1 00:00:00,090 --> 00:00:02,490 The following content is provided under a Creative 2 00:00:02,490 --> 00:00:04,030 Commons license. 3 00:00:04,030 --> 00:00:06,330 Your support will help MIT OpenCourseWare 4 00:00:06,330 --> 00:00:10,720 continue to offer high-quality educational resources for free. 5 00:00:10,720 --> 00:00:13,320 To make a donation or view additional materials 6 00:00:13,320 --> 00:00:17,280 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,280 --> 00:00:18,450 at ocw.mit.edu. 8 00:00:21,000 --> 00:00:24,660 JOHN ESSIGMANN: Let's go now to storyboard 36, 9 00:00:24,660 --> 00:00:28,590 starting with panel A, Regulation of Metabolism. 10 00:00:28,590 --> 00:00:32,490 So far in 5.07, we have looked at metabolic pathways 11 00:00:32,490 --> 00:00:36,060 from the perspective of the cell in organelles within the cell. 12 00:00:36,060 --> 00:00:38,010 Using physiological scenarios, I've 13 00:00:38,010 --> 00:00:40,530 tried to show you how pathways within the cell 14 00:00:40,530 --> 00:00:43,470 respond to a change in the environment, for example, 15 00:00:43,470 --> 00:00:45,870 by running away from a stressor, such as a dog 16 00:00:45,870 --> 00:00:50,100 or dealing with the problems of starvation or diabetes. 17 00:00:50,100 --> 00:00:51,960 We have looked a little bit at the ways 18 00:00:51,960 --> 00:00:55,310 that individual steps and pathways are regulated. 19 00:00:55,310 --> 00:00:57,660 We have not, however, looked at the ways 20 00:00:57,660 --> 00:01:00,660 that individual pathways and individual organs 21 00:01:00,660 --> 00:01:03,300 coordinate their respective activities in order 22 00:01:03,300 --> 00:01:06,106 to accommodate the needs of the entire organism. 23 00:01:06,106 --> 00:01:11,110 Coordinated pathway networking is the focus of this lecture. 24 00:01:11,110 --> 00:01:13,780 Let's turn to panel B. I'm going to start out 25 00:01:13,780 --> 00:01:16,480 by talking a little bit about the seven pathways 26 00:01:16,480 --> 00:01:19,000 that we have studied in detail in 5.07 27 00:01:19,000 --> 00:01:20,710 and look at how they're regulated. 28 00:01:20,710 --> 00:01:22,990 As I've said before, typically pathways 29 00:01:22,990 --> 00:01:25,960 are regulated at their rate-determining steps, that 30 00:01:25,960 --> 00:01:28,780 is, the step at which you'll find an enzyme that 31 00:01:28,780 --> 00:01:31,390 has a large free-energy change associated 32 00:01:31,390 --> 00:01:35,100 with the conversion of substrates to products. 33 00:01:35,100 --> 00:01:37,860 In the first pathway we studied, glycolysis, 34 00:01:37,860 --> 00:01:40,680 there are three irreversible steps, specifically 35 00:01:40,680 --> 00:01:43,920 the glucokinase/hexokinase step; secondly, 36 00:01:43,920 --> 00:01:48,450 the phosphofructokinase-1 step; and thirdly, the last step, 37 00:01:48,450 --> 00:01:49,650 pyruvate kinase. 38 00:01:49,650 --> 00:01:52,980 These are all sites of pathway regulation. 39 00:01:52,980 --> 00:01:56,100 Our second pathway, the tricarboxylic acid cycle, 40 00:01:56,100 --> 00:02:00,550 or Krebs cycle, was regulated at every step at which NADH 41 00:02:00,550 --> 00:02:03,990 is produced, that is, citrate dehydrogenase, 42 00:02:03,990 --> 00:02:05,970 alpha-ketoglutarate dehydrogenase, 43 00:02:05,970 --> 00:02:08,160 and malate dehydrogenase. 44 00:02:08,160 --> 00:02:11,310 While pyruvate dehydrogenase is not formally 45 00:02:11,310 --> 00:02:13,890 part of the TCA cycle, I'll point out here 46 00:02:13,890 --> 00:02:17,430 that it is also regulated by NADH. 47 00:02:17,430 --> 00:02:21,060 In all four cases, NADH feedback inhibits 48 00:02:21,060 --> 00:02:22,860 the enzymes that produce it. 49 00:02:22,860 --> 00:02:25,980 Under conditions of excessive TCA cycle activity, 50 00:02:25,980 --> 00:02:28,550 you'll find that NADH levels will drop, 51 00:02:28,550 --> 00:02:31,770 and accordingly NAD+ levels will increase. 52 00:02:31,770 --> 00:02:34,200 The reduction in NADH will result 53 00:02:34,200 --> 00:02:36,240 in activation of the pathway. 54 00:02:36,240 --> 00:02:38,880 In other words, the disappearance of NADH 55 00:02:38,880 --> 00:02:43,040 results in the uninhibition of the pathway. 56 00:02:43,040 --> 00:02:45,240 Our third pathway, gluconeogenesis, 57 00:02:45,240 --> 00:02:48,360 is regulated at the pyruvate carboxylase step 58 00:02:48,360 --> 00:02:51,240 at the fructose 1,6-bisphosphatase step 59 00:02:51,240 --> 00:02:54,510 and at the glycogen synthase/glycogen phosphorylase 60 00:02:54,510 --> 00:02:55,620 steps. 61 00:02:55,620 --> 00:02:58,350 We're going to be looking at this pathway in some detail 62 00:02:58,350 --> 00:03:00,260 in a little while. 63 00:03:00,260 --> 00:03:03,410 Our fourth pathway, fatty acid catabolism, 64 00:03:03,410 --> 00:03:07,460 is regulated at the CAT, or Carnitine Acyltransferase step, 65 00:03:07,460 --> 00:03:11,900 which is inhibited by malonyl coenzyme A. Malonyl CoA 66 00:03:11,900 --> 00:03:15,890 is one of the key precursors for fatty acid biosynthesis. 67 00:03:15,890 --> 00:03:17,930 It makes sense that the concentration 68 00:03:17,930 --> 00:03:20,120 of malonyl coenzyme A, if high, would 69 00:03:20,120 --> 00:03:21,980 inhibit the uptake of fatty acids 70 00:03:21,980 --> 00:03:23,720 into the mitochondrial matrix. 71 00:03:23,720 --> 00:03:28,220 Keep in mind that uptake into the matrix is the job of CAT1. 72 00:03:28,220 --> 00:03:32,510 Shutting down CAT1 by the high concentration of malonyl CoA 73 00:03:32,510 --> 00:03:36,050 prevents entry of fatty acids into the mitochondrial matrix, 74 00:03:36,050 --> 00:03:39,960 where they otherwise would be subjected to beta-oxidation. 75 00:03:39,960 --> 00:03:43,020 By turning off CAT1, malonyl coenzyme A 76 00:03:43,020 --> 00:03:46,470 prevents the futile cycle of simultaneous fatty acid 77 00:03:46,470 --> 00:03:49,380 degradation and biosynthesis. 78 00:03:49,380 --> 00:03:52,620 The fifth pathway is fatty acid biosynthesis. 79 00:03:52,620 --> 00:03:56,550 Acetyl-CoA Carboxylase, or ACC, is the enzyme 80 00:03:56,550 --> 00:04:00,330 that makes malonyl coenzyme A out of its precursor, 81 00:04:00,330 --> 00:04:05,040 acetyl coenzyme A. This enzyme, ACC, is activated by insulin. 82 00:04:05,040 --> 00:04:07,950 As we've seen before, insulin detects the fed state 83 00:04:07,950 --> 00:04:09,180 in the organism. 84 00:04:09,180 --> 00:04:12,390 Hence, following a meal, insulin levels rise, 85 00:04:12,390 --> 00:04:14,640 and that's the signal that tells the cells of the body 86 00:04:14,640 --> 00:04:16,970 to take up nutrients. 87 00:04:16,970 --> 00:04:18,329 That's just one example. 88 00:04:18,329 --> 00:04:21,390 After a meal, glucose levels will rise in the blood. 