good nucleophile 100 00:05:48,940 --> 00:05:50,650 and is going to attack the carbonyl. 101 00:05:50,650 --> 00:05:55,990 The reaction is once again initiated by this amine group. 102 00:05:55,990 --> 00:06:00,550 The electrons move here and this attacks the carbonyl. 103 00:06:00,550 --> 00:06:05,140 And we can protonate it with a general acid. 104 00:06:05,140 --> 00:06:10,120 So what we obtain here is now a molecule that 105 00:06:10,120 --> 00:06:16,250 has from two portions of three, is going to have six carbons. 106 00:06:16,250 --> 00:06:17,890 And if you look closely, this is really 107 00:06:17,890 --> 00:06:20,590 the fructose 1,6 bisphosphate bound 108 00:06:20,590 --> 00:06:24,160 to the active site of the enzyme as a Schiff space. 109 00:06:24,160 --> 00:06:26,140 So now all that needs to happen now 110 00:06:26,140 --> 00:06:30,490 is to hydrolyze the Schiff space and release our fructose 1,6 111 00:06:30,490 --> 00:06:32,530 bisphosphate product. 112 00:06:32,530 --> 00:06:37,880 So, we're going to need one water molecule. 113 00:06:37,880 --> 00:06:39,820 It's going to be activated by a general base. 114 00:06:39,820 --> 00:06:45,490 Then it is going to attack this imine 115 00:06:45,490 --> 00:06:48,100 and the electrons will move to the nitrogen. 116 00:06:48,100 --> 00:06:56,490 We get to this step and here we need a step in catalysis 117 00:06:56,490 --> 00:07:01,020 to regenerate the enzyme to its freed lysine form 118 00:07:01,020 --> 00:07:04,110 and then release the fructose 1,6 bisphosphate 119 00:07:04,110 --> 00:07:05,920 product of the reaction. 120 00:07:05,920 --> 00:07:09,570 So once again, we started with dihydroxyacetone phosphate 121 00:07:09,570 --> 00:07:12,497 and GAP and you used this covalent catalysis 122 00:07:12,497 --> 00:07:14,580 where the substrates were bound in the active site 123 00:07:14,580 --> 00:07:17,620 of the enzyme to form our product, 124 00:07:17,620 --> 00:07:19,900 fructose 1,6 bisphosphate. 125 00:07:19,900 --> 00:07:22,600 Now this, as we discussed in the carbonyl chemistry, 126 00:07:22,600 --> 00:07:25,310 is an example of direct adol reaction. 127 00:07:25,310 --> 00:07:27,650 And as the name of the enzyme suggests, 128 00:07:27,650 --> 00:07:30,990 aldolase this is the reaction going 129 00:07:30,990 --> 00:07:33,360 in that gluconeogenic mode. 130 00:07:36,850 --> 00:07:38,470 Now in Part B of the question, we're 131 00:07:38,470 --> 00:07:42,070 going to look at another important reaction that 132 00:07:42,070 --> 00:07:44,440 can run both ways-- 133 00:07:44,440 --> 00:07:46,870 glycolysis and gluconeogenesis. 134 00:07:46,870 --> 00:07:49,600 That will be specifically going from glyceraldehyde 135 00:07:49,600 --> 00:07:54,190 3-phosphate, or GAP, to 1,3 bisphosphoglycerate. 136 00:07:54,190 --> 00:07:58,930 This is the reaction catalyzed by GAP dehydrogenase. 137 00:07:58,930 --> 00:08:03,400 Here is a representation of the reaction. 138 00:08:03,400 --> 00:08:05,680 We have glyceraldehyde 3-phosphate 139 00:08:05,680 --> 00:08:11,140 that is converted by GAP DH to the 1,3 bisphosphoglycerate. 