1 00:00:00,500 --> 00:00:02,820 The following content is provided under a Creative 2 00:00:02,820 --> 00:00:04,360 Commons license. 3 00:00:04,360 --> 00:00:06,660 Your support will help MIT OpenCourseWare 4 00:00:06,660 --> 00:00:11,020 continue to offer high quality educational resources for free. 5 00:00:11,020 --> 00:00:13,650 To make a donation, or view additional materials 6 00:00:13,650 --> 00:00:17,600 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,600 --> 00:00:18,550 at ocw.mit.edu. 8 00:00:25,223 --> 00:00:27,640 JOANNE STUBBE: We're talking about the purine biosynthetic 9 00:00:27,640 --> 00:00:28,140 pathway. 10 00:00:28,140 --> 00:00:30,312 Here's the pathway. 11 00:00:30,312 --> 00:00:32,770 I told you, in this part of it we were going to go through, 12 00:00:32,770 --> 00:00:35,080 so at least you saw what the steps in the pathway are. 13 00:00:35,080 --> 00:00:39,580 The key thing is you start out with the ribose 5-phosphate, 14 00:00:39,580 --> 00:00:41,260 and then you build up the base a step 15 00:00:41,260 --> 00:00:43,780 at a time, which is completely different from pyrimidines, 16 00:00:43,780 --> 00:00:45,820 where you make the base, and then stick 17 00:00:45,820 --> 00:00:48,418 on the ribose 5-phosphate. 18 00:00:48,418 --> 00:00:49,960 And I told you at the very beginning, 19 00:00:49,960 --> 00:00:51,940 there were a few interesting steps 20 00:00:51,940 --> 00:00:54,580 in this pathway that are universal in almost 21 00:00:54,580 --> 00:00:57,040 all metabolic pathways. 22 00:00:57,040 --> 00:00:59,695 And one of them we were going over-- two of them 23 00:00:59,695 --> 00:01:00,570 we already went over. 24 00:01:00,570 --> 00:01:02,470 I'm going to briefly go back over this, 25 00:01:02,470 --> 00:01:07,840 but the role of glutamine in the purine and pyrimidine pathway 26 00:01:07,840 --> 00:01:11,470 as the source of nitrogen. There were five of these enzymes. 27 00:01:11,470 --> 00:01:13,270 That's not an accident. 28 00:01:13,270 --> 00:01:18,280 Glutamine is one of the major ways you deliver ammonia 29 00:01:18,280 --> 00:01:20,170 into molecules. 30 00:01:20,170 --> 00:01:24,310 And purines and pyrimidines both have a lot of nitrogens. 31 00:01:24,310 --> 00:01:26,000 The second thing we were talking about, 32 00:01:26,000 --> 00:01:29,890 and we had gone through the first few steps here, 33 00:01:29,890 --> 00:01:35,050 was the second enzyme in the pathway, where we use ATP, 34 00:01:35,050 --> 00:01:38,350 and in this particular pathway, this 35 00:01:38,350 --> 00:01:40,930 is the mammalian version of the pathway, which 36 00:01:40,930 --> 00:01:42,670 is pretty similar to the bacterial, 37 00:01:42,670 --> 00:01:48,670 but there were five different steps that require ATP. 38 00:01:48,670 --> 00:01:52,240 This pathway demonstrates how you 39 00:01:52,240 --> 00:01:56,590 see ATP use over and over and over and over again. 40 00:01:56,590 --> 00:02:02,440 There are defined structures for the binding sites of the ATPs. 41 00:02:02,440 --> 00:02:05,380 Once you have these in your brain, it becomes easy. 42 00:02:05,380 --> 00:02:08,740 You might not know which one of these mechanisms it is, 43 00:02:08,740 --> 00:02:11,830 but after you do a little bit of reading, or bioinformatics, 44 00:02:11,830 --> 00:02:16,780 you can immediately tell what the structure of the enzymes 45 00:02:16,780 --> 00:02:18,820 actually are. 46 00:02:18,820 --> 00:02:23,050 The other thing we talked about already was the role of folate. 