89 00:04:21,390 --> 00:04:23,900 Insulin will be released and drive the glucose 90 00:04:23,900 --> 00:04:25,380 into the cell. 91 00:04:25,380 --> 00:04:27,260 The cell will then activate pathways 92 00:04:27,260 --> 00:04:30,590 by which the glucose is converted to acetyl-CoA. 93 00:04:30,590 --> 00:04:34,250 Then the acetyl-CoA will be converted into fatty acids, 94 00:04:34,250 --> 00:04:36,680 and then ultimately, into triacylglycerides 95 00:04:36,680 --> 00:04:39,040 for energy storage. 96 00:04:39,040 --> 00:04:43,600 Our sixth regulated pathway is the pentose phosphate pathway. 97 00:04:43,600 --> 00:04:45,730 Glucose 6-phosphate dehydrogenase 98 00:04:45,730 --> 00:04:48,070 is the first step in the pathway. 99 00:04:48,070 --> 00:04:50,950 And as we often see, an early or committed step 100 00:04:50,950 --> 00:04:53,170 is where regulation happens. 101 00:04:53,170 --> 00:04:55,150 One of the products of the pentose phosphate 102 00:04:55,150 --> 00:04:58,090 pathway is NADPH. 103 00:04:58,090 --> 00:05:00,840 If the cell has used a lot of NADPH, 104 00:05:00,840 --> 00:05:04,260 for example for biosynthesis, its levels will drop. 105 00:05:04,260 --> 00:05:07,900 Coordinately, there will be an increase in NADP+. 106 00:05:07,900 --> 00:05:10,540 In the case of glucose 6-phosphate dehydrogenase, 107 00:05:10,540 --> 00:05:15,070 we find that NADP+, which is the product of excessive reductive 108 00:05:15,070 --> 00:05:17,680 biosynthesis, as well as other activities, 109 00:05:17,680 --> 00:05:20,320 such as combating oxidative stress, 110 00:05:20,320 --> 00:05:23,710 activates the dehydrogenase in order to enable the synthesis 111 00:05:23,710 --> 00:05:28,540 of additional NADPH in order to sustain biosynthesis. 112 00:05:28,540 --> 00:05:31,390 The last pathway, the seventh, is electron transport 113 00:05:31,390 --> 00:05:33,400 and oxidative phosphorylation. 114 00:05:33,400 --> 00:05:37,420 This is our most robust pathway for the production of ATP. 115 00:05:37,420 --> 00:05:39,920 When there's a lot of demand for ATP, 116 00:05:39,920 --> 00:05:43,160 ADP levels will rise in the cell. 117 00:05:43,160 --> 00:05:45,640 And it's ultimately the level of ADP 118 00:05:45,640 --> 00:05:47,440 that regulates electron transport 119 00:05:47,440 --> 00:05:49,330 in oxidative phosphorylation. 120 00:05:49,330 --> 00:05:52,680 More about that in a few minutes. 121 00:05:52,680 --> 00:05:55,670 Let's now look at panel C. Organs communicate 122 00:05:55,670 --> 00:05:58,220 with one another by the nervous system, of course, 123 00:05:58,220 --> 00:06:01,190 but also by hormones, small molecules that 124 00:06:01,190 --> 00:06:03,320 are secreted by one organ that will 125 00:06:03,320 --> 00:06:05,480 have an effect at one or more organs 126 00:06:05,480 --> 00:06:07,640 distal to the first organ. 127 00:06:07,640 --> 00:06:09,710 Changes in our environment are detected 128 00:06:09,710 --> 00:06:13,460 by the brain with input from our sensory organs. 129 00:06:13,460 --> 00:06:17,660 Our internal organs, such as the liver, pancreas, kidneys, 130 00:06:17,660 --> 00:06:20,330 adrenal glands, and muscles will detect signals, 131 00:06:20,330 --> 00:06:23,870 either independently of the brain or after an instruction 132 00:06:23,870 --> 00:06:26,150 set is received from the brain. 133 00:06:26,150 --> 00:06:29,510 The resulting signal network will allow the entire organism 134 00:06:29,510 --> 00:06:32,150 to be able to adapt to the new environment, 135 00:06:32,150 --> 00:06:35,510 be it one of stress, for example, being chased by a dog, 136 00:06:35,510 --> 00:06:38,430 or one of, for example, hunger. 137 00:06:38,430 --> 00:06:40,470 We'll talk later about the adrenal glands, which 138 00:06:40,470 --> 00:06:42,480 are going to respond to signals that 139 00:06:42,480 --> 00:06:45,330 come in from sensory organs that tell our muscles to start 140 00:06:45,330 --> 00:06:47,880 running and to tell the liver to start to provide 141 00:06:47,880 --> 00:06:50,190 the muscles with the glucose they need in order 142 00:06:50,190 --> 00:06:52,630 to sustain running. 143 00:06:52,630 --> 00:06:54,550 Later we'll be looking in some detail 144 00:06:54,550 --> 00:06:57,760 at the adrenal-produced hormone epinephrine, also called 145 00:06:57,760 --> 00:06:58,900 adrenaline. 146 00:06:58,900 --> 00:07:02,350 Adrenaline will have a profound impact, both in the muscles 147 00:07:02,350 --> 00:07:04,420 and in the liver, to allow these organs 148 00:07:04,420 --> 00:07:06,880 to do their respective jobs. 149 00:07:06,880 --> 00:07:09,150 The pancreas is a very important organ 150 00:07:09,150 --> 00:07:11,650 in that it provides exocrine functions that 151 00:07:11,650 --> 00:07:14,320 help with digestion and endocrine 152 00:07:14,320 --> 00:07:17,020 functions that enable us to regulate or balance 153 00:07:17,020 --> 00:07:19,070 fuel metabolism. 154 00:07:19,070 --> 00:07:22,170 The alpha cells of the pancreas produce glucagon. 155 00:07:22,170 --> 00:07:24,200 The alpha cells sense hunger. 156 00:07:24,200 --> 00:07:26,580 They secrete glucagon into the blood, 157 00:07:26,580 --> 00:07:30,530 which travels to organs that represent our fuel depots. 158 00:07:30,530 --> 00:07:33,170 And fuel from those depots, for example, fatty acids 159 00:07:33,170 --> 00:07:35,000 and glucose, will then be supplied 160 00:07:35,000 --> 00:07:39,230 to other tissues of the body that are in need of nutrition. 161 00:07:39,230 --> 00:07:41,420 The beta cells of the pancreas sense 162 00:07:41,420 --> 00:07:45,020 what we call the fed state, and they produce insulin. 163 00:07:45,020 --> 00:07:47,390 Insulin instructs the various organs 164 00:07:47,390 --> 00:07:49,670 of the body to take up fuel in the wake of us 165 00:07:49,670 --> 00:07:52,060 having eaten a meal. 166 00:07:52,060 --> 00:07:53,920 Now let's look at panel D. There are 167 00:07:53,920 --> 00:07:58,150 three general paradigms by which pathways are regulated. 168 00:07:58,150 --> 00:08:00,760 The three hormones that I've just mentioned typically 169 00:08:00,760 --> 00:08:04,120 will cause the activation of a kinase that will phosphorylate 170 00:08:04,120 --> 00:08:07,240 a target protein, resulting in either increased or decreased 171 00:08:07,240 --> 00:08:09,130 activity of that protein. 172 00:08:09,130 --> 00:08:12,760 We call this hormonal or, more properly, covalent control, 173 00:08:12,760 --> 00:08:16,060 because there will be a covalent modification of a protein that 174 00:08:16,060 --> 00:08:19,240 will be responsible for pathway regulation. 