140 00:08:11,140 --> 00:08:12,820 Now in the glycolysis step, we know 141 00:08:12,820 --> 00:08:17,320 we need one NAD equivalent, and one inorganic 142 00:08:17,320 --> 00:08:19,600 phosphate to go into the enzyme to be 143 00:08:19,600 --> 00:08:24,390 able to oxidize our aldehyde to an acidic anhydride in 1,3 144 00:08:24,390 --> 00:08:26,140 bisphosphoglycerate. 145 00:08:26,140 --> 00:08:27,550 Now in the gluconeogenesis, we're 146 00:08:27,550 --> 00:08:29,890 going to be going from right to left. 147 00:08:29,890 --> 00:08:32,150 We're starting with 1,3 bisphosphoglycerate We're 148 00:08:32,150 --> 00:08:34,870 going to need NADH and we're going 149 00:08:34,870 --> 00:08:38,260 to generate GAP and inorganic phosphate. 150 00:08:38,260 --> 00:08:41,640 Now let's take a closer look at the mechanism of this reaction 151 00:08:41,640 --> 00:08:44,620 as it would run in gluconeogenesis. 152 00:08:44,620 --> 00:08:48,730 Here we have a representation of the active site of the GAPDH, 153 00:08:48,730 --> 00:08:53,650 where we highlighted the cysteine group, SH. 154 00:08:53,650 --> 00:08:56,740 And we also have a general base in the active site 155 00:08:56,740 --> 00:08:59,729 and we're going to call it B. And here 156 00:08:59,729 --> 00:09:01,770 is our starting material 1,3 bisphosphoglycerate. 157 00:09:04,340 --> 00:09:07,090 Now if you remember how GAPDH works 158 00:09:07,090 --> 00:09:10,720 in the direct reaction in the glycolysis, 159 00:09:10,720 --> 00:09:13,690 we're going to have to form a covalent intermediate, 160 00:09:13,690 --> 00:09:17,400 in which the substrate is going to bind to the enzyme. 161 00:09:17,400 --> 00:09:21,820 That's going to form a thioester with that thiol group 162 00:09:21,820 --> 00:09:23,870 of the cysteine. 163 00:09:23,870 --> 00:09:27,880 So this is what's going to happen here. 164 00:09:27,880 --> 00:09:29,670 So the base in the active site actually 165 00:09:29,670 --> 00:09:33,590 is going to assist the protonation of the cysteine. 166 00:09:33,590 --> 00:09:35,850 Then the thiol now is activated and can 167 00:09:35,850 --> 00:09:41,544 attack the phosphoanhydride of the 1,3 biphosphoglycerate. 168 00:09:41,544 --> 00:09:42,960 And then the first step, it's just 169 00:09:42,960 --> 00:09:46,590 going to form a tetrahedral intermediate. 170 00:09:46,590 --> 00:09:47,500 There you have it. 171 00:09:47,500 --> 00:09:52,140 Now we have a negative charge on the oxygen 172 00:09:52,140 --> 00:09:55,881 and we spelled out the phosphate with all the atoms here. 173 00:09:55,881 --> 00:09:57,630 As you know, this tetrahedral intermediate 174 00:09:57,630 --> 00:10:02,620 is now going to fall apart, releasing the best leaving 175 00:10:02,620 --> 00:10:03,120 group. 176 00:10:03,120 --> 00:10:05,750 In this case it is going to be the inorganic phosphate. 177 00:10:05,750 --> 00:10:07,390 So the electrons come down. 178 00:10:07,390 --> 00:10:11,630 They're going to be transferred onto the phosphate 179 00:10:11,630 --> 00:10:13,230 and presumably protonated. 180 00:10:16,310 --> 00:10:19,250 So after the phosphate is released, 181 00:10:19,250 --> 00:10:24,110 now we have formed finally the thioester of our substrate 182 00:10:24,110 --> 00:10:26,480 in the active site of GAPDH. 