47 00:02:23,050 --> 00:02:26,392 Those are the three things I want you to get out of this, 48 00:02:26,392 --> 00:02:28,600 and we're going to go through the rest of that today, 49 00:02:28,600 --> 00:02:31,540 and then, after we finish that, we'll come back 50 00:02:31,540 --> 00:02:33,460 to the purinosomes, which is the reason 51 00:02:33,460 --> 00:02:36,550 I chose this topic a long time ago, 52 00:02:36,550 --> 00:02:40,270 because it speaks to the question of the importance 53 00:02:40,270 --> 00:02:44,830 of transient protein-protein interactions in metabolism 54 00:02:44,830 --> 00:02:47,950 inside the cell, which has been something that people have been 55 00:02:47,950 --> 00:02:51,430 interested in for decades, and this paper in 2008 56 00:02:51,430 --> 00:02:54,430 that you read for recitation was very 57 00:02:54,430 --> 00:02:57,070 interesting to a lot of people, and we'll come back and talk 58 00:02:57,070 --> 00:03:01,330 about that at the end. 59 00:03:01,330 --> 00:03:04,540 The first enzyme-- the names are horrible. 60 00:03:04,540 --> 00:03:06,900 I gave you the names of all these things. 61 00:03:06,900 --> 00:03:09,670 If you look at last year's exam, you 62 00:03:09,670 --> 00:03:12,500 will have the purine pathway with the name stuck at the end. 63 00:03:12,500 --> 00:03:17,710 I don't expect you to remember this, but we go from PRPP-- 64 00:03:17,710 --> 00:03:20,680 we've already gone through this step-- 65 00:03:20,680 --> 00:03:22,690 and the enzyme is PurF-- 66 00:03:22,690 --> 00:03:24,400 I'm not going to write it out-- 67 00:03:24,400 --> 00:03:27,670 goes to PRA. 68 00:03:27,670 --> 00:03:32,260 The reason I'm writing that again is because a key reason 69 00:03:32,260 --> 00:03:34,450 that Bankovic's lab and my lab, many years ago, 70 00:03:34,450 --> 00:03:36,580 was focused on this is because of the instability 71 00:03:36,580 --> 00:03:38,260 of the intermediates in this pathway. 72 00:03:38,260 --> 00:03:43,240 This guy has a half life of 15 seconds at 37 degrees, 73 00:03:43,240 --> 00:03:45,760 so this is chemically unstable. 74 00:03:45,760 --> 00:03:49,300 This is enzyme 1, and this is the first place 75 00:03:49,300 --> 00:03:55,510 we saw glutamine going to glutamate 76 00:03:55,510 --> 00:03:58,090 as the source of ammonia. 77 00:03:58,090 --> 00:04:04,840 And I wanted to go back and say one more thing about that. 78 00:04:04,840 --> 00:04:07,820 Again, there are two enzymes that use glutamine 79 00:04:07,820 --> 00:04:09,370 as a source of ammonia. 80 00:04:09,370 --> 00:04:13,070 This one is simply, if you look at the pathway displacing 81 00:04:13,070 --> 00:04:15,910 pyrophosphate ammonia, you have a nucleophile displacing 82 00:04:15,910 --> 00:04:18,700 pyrophosphate which, when complexed to magnesium, 83 00:04:18,700 --> 00:04:21,130 is a good leaving group. 84 00:04:21,130 --> 00:04:24,430 The idea here is that all of these proteins, 85 00:04:24,430 --> 00:04:27,640 and there were five of them, in the purine and pyrimidine 86 00:04:27,640 --> 00:04:31,300 pathway, have two domains. 87 00:04:31,300 --> 00:04:34,190 Sometimes the domains are separate polypeptides. 88 00:04:34,190 --> 00:04:37,030 Often they're linked together. 