175 00:08:19,240 --> 00:08:22,630 The second major paradigm by which pathways are controlled 176 00:08:22,630 --> 00:08:24,760 is allosteric regulation. 177 00:08:24,760 --> 00:08:27,190 Earlier in 5.07, JoAnne Stubbe showed us 178 00:08:27,190 --> 00:08:29,110 how hemoglobin, the molecule that 179 00:08:29,110 --> 00:08:31,480 carries oxygen in the blood, is controlled 180 00:08:31,480 --> 00:08:34,090 by bisphosphoglycerate and protons, which 181 00:08:34,090 --> 00:08:36,400 act as allosteric effectors. 182 00:08:36,400 --> 00:08:38,919 Similarly, other small molecule effectors 183 00:08:38,919 --> 00:08:42,320 will control the major pathways of metabolism. 184 00:08:42,320 --> 00:08:44,620 We're going to be looking at phosphofructokinase-1 185 00:08:44,620 --> 00:08:46,810 as our prime example. 186 00:08:46,810 --> 00:08:48,580 The third strategy of regulation is 187 00:08:48,580 --> 00:08:50,770 what I call "acceptor control." 188 00:08:50,770 --> 00:08:53,470 This is the way that electron transport and oxidative 189 00:08:53,470 --> 00:08:55,480 phosphorylation are regulated. 190 00:08:55,480 --> 00:08:58,870 To the right side of panel D is an abbreviated metabolic 191 00:08:58,870 --> 00:09:02,020 pathway that I'm going to use to describe each of these three 192 00:09:02,020 --> 00:09:02,950 paradigms. 193 00:09:02,950 --> 00:09:05,380 At the upper left is a box that contains 194 00:09:05,380 --> 00:09:08,440 glycogen synthase and glycogen phosphorylase. 195 00:09:08,440 --> 00:09:11,590 Earlier in 5.07, I told you how phosphorylation 196 00:09:11,590 --> 00:09:14,080 of a specific C-ring residue on glycogen 197 00:09:14,080 --> 00:09:17,140 phosphorylase results in a dramatic increase 198 00:09:17,140 --> 00:09:19,390 in the activity of that enzyme. 199 00:09:19,390 --> 00:09:22,360 A little later, we'll see that covalent modification, again, 200 00:09:22,360 --> 00:09:26,170 by serine phosphorylation of glycogen synthase, results 201 00:09:26,170 --> 00:09:29,260 in a decrease in activity of that enzyme. 202 00:09:29,260 --> 00:09:32,450 Hence, covalent modification of these two proteins 203 00:09:32,450 --> 00:09:35,130 enables, in one case, activation of the enzyme 204 00:09:35,130 --> 00:09:38,080 and, in the other case, inhibition of the enzyme. 205 00:09:38,080 --> 00:09:42,750 This reciprocal control prevents futile cycling. 206 00:09:42,750 --> 00:09:45,030 Now let's look at the box in the center 207 00:09:45,030 --> 00:09:47,430 of this abbreviated metabolic pathway. 208 00:09:47,430 --> 00:09:51,210 The glycolytic enzyme, PFK-1, Phosphofructokinase-1, 209 00:09:51,210 --> 00:09:56,040 converts fructose 6-phosphate to fructose 1,6-bisphosphate. 210 00:09:56,040 --> 00:09:59,370 We're going to see that a small molecule derivative of fructose 211 00:09:59,370 --> 00:10:04,320 6-phosphate, specifically fructose 2,6-bisphosphate, 212 00:10:04,320 --> 00:10:09,400 will act as a powerful allosteric stimulator of PFK-1. 213 00:10:09,400 --> 00:10:12,480 I'll also point out that AMP, Adenosine Monophosphate, 214 00:10:12,480 --> 00:10:14,660 will also stimulate this enzyme. 215 00:10:14,660 --> 00:10:18,540 I'll also point out here that PFK-1 is a tetramer. 216 00:10:18,540 --> 00:10:21,870 Oftentimes, as JoAnne taught us, proteins that are multimers 217 00:10:21,870 --> 00:10:26,010 are the ones that are subject to allosteric regulation. 218 00:10:26,010 --> 00:10:29,220 Our third paradigm of regulation is accepter control, 219 00:10:29,220 --> 00:10:32,160 shown in the box at the bottom of panel D. 220 00:10:32,160 --> 00:10:35,070 During periods of intense physical activity, 221 00:10:35,070 --> 00:10:38,190 ATP is consumed and converted to ADP, 222 00:10:38,190 --> 00:10:41,670 and NADH is converted to NAD+. 223 00:10:41,670 --> 00:10:44,790 Rising levels of ADP in the mitochondrial matrix 224 00:10:44,790 --> 00:10:47,770 activate the proton-translocating ATP 225 00:10:47,770 --> 00:10:49,380 synathase. 226 00:10:49,380 --> 00:10:54,000 As more protons flow through the synthase, more ATP is made. 227 00:10:54,000 --> 00:10:57,750 They call this accepter control, because the regulatory molecule 228 00:10:57,750 --> 00:11:00,330 is ADP, which is the, quote unquote, "acceptor 229 00:11:00,330 --> 00:11:03,630 of phosphate" in the synthesis of ATP. 230 00:11:03,630 --> 00:11:05,670 Because we covered accepter control 231 00:11:05,670 --> 00:11:08,490 in some detail in the lecture on electron transport 232 00:11:08,490 --> 00:11:10,530 and oxidative phosphorylation, I'm 233 00:11:10,530 --> 00:11:12,900 going to leave that topic for now 234 00:11:12,900 --> 00:11:17,410 and focus on covalent control and allosteric control. 235 00:11:17,410 --> 00:11:20,500 Let's turn now to storyboard 37, starting 236 00:11:20,500 --> 00:11:25,390 with panel A. With regard to the paradigm of covalent control, 237 00:11:25,390 --> 00:11:28,120 I'm going to focus on the pair of enzymes, glycogen 238 00:11:28,120 --> 00:11:30,010 synthase and glycogen phosphorylase, 239 00:11:30,010 --> 00:11:32,980 which reciprocally regulate glycogen synthesis 240 00:11:32,980 --> 00:11:35,050 and glycogenolysis. 241 00:11:35,050 --> 00:11:36,700 Let's consider a scenario in which 242 00:11:36,700 --> 00:11:38,380 there's some kind of stressor. 243 00:11:38,380 --> 00:11:40,540 The muscles have to be activated in order 244 00:11:40,540 --> 00:11:42,450 to be able to deal with the stress, 245 00:11:42,450 --> 00:11:45,340 and the liver has to be able to provide the resources 246 00:11:45,340 --> 00:11:48,220 to the muscles that will allow the muscles to continue 247 00:11:48,220 --> 00:11:50,880 intense physical activity. 248 00:11:50,880 --> 00:11:53,470 Now let's look at panel B. In our scenario, 249 00:11:53,470 --> 00:11:55,270 you've just seen something frightening. 250 00:11:55,270 --> 00:11:57,520 Your brain then sends an electrical signal 251 00:11:57,520 --> 00:11:59,680 to the adrenal glands, which then 252 00:11:59,680 --> 00:12:02,980 send a chemical signal, specifically adrenaline, 253 00:12:02,980 --> 00:12:05,020 to the liver and to the muscles. 254 00:12:05,020 --> 00:12:07,570 At the top part of this response scenario, 255 00:12:07,570 --> 00:12:09,610 both the liver and muscles are going 256 00:12:09,610 --> 00:12:12,610 to be responding in a very similar way. 257 00:12:12,610 --> 00:12:15,790 However, at the bottom part of the response scenario, 258 00:12:15,790 --> 00:12:17,830 the liver and muscles are going to be activating 259 00:12:17,830 --> 00:12:20,530 very different pathways. 260 00:12:20,530 --> 00:12:23,570 Specifically, we're going to see that muscles will strongly 261 00:12:23,570 --> 00:12:26,170 activate glycolytic pathways in order 262 00:12:26,170 --> 00:12:30,220 to enable the production of ATP to keep the muscles running. 