183 00:10:26,480 --> 00:10:28,310 As you remember, this is a redox reaction 184 00:10:28,310 --> 00:10:30,770 so it involves the co-factor NAD. 185 00:10:30,770 --> 00:10:33,230 In this case, running the reaction from right to left, 186 00:10:33,230 --> 00:10:38,240 we're going to use NADH to reduce the bisphosphoglycerate 187 00:10:38,240 --> 00:10:41,760 to the aldehyde group. 188 00:10:41,760 --> 00:10:44,940 So here is our representation of the NADH. 189 00:10:44,940 --> 00:10:49,430 As you remember, one of these hydrogens together 190 00:10:49,430 --> 00:10:51,560 with its electron pair, it's is to be donated 191 00:10:51,560 --> 00:10:55,670 as a hydride or H minus. 192 00:10:55,670 --> 00:11:01,640 So the reaction proceeds by rearranging the electrons 193 00:11:01,640 --> 00:11:06,700 on the pyradine ring of NAD and the H minus 194 00:11:06,700 --> 00:11:08,450 is going to be the nucleophile that's 195 00:11:08,450 --> 00:11:10,440 attacking now the thioester. 196 00:11:10,440 --> 00:11:12,490 And once again, I'm going to be forming 197 00:11:12,490 --> 00:11:16,670 a tetrahedral intermediate, which is shown here. 198 00:11:16,670 --> 00:11:22,700 Now this hydride here is the one that came from NADH. 199 00:11:22,700 --> 00:11:30,710 And in the process the NADH co-factor 200 00:11:30,710 --> 00:11:32,580 is converted to NAD-plus. 201 00:11:35,210 --> 00:11:38,180 So now we can see we're just one step away. 202 00:11:38,180 --> 00:11:41,290 This is now like a hemiacetal like molecule. 203 00:11:41,290 --> 00:11:43,470 So it's one step away from forming 204 00:11:43,470 --> 00:11:45,030 glyceraldehyde 3-phosphate. 205 00:11:45,030 --> 00:11:47,930 All we need to do is release the enzyme 206 00:11:47,930 --> 00:11:50,270 in its original confirmation. 207 00:11:50,270 --> 00:11:55,340 So the electrons will flow now to reform the carbonyl, 208 00:11:55,340 --> 00:11:58,310 while the sulfur is going to get its electrons 209 00:11:58,310 --> 00:12:03,950 from the base and this regenerates the enzyme 210 00:12:03,950 --> 00:12:05,630 as we had it in the beginning. 211 00:12:05,630 --> 00:12:09,540 And we're releasing GAP, the product of the reaction. 212 00:12:09,540 --> 00:12:11,810 So with this mechanistic insight, 213 00:12:11,810 --> 00:12:14,660 we have actually addressed how the GAPDH reaction 214 00:12:14,660 --> 00:12:18,170 runs in the gluconeogenic mode from bisphosphoglycerate 215 00:12:18,170 --> 00:12:19,310 to GAP. 216 00:12:19,310 --> 00:12:21,590 Now Part A of the problem also asked 217 00:12:21,590 --> 00:12:24,020 us to look up the free energy value 218 00:12:24,020 --> 00:12:28,820 for the aldolase reaction that is that delta G naught prime. 219 00:12:28,820 --> 00:12:31,340 Now, if you're going to look in your favorite chemistry 220 00:12:31,340 --> 00:12:36,590 textbook, or I have here the Voet and Voet Third Edition, 221 00:12:36,590 --> 00:12:39,930 the book that we use in this course. 222 00:12:39,930 --> 00:12:43,740 Now you're going to find on page 511, 223 00:12:43,740 --> 00:12:46,310 you're going to see a table with all the free energies 224 00:12:46,310 --> 00:12:48,010 of glycolysis reactions. 225 00:12:48,010 --> 00:12:49,430 And for the aldolase step, you're 226 00:12:49,430 --> 00:12:52,100 going to see that it's a positive free energy, that 227 00:12:52,100 --> 00:12:54,960 is the reaction is spontaneous in the reverse 228 00:12:54,960 --> 00:12:57,590 in the gluconeogenic direction. 