263 00:12:30,220 --> 00:12:33,580 The liver, by contrast, is going to be activating pathways that 264 00:12:33,580 --> 00:12:36,670 are more like those of gluconeogenesis, that is, 265 00:12:36,670 --> 00:12:39,820 spilling out fuel from the liver to provide 266 00:12:39,820 --> 00:12:42,370 the muscles with the energy-rich resources 267 00:12:42,370 --> 00:12:46,040 that it needs to keep us running away from our stressor. 268 00:12:46,040 --> 00:12:49,310 Adrenaline, or epinephrine, travels from the adrenal 269 00:12:49,310 --> 00:12:50,900 glands through the blood. 270 00:12:50,900 --> 00:12:53,940 It takes only a second or two for this to happen. 271 00:12:53,940 --> 00:12:56,120 It's received by the liver and the muscles 272 00:12:56,120 --> 00:12:59,090 at the beta-adrenergic receptor. 273 00:12:59,090 --> 00:13:02,120 The blood concentration of epinephrine is very low, 274 00:13:02,120 --> 00:13:04,700 something of the order of 10 to the minus 10 275 00:13:04,700 --> 00:13:05,900 molar at this point. 276 00:13:05,900 --> 00:13:08,060 There's going to be a very substantial signal 277 00:13:08,060 --> 00:13:09,980 amplification as we move ahead. 278 00:13:09,980 --> 00:13:12,770 Keep in mind for later that the initial triggering 279 00:13:12,770 --> 00:13:16,950 signal is in the 10 to the minus 10 molar range. 280 00:13:16,950 --> 00:13:19,440 The arrival of epinephrine at the receptor 281 00:13:19,440 --> 00:13:22,710 results in structural changes in the transmembrane domain 282 00:13:22,710 --> 00:13:24,990 of the beta-adrenergic receptor. 283 00:13:24,990 --> 00:13:27,510 The signal is received by a heterotrimeric G 284 00:13:27,510 --> 00:13:30,720 protein, shown as the circle with alpha, beta, and gamma 285 00:13:30,720 --> 00:13:32,010 subunits. 286 00:13:32,010 --> 00:13:34,500 This G protein, in its inactive state, 287 00:13:34,500 --> 00:13:41,280 has a bound molecule of GDP, that is, guanosine diphosphate. 288 00:13:41,280 --> 00:13:45,300 Upon receipt of the signal from the beta-adrenergic receptor, 289 00:13:45,300 --> 00:13:49,090 the heterotrimeric G protein ejects the beta and gamma 290 00:13:49,090 --> 00:13:53,400 subunits, and the GDP molecule, which is non-covalently bound, 291 00:13:53,400 --> 00:13:54,840 is released. 292 00:13:54,840 --> 00:14:00,150 The GDP is replaced by a GTP, guanosine triphosphate. 293 00:14:00,150 --> 00:14:02,430 This replacement results in the formation 294 00:14:02,430 --> 00:14:07,770 of the alpha subunit with a non-covalently bound GTP. 295 00:14:07,770 --> 00:14:11,400 This is the active form of the enzyme. 296 00:14:11,400 --> 00:14:14,180 It translocates along the inner surface of the membrane 297 00:14:14,180 --> 00:14:17,810 until it encounters AC, or adenyl cyclase. 298 00:14:17,810 --> 00:14:23,060 The GTP-bound G protein activates adenyl cyclase. 299 00:14:23,060 --> 00:14:25,820 The activated AC dynamically starts 300 00:14:25,820 --> 00:14:28,930 converting ATP, which is very abundant in the cell, 301 00:14:28,930 --> 00:14:30,560 into cyclic AMP. 302 00:14:30,560 --> 00:14:34,380 We call cyclic AMP the second messenger. 303 00:14:34,380 --> 00:14:36,660 In our scenario, the first messenger 304 00:14:36,660 --> 00:14:38,790 is epinephrine, which interacted with 305 00:14:38,790 --> 00:14:40,920 the beta-adrenergic receptor. 306 00:14:40,920 --> 00:14:43,590 The second messenger is cyclic AMP, 307 00:14:43,590 --> 00:14:47,160 which is produced by the activation of adenyl cyclase, 308 00:14:47,160 --> 00:14:48,870 which is asymmetrically associated 309 00:14:48,870 --> 00:14:52,550 with the inner surface of the cell's membrane. 310 00:14:52,550 --> 00:14:56,720 Let's turn now to panel C. In the upper left of panel C, 311 00:14:56,720 --> 00:14:58,290 you'll see a box. 312 00:14:58,290 --> 00:15:00,530 This box depicts the chemical mechanism 313 00:15:00,530 --> 00:15:03,590 leading to the production of cyclic AMP. 314 00:15:03,590 --> 00:15:05,960 Let's now look at the main part of the panel. 315 00:15:05,960 --> 00:15:07,640 In a very short period of time, a matter 316 00:15:07,640 --> 00:15:11,120 of seconds, the concentration of cyclic AMP within the cell 317 00:15:11,120 --> 00:15:15,230 goes from about 1 micromolar up to about 5 micromolar. 318 00:15:15,230 --> 00:15:18,560 So the presence of epinephrine at the 10 to the minus 10 319 00:15:18,560 --> 00:15:20,840 molar concentration outside the cell 320 00:15:20,840 --> 00:15:23,930 results in a perturbation of cyclic AMP concentrations 321 00:15:23,930 --> 00:15:27,920 inside the cell, bringing cyclic AMP concentration to about 10 322 00:15:27,920 --> 00:15:29,660 to the minus 6 molar. 323 00:15:29,660 --> 00:15:31,580 This is a very substantial increase 324 00:15:31,580 --> 00:15:35,180 in signal, which an engineer would call gain. 325 00:15:35,180 --> 00:15:38,060 The increasing cyclic AMP concentration 326 00:15:38,060 --> 00:15:40,780 is going to activate a kinase cascade. 327 00:15:40,780 --> 00:15:42,905 And the initial kinase that's going to be activated 328 00:15:42,905 --> 00:15:45,990 is called Protein Kinase A, or PKA, 329 00:15:45,990 --> 00:15:48,590 which is represented symbolically as a C 330 00:15:48,590 --> 00:15:51,470 with a circle around it in my drawing. 331 00:15:51,470 --> 00:15:54,230 In its inactive state, protein kinase A 332 00:15:54,230 --> 00:15:57,890 is in a complex involving two molecules of itself 333 00:15:57,890 --> 00:16:00,830 and two molecules of a regulatory protein, which 334 00:16:00,830 --> 00:16:03,860 I've indicated as an R in the middle of a circle. 335 00:16:03,860 --> 00:16:07,440 Cyclic AMP forms a tight complex with R. 336 00:16:07,440 --> 00:16:10,190 This basically causes R to dissociate 337 00:16:10,190 --> 00:16:13,040 from the active subunit C, that is, 338 00:16:13,040 --> 00:16:15,800 the catalytic portion of protein kinase 339 00:16:15,800 --> 00:16:18,425 A. The catalytic portion of protein kinase A 340 00:16:18,425 --> 00:16:21,470 is now free to act as a kinase to phosphorylate 341 00:16:21,470 --> 00:16:23,720 the next kinase in the cascade, which 342 00:16:23,720 --> 00:16:27,950 is called SPK, or Synthase Phosphorylase Kinase. 343 00:16:27,950 --> 00:16:31,440 A serine residue on SPK is phosphorylated, 344 00:16:31,440 --> 00:16:34,860 and that event converts this kinase into its active form. 345 00:16:34,860 --> 00:16:37,320 I'll also point out that one of the subunits, 346 00:16:37,320 --> 00:16:41,240 the delta subunit of SPK, is the calcium-binding protein 347 00:16:41,240 --> 00:16:44,880 calmodulin, which gives a second level of regulation 348 00:16:44,880 --> 00:16:46,080 to this enzyme. 349 00:16:46,080 --> 00:16:48,270 Specifically, this enzyme is regulated 350 00:16:48,270 --> 00:16:50,880 not only by the concentration of cyclic AMP, 351 00:16:50,880 --> 00:16:54,450 but also by the levels of calcium within the cell. 352 00:16:54,450 --> 00:16:58,020 The activated SPK, or synthase phosphorylase kinase, 353 00:16:58,020 --> 00:17:01,770 is now going to phosphorylate two other proteins, glycogen 354 00:17:01,770 --> 00:17:05,150 phosphorylase and glycogen synthase. 355 00:17:05,150 --> 00:17:07,640 As I mentioned earlier, phosphorylation of glycogen 356 00:17:07,640 --> 00:17:11,510 phosphorylase results in the enzyme becoming more active. 357 00:17:11,510 --> 00:17:15,050 That is, it starts breaking down glycogen by phosphorolysis 358 00:17:15,050 --> 00:17:17,480 to produce glucose 1-phosphate, which 359 00:17:17,480 --> 00:17:21,359 will then be available for further metabolic processing. 360 00:17:21,359 --> 00:17:23,390 In the muscle, glucose 1-phosphate 361 00:17:23,390 --> 00:17:25,700 will be converted by phosphoglucomutase 362 00:17:25,700 --> 00:17:29,180 into glucose 6-phosphate, which will then be a substrate 363 00:17:29,180 --> 00:17:32,360 to initiate glycolysis, with the ultimate generation of lots 364 00:17:32,360 --> 00:17:34,940 of ATP for the muscle. 365 00:17:34,940 --> 00:17:37,610 In the liver, phosphoglucomutase will 366 00:17:37,610 --> 00:17:40,820 convert glucose 1-phosphate into glucose 6-phosphate, 367 00:17:40,820 --> 00:17:43,400 but in this case, gluconeogenesis 368 00:17:43,400 --> 00:17:45,260 will be activated. 369 00:17:45,260 --> 00:17:48,200 Glucose 6-phosphatase will remove the phosphate 370 00:17:48,200 --> 00:17:51,870 from glucose 6-phosphate, converting it to glucose. 371 00:17:51,870 --> 00:17:54,590 The glucose will then be secreted from the liver, 372 00:17:54,590 --> 00:17:56,420 go out into the blood, and then go 373 00:17:56,420 --> 00:18:00,230 to the muscle to help the muscle carry out glycolysis. 374 00:18:00,230 --> 00:18:02,650 Now let's look at the other target of SPK, 375 00:18:02,650 --> 00:18:05,350 synthase phosphorylase kinase, specifically, 376 00:18:05,350 --> 00:18:07,450 glycogen synthase. 377 00:18:07,450 --> 00:18:10,900 In this case, glycogen synthase phosphorylation results 378 00:18:10,900 --> 00:18:14,680 in a less active protein, and hence, glycogen synthesis 379 00:18:14,680 --> 00:18:15,880 will cease. 380 00:18:15,880 --> 00:18:18,880 This makes sense, because a cell under stress 381 00:18:18,880 --> 00:18:22,300 would not want to be making glycogen. It wants to be using 382 00:18:22,300 --> 00:18:25,410 energy and not storing fuel. 383 00:18:25,410 --> 00:18:28,680 Let's step back and look at the big picture at this point. 384 00:18:28,680 --> 00:18:30,930 The interaction of a hormone resulted 385 00:18:30,930 --> 00:18:33,820 in the covalent modification of proteins. 386 00:18:33,820 --> 00:18:36,240 One of those phosphorylated proteins, 387 00:18:36,240 --> 00:18:40,000 glycogen phosphorylase, triggered glycogen breakdown. 388 00:18:40,000 --> 00:18:43,680 Phosphorylation of a second protein, glycogen synthase, 389 00:18:43,680 --> 00:18:45,460 turned it off. 390 00:18:45,460 --> 00:18:47,620 Turning off glycogen synthase helped 391 00:18:47,620 --> 00:18:51,430 us avoid the otherwise futile simultaneous synthesis 392 00:18:51,430 --> 00:18:53,760 and breakdown of glycogen. 393 00:18:53,760 --> 00:18:57,560 So overall, this is an example of a hormone causing 394 00:18:57,560 --> 00:19:00,860 covalent modification of proteins in such a way 395 00:19:00,860 --> 00:19:03,290 that it altered the activity of those proteins 396 00:19:03,290 --> 00:19:05,120 in a manner that made sense given 397 00:19:05,120 --> 00:19:09,340 the physiological challenges to the organism. 398 00:19:09,340 --> 00:19:11,720 Next, let's turn to storyboard 38, 399 00:19:11,720 --> 00:19:14,520 panel A. Our next regulatory paradigm 400 00:19:14,520 --> 00:19:16,530 will be the use of allosteric effectors 401 00:19:16,530 --> 00:19:18,940 to regulate a pathway. 402 00:19:18,940 --> 00:19:22,600 As before, I'm going to use the physiological scenario 403 00:19:22,600 --> 00:19:23,632 of stress. 404 00:19:23,632 --> 00:19:25,090 But in this case, I'm going to show 405 00:19:25,090 --> 00:19:26,800 how stress will cause the production 406 00:19:26,800 --> 00:19:29,500 of small-molecule allosteric effectors 407 00:19:29,500 --> 00:19:32,140 that will activate glycolysis in the muscle 408 00:19:32,140 --> 00:19:35,730 and activate gluconeogenesis in the liver. 409 00:19:35,730 --> 00:19:40,170 Our focal point is going to be PFK-1, phosphofructokinase-1, 410 00:19:40,170 --> 00:19:41,940 of glycolysis. 411 00:19:41,940 --> 00:19:44,520 Of central importance is going to be a small molecule, 412 00:19:44,520 --> 00:19:48,870 fructose 2,6-bisphosphate, which we'll see is going to be a very 413 00:19:48,870 --> 00:19:53,310 powerful allosteric stimulant of PFK-1. 414 00:19:53,310 --> 00:19:57,210 Fructose 2,6-bisphosphate is made by the enzyme PFK-2, 415 00:19:57,210 --> 00:19:58,870 phosphofructokinase-2. 416 00:19:58,870 --> 00:20:01,440 Let's start out by looking at the very 417 00:20:01,440 --> 00:20:05,370 short biochemical pathway shown in panel A. We see glucose 418 00:20:05,370 --> 00:20:07,860 converted by hexokinase or glucokinase 419 00:20:07,860 --> 00:20:10,830 to glucose 6-phosphate and then some equilibrium 420 00:20:10,830 --> 00:20:15,000 steps up until we get fructose 6-phosphate. 421 00:20:15,000 --> 00:20:17,550 Ordinarily, we think of fructose 6-phosphate 422 00:20:17,550 --> 00:20:19,960 as continuing in the glycolytic pathway, 423 00:20:19,960 --> 00:20:22,560 but let's not think of it that way right now. 424 00:20:22,560 --> 00:20:24,810 The chemical structure of fructose 6-phosphate 425 00:20:24,810 --> 00:20:27,220 is shown slightly to the right. 426 00:20:27,220 --> 00:20:28,980 Note that fructose 6-phosphate has 427 00:20:28,980 --> 00:20:32,160 a phosphate on the 6-hydroxyl group, 428 00:20:32,160 --> 00:20:34,290 and that the 1-hydroxyl functionality 429 00:20:34,290 --> 00:20:36,550 has only a hydrogen. 430 00:20:36,550 --> 00:20:38,700 This is the alpha anomer of fructose, 431 00:20:38,700 --> 00:20:42,360 as evidenced by the fact that the hydroxyl group on carbon-2, 432 00:20:42,360 --> 00:20:46,410 the anomeric position, is down, or under the furanose ring. 433 00:20:46,410 --> 00:20:48,210 With a little bit of electron pushing, 434 00:20:48,210 --> 00:20:49,950 you can change the stereochemistry 435 00:20:49,950 --> 00:20:53,400 at the 2-carbon such that the hydroxyl group would be up, 436 00:20:53,400 --> 00:20:55,740 or in the beta configuration. 