229 00:12:57,590 --> 00:13:00,800 So now that may be a little surprising, but keep in mind 230 00:13:00,800 --> 00:13:03,830 that the reactions in these pathways 231 00:13:03,830 --> 00:13:05,930 are more often than not, governed 232 00:13:05,930 --> 00:13:09,680 by mass action, that is if we have an excess of the starting 233 00:13:09,680 --> 00:13:12,740 materials, the reaction would proceed towards the product, 234 00:13:12,740 --> 00:13:14,570 while if we have an excess of the products, 235 00:13:14,570 --> 00:13:16,340 the reaction will proceed backward 236 00:13:16,340 --> 00:13:18,120 towards the starting materials. 237 00:13:18,120 --> 00:13:21,480 So both the aldolase reaction and the GAP 238 00:13:21,480 --> 00:13:23,990 dehydrogenase reactions are very susceptible 239 00:13:23,990 --> 00:13:25,470 to this mass action. 240 00:13:25,470 --> 00:13:28,082 So they will be controlled- the direction, the spontaneity 241 00:13:28,082 --> 00:13:29,540 of this reaction will be controlled 242 00:13:29,540 --> 00:13:31,850 by which of the starting materials or products 243 00:13:31,850 --> 00:13:32,440 are in excess. 244 00:13:36,190 --> 00:13:39,280 Part C of this problem asked us to evaluate 245 00:13:39,280 --> 00:13:41,440 the spontaneity of gluconeogenesis 246 00:13:41,440 --> 00:13:43,600 at the level of the whole pathway. 247 00:13:43,600 --> 00:13:47,140 Now we know glycolysis is a spontaneous process. 248 00:13:47,140 --> 00:13:52,570 Not only when it goes from glucose to pyruvate, 249 00:13:52,570 --> 00:13:55,530 but also it generates some high energy 250 00:13:55,530 --> 00:13:58,030 intermediates like ATP in the process-- 251 00:13:58,030 --> 00:14:00,920 like we get two molecules of ATP per glucose. 252 00:14:00,920 --> 00:14:02,950 Now if we want to go backwards from pyruvate 253 00:14:02,950 --> 00:14:05,920 to glucose, in a gluconeogenesis pathway, 254 00:14:05,920 --> 00:14:09,070 can this process be spontaneous? 255 00:14:09,070 --> 00:14:12,610 First let's look at glycolysis. 256 00:14:12,610 --> 00:14:15,160 Here is a schematic of a glycolysis. 257 00:14:15,160 --> 00:14:17,470 We're starting here from glucose and we're 258 00:14:17,470 --> 00:14:20,350 going to need a couple of molecules of ATP 259 00:14:20,350 --> 00:14:23,620 to activate it, to get to fructose 1,6 bisphosphate. 260 00:14:23,620 --> 00:14:26,530 And from there on, we're going to generate 261 00:14:26,530 --> 00:14:30,040 actually, two molecules of ATP at this step 262 00:14:30,040 --> 00:14:34,630 and then two molecules of ATP in the final pyruvate kinase step. 263 00:14:34,630 --> 00:14:44,320 And of course, we're also going to need an NAD- plus going 264 00:14:44,320 --> 00:14:46,860 to NADH here at the GAP step. 265 00:14:46,860 --> 00:14:50,820 Well, there's going to be two of these NAD-plus needed 266 00:14:50,820 --> 00:14:54,330 to oxidize GAP to 1,3 bisphosphoglycerate. 267 00:14:54,330 --> 00:14:59,250 And as we just said, even though we have some energy cost 268 00:14:59,250 --> 00:15:02,610 in early on, we're actually generating more ATP 269 00:15:02,610 --> 00:15:05,370 by the time we reach pyruvate so the pathway is 270 00:15:05,370 --> 00:15:07,260 in fact spontaneous. 271 00:15:07,260 --> 00:15:09,920 Now what about gluconeogenesis. 