437 00:20:55,740 --> 00:20:58,590 In the beta configuration, the hydroxymethyl group 438 00:20:58,590 --> 00:21:02,280 of fructose 6-phosphate, which includes its 1-carbon, 439 00:21:02,280 --> 00:21:04,370 would be down. 440 00:21:04,370 --> 00:21:07,670 The beta and alpha-anomers of fructose 6-phosphate 441 00:21:07,670 --> 00:21:09,020 are in equilibrium. 442 00:21:09,020 --> 00:21:12,160 That is, they both exist at the same time. 443 00:21:12,160 --> 00:21:14,990 The kinase, phosphofructokinase-2, 444 00:21:14,990 --> 00:21:18,560 catches the beta anomer of fructose 6-phosphate 445 00:21:18,560 --> 00:21:21,530 and phosphorylates it, producing the molecule at the lower 446 00:21:21,530 --> 00:21:22,880 right of this panel. 447 00:21:22,880 --> 00:21:26,810 That's the actual structure of fructose 2,6-bisphosphate. 448 00:21:26,810 --> 00:21:28,220 This is the molecule that's going 449 00:21:28,220 --> 00:21:30,350 to serve as the allosteric effector 450 00:21:30,350 --> 00:21:32,860 of the glycolysis pathway. 451 00:21:32,860 --> 00:21:35,740 The PFK-2, or phosphofructokinase-2 enzyme, 452 00:21:35,740 --> 00:21:37,000 is shown to the left. 453 00:21:37,000 --> 00:21:39,190 It's a complex enzyme having lots 454 00:21:39,190 --> 00:21:41,410 of different functionalities. 455 00:21:41,410 --> 00:21:44,410 The hatched circle within the larger circle at the top 456 00:21:44,410 --> 00:21:47,270 represents the phosphofructokinase domain, 457 00:21:47,270 --> 00:21:50,010 which converts fructose 6-phosphate to fructose 458 00:21:50,010 --> 00:21:51,550 2,6-bisphosphate. 459 00:21:51,550 --> 00:21:54,690 That is the structure at the lower right. 460 00:21:54,690 --> 00:21:59,470 The hatched domain at the bottom is the fructose bisphosphatase 461 00:21:59,470 --> 00:22:00,520 domain. 462 00:22:00,520 --> 00:22:02,500 This is an unusual enzyme. 463 00:22:02,500 --> 00:22:06,700 In the top domain, it acts as a kinase, in the bottom domain, 464 00:22:06,700 --> 00:22:08,820 acts as a phosphatase. 465 00:22:08,820 --> 00:22:13,150 PFK-2 has two hydroxylated amino acids, 466 00:22:13,150 --> 00:22:16,360 one at about 3 o'clock as drawn and the other at about 6 467 00:22:16,360 --> 00:22:17,680 o'clock. 468 00:22:17,680 --> 00:22:20,380 When the hydroxyl group at the bottom 469 00:22:20,380 --> 00:22:23,770 is present as a pure, unmodified hydroxyl group, 470 00:22:23,770 --> 00:22:26,350 the protein acts as a kinase. 471 00:22:26,350 --> 00:22:28,750 When the 6 o'clock domain is phosphorylated, 472 00:22:28,750 --> 00:22:32,040 the enzyme acts as a phosphatase. 473 00:22:32,040 --> 00:22:35,760 Protein kinase A, or PKA, from the previous storyboard, 474 00:22:35,760 --> 00:22:38,210 where we talked about hormonal regulation, 475 00:22:38,210 --> 00:22:40,230 is the kinase that phosphorylates 476 00:22:40,230 --> 00:22:43,720 the hydroxyl group at 6 o'clock on the protein. 477 00:22:43,720 --> 00:22:46,380 As we'll see later, the hydroxyl group at 3 o'clock 478 00:22:46,380 --> 00:22:51,040 is targeted by a kinase called AMP kinase in the muscle. 479 00:22:51,040 --> 00:22:54,730 Before we go on, I want you to look carefully at the structure 480 00:22:54,730 --> 00:22:59,960 of fructose 2,6-bisphosphate at the bottom right of panel A. 481 00:22:59,960 --> 00:23:02,470 Compare that structure with the structure of fructose 482 00:23:02,470 --> 00:23:07,090 1,6-bisphosphate at the right in panel B. 483 00:23:07,090 --> 00:23:09,850 If you squint and look at these two molecules, 484 00:23:09,850 --> 00:23:13,810 you'll see that they look remarkably alike. 485 00:23:13,810 --> 00:23:15,640 In both cases, the phosphate group 486 00:23:15,640 --> 00:23:18,800 is above the plane of the furanose sugar. 487 00:23:18,800 --> 00:23:21,890 However, in the case of fructose 2,6-bisphosphate, 488 00:23:21,890 --> 00:23:24,250 the phosphate is on the 2-carbon, 489 00:23:24,250 --> 00:23:27,190 whereas with fructose 1,6-bisphosphate, 490 00:23:27,190 --> 00:23:29,650 the phosphate is on the 1-carbon. 491 00:23:29,650 --> 00:23:32,050 The way these molecules look will become important 492 00:23:32,050 --> 00:23:34,040 in a few minutes. 493 00:23:34,040 --> 00:23:35,710 Now let's take a look at the pathway 494 00:23:35,710 --> 00:23:39,280 as shown in panel B. Panel B reflects 495 00:23:39,280 --> 00:23:42,760 the liver in its normal, that is, non-stress state. 496 00:23:42,760 --> 00:23:47,410 So this is just the liver doing everyday liver business. 497 00:23:47,410 --> 00:23:50,200 Glucokinase of the liver is converting glucose 498 00:23:50,200 --> 00:23:52,210 to glucose 6-phosphate. 499 00:23:52,210 --> 00:23:56,770 The alpha anomer of fructose 6-phosphate is produced. 500 00:23:56,770 --> 00:23:59,500 Most of the fructose 6-phosphate is converted to fructose 501 00:23:59,500 --> 00:24:02,000 1,6-bisphosphate. 502 00:24:02,000 --> 00:24:03,700 In the liver cell at this time, there's 503 00:24:03,700 --> 00:24:08,230 probably some low level of glycolytic activity going on. 504 00:24:08,230 --> 00:24:11,620 However, because of natural anomerization, 505 00:24:11,620 --> 00:24:14,560 some of the alpha-fructose 6-phosphate 506 00:24:14,560 --> 00:24:17,680 is converted to its beta-anomer. 507 00:24:17,680 --> 00:24:22,700 Phosphofructokinase-2, or PFK-2, is shown to the lower left, 508 00:24:22,700 --> 00:24:24,330 and it's active. 509 00:24:24,330 --> 00:24:28,320 Note that neither of its hydroxyl groups, at this point, 510 00:24:28,320 --> 00:24:29,850 are phosphorylated. 511 00:24:29,850 --> 00:24:33,990 And its kinase site will be converting the beta-fructose 512 00:24:33,990 --> 00:24:37,770 6-phosphate anomer to its phosphorylated form, that is, 513 00:24:37,770 --> 00:24:42,570 beta-fructose 2,6-bisphosphate, which has the structure shown 514 00:24:42,570 --> 00:24:45,810 at the bottom right of the previous panel. 515 00:24:45,810 --> 00:24:50,280 Fructose 2,6-bisphosphate is a positive allosteric effector 516 00:24:50,280 --> 00:24:52,290 of phosphofructokinase-1. 517 00:24:52,290 --> 00:24:54,690 So it facilitates the glycolytic pathway 518 00:24:54,690 --> 00:24:58,020 that is enabling net flux from left to right in the pathway 519 00:24:58,020 --> 00:24:59,610 as shown. 520 00:24:59,610 --> 00:25:02,460 In addition to stimulating glycolysis, 521 00:25:02,460 --> 00:25:05,850 fructose 2,6-bisphosphate strongly inhibits 522 00:25:05,850 --> 00:25:07,740 gluconeogenesis. 