272 00:15:09,920 --> 00:15:12,810 In gluconeogenesis, we are starting with pyruvate 273 00:15:12,810 --> 00:15:15,760 and we want to go back to glucose. 274 00:15:15,760 --> 00:15:18,800 Now if we were to reverse exactly every single step 275 00:15:18,800 --> 00:15:22,620 in glycolysis, that pathway is not going to be spontaneous. 276 00:15:22,620 --> 00:15:27,450 Step four in gluconeogenesis uses these alternate pathways 277 00:15:27,450 --> 00:15:29,310 in a couple of the steps in order 278 00:15:29,310 --> 00:15:31,560 to make the process spontaneous. 279 00:15:31,560 --> 00:15:34,890 For example, going from pyruvate to phosphoenolpyruvate 280 00:15:34,890 --> 00:15:38,940 in gluconeogenesis is not a reverse of the pyruvate kinase 281 00:15:38,940 --> 00:15:39,690 reaction. 282 00:15:39,690 --> 00:15:41,145 It rather occurs in two steps. 283 00:15:44,189 --> 00:15:46,230 So the first step we take pyruvate and convert it 284 00:15:46,230 --> 00:15:49,410 to oxaloacetate, using pyruvate carboxylase, 285 00:15:49,410 --> 00:15:52,660 so it's going to need a molecule of CO2. 286 00:15:52,660 --> 00:15:54,630 And oxaloacetate has four carbons. 287 00:15:54,630 --> 00:15:58,120 And it's also going to need energy. 288 00:15:58,120 --> 00:16:03,090 So we're going to need a molecule of ATP going to ADP. 289 00:16:03,090 --> 00:16:09,780 Now oxaloacetate can now be processed 290 00:16:09,780 --> 00:16:11,610 inside the mitochondria or it can 291 00:16:11,610 --> 00:16:15,180 be taken out of the mitochondria into the cytosol. 292 00:16:15,180 --> 00:16:18,720 And let's say that's the course of the reaction. 293 00:16:18,720 --> 00:16:23,010 Where it's going to find an enzyme called 294 00:16:23,010 --> 00:16:26,730 PEP carboxylic kinase, or PEPCK, that 295 00:16:26,730 --> 00:16:29,670 can take oxaloacetate to PEP. 296 00:16:29,670 --> 00:16:33,330 This enzyme once again requires energy. 297 00:16:33,330 --> 00:16:36,570 This time in the form of GTP going to GDP. 298 00:16:36,570 --> 00:16:39,120 And here we're going to lose that carboxyl group 299 00:16:39,120 --> 00:16:42,720 that we added on earlier. 300 00:16:42,720 --> 00:16:47,910 Now while this might seem like a cumbersome way 301 00:16:47,910 --> 00:16:53,140 to reverse one reaction, this allows both the part 302 00:16:53,140 --> 00:16:56,730 of a kinase reaction and going from pyruvate going back 303 00:16:56,730 --> 00:17:00,870 to PEP, to be controlled in different ways 304 00:17:00,870 --> 00:17:03,300 and therefore allow both of these processes 305 00:17:03,300 --> 00:17:06,510 to happen spontaneously. 306 00:17:06,510 --> 00:17:12,990 The rest of gluconeogenesis will have exactly the reverse 307 00:17:12,990 --> 00:17:15,420 of these steps in glycolsis. 308 00:17:15,420 --> 00:17:18,190 For example, PEP going to 2-phosphoglycerate. 309 00:17:18,190 --> 00:17:21,501 2-Phosphoglycerate going to 3-phosphoglycerate. 310 00:17:21,501 --> 00:17:24,510 3-phosphoglycerate going to 1,3 bisphosphoglycerate. 311 00:17:24,510 --> 00:17:28,079 Here, since in glycolysis we generated ATP here, 312 00:17:28,079 --> 00:17:33,540 we're going to need the ATP to come in and go 313 00:17:33,540 --> 00:17:38,630 to ADP in order to accomplish this step in gluconeogenesis. 314 00:17:38,630 --> 00:17:41,154 1,3 bisphosphoglycerate going to GAP, 315 00:17:41,154 --> 00:17:42,570 this is the step we just discussed 316 00:17:42,570 --> 00:17:45,630 in Part 2 of the problem. 317 00:17:45,630 --> 00:17:51,150 As we said, we're going to need NADH going to NAD-plus. 318 00:17:51,150 --> 00:17:55,410 Then GAP and DHAP going to fructose 1,6 bisphosphate, this 319 00:17:55,410 --> 00:17:59,070 is the step we discussed in Part A of this problem, the reverse 320 00:17:59,070 --> 00:18:00,990 of the aldolase reaction. 321 00:18:00,990 --> 00:18:05,040 Now going from fructose 1,6 bisphosphate to glucose, 322 00:18:05,040 --> 00:18:08,400 we're not going to do these kinase reactions in reverse, 323 00:18:08,400 --> 00:18:11,220 where we would be generating ATP and therefore 324 00:18:11,220 --> 00:18:14,430 that would be not spontaneous in the reverse direction. 325 00:18:14,430 --> 00:18:17,700 But rather, we're going to use alternate enzymes called 326 00:18:17,700 --> 00:18:21,540 phosphatases where we lose the phosphates without regenerating 327 00:18:21,540 --> 00:18:22,800 an ATP molecules. 328 00:18:25,330 --> 00:18:28,800 So therefore, by using these two tricks 329 00:18:28,800 --> 00:18:32,460 we can go back to glucose without regenerating these ATP 330 00:18:32,460 --> 00:18:36,910 molecules, and therefore the pathway can be spontaneous. 331 00:18:36,910 --> 00:18:40,080 Now the bottom line here is that both glycolysis 332 00:18:40,080 --> 00:18:42,960 and gluconeogenesis are spontaneous, 333 00:18:42,960 --> 00:18:46,590 but gluconeogenesis uses most but not all of the glycolysis 334 00:18:46,590 --> 00:18:48,930 steps to run in reverse. 335 00:18:48,930 --> 00:18:51,960 And while glycolysis generates energy, 336 00:18:51,960 --> 00:18:55,590 we get a net of 2 molecules of ATP per molecule of glucose 337 00:18:55,590 --> 00:18:59,670 used, gluconeogenesis as you guys have seen here, 338 00:18:59,670 --> 00:19:04,320 actually uses energy to run spontaneously. 339 00:19:04,320 --> 00:19:07,430 That is, we need ATP molecules. 340 00:19:07,430 --> 00:19:09,120 Now if you look at our diagram, we 341 00:19:09,120 --> 00:19:12,060 need ATP molecules to convert pyruvate 342 00:19:12,060 --> 00:19:15,080 to phosphoenolpyruvate, actually 2 of them. 343 00:19:15,080 --> 00:19:16,830 And then we're going to need more ATP here 344 00:19:16,830 --> 00:19:19,020 to form 1,3 bisphosphoglycerate. 345 00:19:19,020 --> 00:19:23,210 In addition to the redox, like NADH 346 00:19:23,210 --> 00:19:26,200 co-factor, to run the GAP dehydrogenase 347 00:19:26,200 --> 00:19:28,560 reaction in reverse. 348 00:19:28,560 --> 00:19:32,100 So given these considerations, gluconeogenesis 349 00:19:32,100 --> 00:19:35,070 is in fact spontaneous, but it's going to cost us 350 00:19:35,070 --> 00:19:36,890 several ATP equivalents. 351 00:19:36,890 --> 00:19:41,190 While glycolysis is spontaneous and generates ATP. 352 00:19:41,190 --> 00:19:45,290 Well, that solves problem 3 of problem set 10. 353 00:19:45,290 --> 00:19:48,180 Here, I hope you got a better understanding of why 354 00:19:48,180 --> 00:19:50,790 both glycolysis and gluconeogenesis are 355 00:19:50,790 --> 00:19:54,090 both spontaneous pathways inside the cell. 356 00:19:54,090 --> 00:19:55,830 Thank you.