523 00:25:07,740 --> 00:25:11,360 It does this by binding at the active site of fructose 524 00:25:11,360 --> 00:25:15,570 1,6-bisphosphatase, the gluconeogenic enzyme, 525 00:25:15,570 --> 00:25:18,720 clogging up the active site, and thus disabling 526 00:25:18,720 --> 00:25:21,120 the gluconeogenic pathway. 527 00:25:21,120 --> 00:25:24,720 The reason that it's such a good inhibitor of gluconeogenesis 528 00:25:24,720 --> 00:25:26,910 stems from an examination, once again, 529 00:25:26,910 --> 00:25:30,870 of the structures of fructose 2,6-bisphosphate in the bottom 530 00:25:30,870 --> 00:25:35,820 right of the previous panel and fructose 1,6-bisphosphate shown 531 00:25:35,820 --> 00:25:38,520 to the right on this panel. 532 00:25:38,520 --> 00:25:41,820 Keep in mind that fructose 1,6-bisphosphate, 533 00:25:41,820 --> 00:25:44,880 while it's the product in glycolysis, 534 00:25:44,880 --> 00:25:49,320 is the substrate for the gluconeogenic reaction. 535 00:25:49,320 --> 00:25:53,190 If the active site of the fructose 1,6-bisphosphatase is 536 00:25:53,190 --> 00:25:56,670 clogged by fructose 2,6-bisphosphate, 537 00:25:56,670 --> 00:25:59,710 then it's not going to be able to process the fructose 538 00:25:59,710 --> 00:26:03,720 1,6-bisphosphate in the gluconeogenic direction. 539 00:26:03,720 --> 00:26:07,950 Hence, gluconeogenesis is strongly inhibited. 540 00:26:07,950 --> 00:26:12,000 All of the above shows us that fructose 2,6-bisphosphate is 541 00:26:12,000 --> 00:26:13,920 really important. 542 00:26:13,920 --> 00:26:16,020 It stimulates the forward direction 543 00:26:16,020 --> 00:26:19,050 in the liver that is causing glycolysis to have 544 00:26:19,050 --> 00:26:21,780 a net flux from left to right. 545 00:26:21,780 --> 00:26:25,200 And in addition, it strongly inhibits the back reaction, 546 00:26:25,200 --> 00:26:29,630 that is, the reaction in the gluconeogenic direction. 547 00:26:29,630 --> 00:26:34,350 Turn at this point to panel C. Now let's ramp up the scenario 548 00:26:34,350 --> 00:26:36,500 and think about a situation in which you're 549 00:26:36,500 --> 00:26:38,720 being chased by a dog. 550 00:26:38,720 --> 00:26:41,930 You want to be able to generate glucose in the liver 551 00:26:41,930 --> 00:26:44,270 and then export that glucose to the muscles 552 00:26:44,270 --> 00:26:47,260 so that you can run away from your foe. 553 00:26:47,260 --> 00:26:50,380 This is a situation in which you would like to have your liver 554 00:26:50,380 --> 00:26:52,480 stop doing glycolysis. 555 00:26:52,480 --> 00:26:55,570 You want the liver to strongly turn to gluconeogenesis 556 00:26:55,570 --> 00:26:58,660 in order to manufacture glucose for the muscle. 557 00:26:58,660 --> 00:27:00,850 At this point, refer back to the storyboard 558 00:27:00,850 --> 00:27:03,900 that dealt with covalent control, where protein kinase 559 00:27:03,900 --> 00:27:05,890 A was activated. 560 00:27:05,890 --> 00:27:07,870 Recall the part of the lecture when 561 00:27:07,870 --> 00:27:10,000 I talked about covalent control, and we 562 00:27:10,000 --> 00:27:12,640 were talking about the glycogen phosphorylase 563 00:27:12,640 --> 00:27:14,630 step in the liver. 564 00:27:14,630 --> 00:27:18,070 In the liver, we generated first glucose 1-phosphate 565 00:27:18,070 --> 00:27:20,500 and then glucose 6-phosphate. 566 00:27:20,500 --> 00:27:23,260 And then the phosphate was lopped off the glucose 567 00:27:23,260 --> 00:27:27,340 6-phosphate to produce glucose that went out into the blood. 568 00:27:27,340 --> 00:27:28,810 In the present case, we're looking 569 00:27:28,810 --> 00:27:33,100 at a step that's further down in the gluconeogenesis pathway. 570 00:27:33,100 --> 00:27:36,100 Specifically, we want to activate the fructose 571 00:27:36,100 --> 00:27:40,900 1,6-bisphosphatase in order to push even more carbohydrate 572 00:27:40,900 --> 00:27:43,530 toward the production of glucose. 573 00:27:43,530 --> 00:27:48,650 So in panel C, we see stress inducing the cyclic AMP cascade 574 00:27:48,650 --> 00:27:52,460 that results in activation of protein kinase A. And protein 575 00:27:52,460 --> 00:27:54,410 kinase A is going to phosphorylate 576 00:27:54,410 --> 00:27:58,280 the southernmost, that is, 6 o'clock domain, on the PFK-2, 577 00:27:58,280 --> 00:28:01,230 or phosphofructokinase-2 protein. 578 00:28:01,230 --> 00:28:03,960 As I indicated earlier, phosphorylation 579 00:28:03,960 --> 00:28:06,840 of the 6 o'clock domain results in conversion 580 00:28:06,840 --> 00:28:11,940 of phosphofructokinase-2 from a kinase into a phosphatase. 581 00:28:11,940 --> 00:28:16,770 The phosphatase produced from PFK-2 will act upon the pool 582 00:28:16,770 --> 00:28:20,610 of fructose 2,6-bisphosphate and convert that pool to fructose 583 00:28:20,610 --> 00:28:21,990 6-phosphate. 584 00:28:21,990 --> 00:28:25,810 That is, the 2-phosphate will be lopped off of the fructose 585 00:28:25,810 --> 00:28:27,930 2,6-bisphosphate molecule. 586 00:28:27,930 --> 00:28:30,490 By doing this biochemistry in the liver, 587 00:28:30,490 --> 00:28:31,630 we've done two things. 588 00:28:31,630 --> 00:28:35,550 First, we destroyed the fructose 2,6-bisphosphate pool. 589 00:28:35,550 --> 00:28:37,650 Hence, this molecule is no longer 590 00:28:37,650 --> 00:28:42,750 available to act as an allosteric stimulator of PFK-1. 591 00:28:42,750 --> 00:28:47,460 Secondly, removing fructose 2,6-bisphosphate from the pool 592 00:28:47,460 --> 00:28:50,910 has resulted in removal of the active-site inhibitor 593 00:28:50,910 --> 00:28:53,910 of fructose 1,6-bisphosphatase. 594 00:28:53,910 --> 00:28:56,250 This enzyme, therefore, becomes active 595 00:28:56,250 --> 00:28:59,190 and is able to push the net flux of carbon 596 00:28:59,190 --> 00:29:02,850 from right to left in the direction of gluconeogenesis, 597 00:29:02,850 --> 00:29:08,410 producing glucose that will then spill out into the blood. 598 00:29:08,410 --> 00:29:10,780 Please turn now to panel D. We just 599 00:29:10,780 --> 00:29:14,554 saw that the liver, post-stress, produces glucose. 600 00:29:14,554 --> 00:29:16,720 Now we're going to take a look at the muscle, which, 601 00:29:16,720 --> 00:29:20,080 of course, is going to be using that glucose. 602 00:29:20,080 --> 00:29:22,720 In response to the stress state, the muscle 603 00:29:22,720 --> 00:29:24,950 is going to activate an enzyme called 604 00:29:24,950 --> 00:29:27,670 the AMP-dependent protein kinase, 605 00:29:27,670 --> 00:29:30,900 or AMP kinase, also known as AMPK. 606 00:29:30,900 --> 00:29:35,350 AMPK activates metabolic pathways that generate ATP. 607 00:29:35,350 --> 00:29:38,890 The 3 o'clock phosphorylated PFK-2 proves to be 608 00:29:38,890 --> 00:29:42,340 an exceptionally competent enzyme for converting fructose 609 00:29:42,340 --> 00:29:46,030 6-phosphate into fructose 2,6-bisphosphate. 610 00:29:46,030 --> 00:29:49,430 This increases further the concentration of fructose 611 00:29:49,430 --> 00:29:53,470 2,6-bisphosphate in the muscle cytoplasmic pool. 612 00:29:53,470 --> 00:29:57,130 And keep in mind that fructose 2,6-bisphosphate itself is 613 00:29:57,130 --> 00:30:01,150 a powerful allosteric stimulator of glycolysis by its effect 614 00:30:01,150 --> 00:30:02,930 on PFK-1. 615 00:30:02,930 --> 00:30:06,670 So in the muscle cell, we're able to achieve extraordinarily 616 00:30:06,670 --> 00:30:09,940 high concentrations of fructose 2,6-bisphosphate, 617 00:30:09,940 --> 00:30:12,610 thus favoring glycolysis. 618 00:30:12,610 --> 00:30:15,550 Let's turn now to storyboard 39. 619 00:30:15,550 --> 00:30:17,380 Let's start with panel A. The way 620 00:30:17,380 --> 00:30:20,680 that AMP kinase stimulates glycolysis in the muscle 621 00:30:20,680 --> 00:30:24,190 post-stress is illustrated in this abbreviated metabolic 622 00:30:24,190 --> 00:30:25,540 pathway. 623 00:30:25,540 --> 00:30:27,700 Starting at the lower left, the stressor 624 00:30:27,700 --> 00:30:30,430 has created the cyclic AMP cascade 625 00:30:30,430 --> 00:30:32,780 that activates AMP kinase. 626 00:30:32,780 --> 00:30:38,050 AMP kinase phosphorylates the 3 o'clock domain on PFK-2, 627 00:30:38,050 --> 00:30:41,270 converting it to its phosphorylated derivative that 628 00:30:41,270 --> 00:30:45,730 shows powerful kinase activity to convert fructose 6-phosphate 629 00:30:45,730 --> 00:30:48,490 into fructose 2,6-bisphosphate. 630 00:30:48,490 --> 00:30:53,350 This enhanced pool of fructose 2,6-bisphosphate will now act 631 00:30:53,350 --> 00:30:57,970 as a powerful allosteric stimulator of PFK-1 to favor 632 00:30:57,970 --> 00:30:59,740 glycolysis. 633 00:30:59,740 --> 00:31:03,490 I want to point out that muscles do not do gluconeogenesis. 634 00:31:03,490 --> 00:31:07,160 Only the liver and renal cortex do this pathway. 635 00:31:07,160 --> 00:31:11,680 So muscles do not have fructose 1,6-bisphosphatase, 636 00:31:11,680 --> 00:31:12,970 the gluconeogenic enzyme. 637 00:31:12,970 --> 00:31:15,490 Accordingly, in muscles we don't have 638 00:31:15,490 --> 00:31:19,610 to worry about gluconeogenesis being turned on to some extent 639 00:31:19,610 --> 00:31:21,970 and thus dampening the glycolytic effect 640 00:31:21,970 --> 00:31:24,460 in the muscle that's needed in order for the muscle 641 00:31:24,460 --> 00:31:27,940 to generate the ATP needed to outrun your foe. 642 00:31:27,940 --> 00:31:29,850 On this panel, we also see that there 643 00:31:29,850 --> 00:31:32,380 are several other effectors that influence 644 00:31:32,380 --> 00:31:34,270 the activity of PFK-1. 645 00:31:34,270 --> 00:31:35,560 One is citrate. 646 00:31:35,560 --> 00:31:36,880 Another is ATP. 647 00:31:36,880 --> 00:31:40,360 Another one is AMP, or Adenosine Monophosphate. 648 00:31:40,360 --> 00:31:43,930 I want to say a couple of words about AMP in this regard. 649 00:31:43,930 --> 00:31:48,980 Let's look at panel B. AMP is not as powerful an allosteric 650 00:31:48,980 --> 00:31:53,660 effector on PFK-1 as is fructose 2,6-bisphosphate. 651 00:31:53,660 --> 00:31:55,907 But we know a lot about its activity 652 00:31:55,907 --> 00:31:57,490 as an allosteric effector because it's 653 00:31:57,490 --> 00:31:59,470 been studied quite thoroughly. 654 00:31:59,470 --> 00:32:01,930 PFK-1 is a tetramer, and its activity 655 00:32:01,930 --> 00:32:04,720 is regulated by cooperativity. 656 00:32:04,720 --> 00:32:07,690 This graph shows the activity of PFK-1 657 00:32:07,690 --> 00:32:09,550 as a function of the concentration of one 658 00:32:09,550 --> 00:32:12,700 of its substrates, fructose 6-phosphate. 659 00:32:12,700 --> 00:32:14,410 When there's no ATP present, which 660 00:32:14,410 --> 00:32:16,690 is not a realistic situation, you 661 00:32:16,690 --> 00:32:19,090 see the regulatory pattern following that 662 00:32:19,090 --> 00:32:20,920 of a rectangular hyperbola. 663 00:32:20,920 --> 00:32:22,690 That's scenario A. 664 00:32:22,690 --> 00:32:24,880 Scenario B shows what would happen 665 00:32:24,880 --> 00:32:26,800 to the activity of the enzyme when 666 00:32:26,800 --> 00:32:29,200 ATP is present at a concentration of about 667 00:32:29,200 --> 00:32:32,170 1 millimolar, which is quite realistic, 668 00:32:32,170 --> 00:32:35,080 and what you see is suppressed activity. 669 00:32:35,080 --> 00:32:38,380 When the heterotropic allosteric effector AMP is added, 670 00:32:38,380 --> 00:32:40,930 you see the curve shift to the left. 671 00:32:40,930 --> 00:32:44,290 That is, you get enhanced activity at a given substrate 672 00:32:44,290 --> 00:32:45,520 concentration. 673 00:32:45,520 --> 00:32:49,030 My guess is that fructose 2,6-bisphosphate would push 674 00:32:49,030 --> 00:32:52,010 the curve even further to the left. 675 00:32:52,010 --> 00:32:55,780 Let's now look at panel C. I think it's useful to use AMP 676 00:32:55,780 --> 00:33:00,190 to help us construct a model for what fructose 2,6-bisphosphate 677 00:33:00,190 --> 00:33:05,720 might be doing as an allosteric stimulator of the PFK-1 enzyme. 678 00:33:05,720 --> 00:33:07,750 The equilibrium diagram that I've drawn 679 00:33:07,750 --> 00:33:10,540 shows the relationship between the relaxed, that 680 00:33:10,540 --> 00:33:14,020 is, more active state of the PFK-1 tetramer, 681 00:33:14,020 --> 00:33:18,160 and the tense, that is, more inactive state of the enzyme. 682 00:33:18,160 --> 00:33:20,680 When ATP is abundant within a cell, 683 00:33:20,680 --> 00:33:23,860 it binds more strongly to the tense state, 684 00:33:23,860 --> 00:33:27,700 and hence, ATP inactivates PFK-1. 685 00:33:27,700 --> 00:33:30,680 This makes sense, because if ATP is abundant, 686 00:33:30,680 --> 00:33:32,980 you do not want to be doing glycolysis 687 00:33:32,980 --> 00:33:35,400 to make even more ATP. 688 00:33:35,400 --> 00:33:37,540 However, when the ATP pool of a cell 689 00:33:37,540 --> 00:33:41,050 is challenged by doing heavy work or biosynthesis, 690 00:33:41,050 --> 00:33:42,820 the AMP is going to bind more tightly 691 00:33:42,820 --> 00:33:45,450 to the R state of PFK-1. 692 00:33:45,450 --> 00:33:47,980 And that's going to help convert the enzyme 693 00:33:47,980 --> 00:33:50,110 into its more active state. 694 00:33:50,110 --> 00:33:53,500 That's a little snapshot of how a small molecule, in this case, 695 00:33:53,500 --> 00:33:56,410 AMP or perhaps fructose 2,6-bisphosphate can 696 00:33:56,410 --> 00:33:59,080 dramatically regulate the activity of an enzyme 697 00:33:59,080 --> 00:34:02,520 at a specific step in metabolism.