89 00:04:37,030 --> 00:04:42,010 The glutaminase domain is in one of these domains, 90 00:04:42,010 --> 00:04:45,400 and the way the chemistry goes, the way 91 00:04:45,400 --> 00:04:48,580 the ammonia is going to displace whatever the leaving group is 92 00:04:48,580 --> 00:04:55,000 in the second domain, requiring a tunnel that 93 00:04:55,000 --> 00:05:00,250 varies from 25 to 40 angstroms to actually mediate 94 00:05:00,250 --> 00:05:02,890 ammonia release. 95 00:05:02,890 --> 00:05:06,920 PurL is the fourth enzyme in the pathway. 96 00:05:06,920 --> 00:05:08,920 Again, here's the glutaminase domain. 97 00:05:08,920 --> 00:05:12,940 It's upside down, and here's where the chemistry 98 00:05:12,940 --> 00:05:16,540 occurs in the other system. 99 00:05:16,540 --> 00:05:18,670 What I wanted to say about that is 100 00:05:18,670 --> 00:05:25,890 that all of these enzymes in the active site have a cysteine. 101 00:05:25,890 --> 00:05:31,307 All of these enzymes have a cysteine in the active site, 102 00:05:31,307 --> 00:05:33,390 and you should go back and look at the PowerPoint, 103 00:05:33,390 --> 00:05:35,970 because I'm not going to write this out on the board. 104 00:05:35,970 --> 00:05:40,200 You've seen this chemistry now, over and over again, 105 00:05:40,200 --> 00:05:43,890 but, in some way, the glutamine is 106 00:05:43,890 --> 00:05:47,490 going to be attached covalently with loss of ammonia 107 00:05:47,490 --> 00:05:49,350 to a cysteine in the active site. 108 00:05:49,350 --> 00:05:53,010 Let me show you what the mechanism of that is. 109 00:05:53,010 --> 00:05:55,200 Here is a generic mechanism, but it 110 00:05:55,200 --> 00:05:57,200 could be a cysteine protease. 111 00:05:57,200 --> 00:05:59,860 These are the same things we've seen over and over again, 112 00:05:59,860 --> 00:06:04,380 so this should now be part of your basic vocabulary. 113 00:06:04,380 --> 00:06:05,580 So the goal, then-- 114 00:06:05,580 --> 00:06:12,240 here's our glutamine-- is simply to liberate ammonia. 115 00:06:12,240 --> 00:06:15,300 The cysteine needs to be activated somehow 116 00:06:15,300 --> 00:06:16,470 for nucleophilic attack. 117 00:06:16,470 --> 00:06:17,700 How is that done, normally? 118 00:06:17,700 --> 00:06:19,350 With a histamine. 119 00:06:19,350 --> 00:06:20,940 This particular enzyme. 120 00:06:20,940 --> 00:06:24,240 There are two superfamilies of enzymes that do this. 121 00:06:24,240 --> 00:06:26,970 This one doesn't use histamine, but it still 122 00:06:26,970 --> 00:06:28,080 needs to be activated. 123 00:06:28,080 --> 00:06:31,830 You go through a tetrahedral transition state, which 124 00:06:31,830 --> 00:06:35,820 collapses to form an acylated enzyme, and, in the end, 125 00:06:35,820 --> 00:06:40,140 you need to hydrolyze this off to give you a glutamic acid. 126 00:06:40,140 --> 00:06:42,190 One of the reasons I wanted to go back to this, 127 00:06:42,190 --> 00:06:44,880 again, is because, in the Bankovic paper, 128 00:06:44,880 --> 00:06:46,650 we talked about, but didn't go through 129 00:06:46,650 --> 00:06:50,280 in any detail, the fact that, in that paper, 130 00:06:50,280 --> 00:06:54,420 to study whether these purinosomes could assemble 131 00:06:54,420 --> 00:06:57,600 and disassemble, they use an inhibitor 132 00:06:57,600 --> 00:07:00,990 of the purine pathway, which then should 133 00:07:00,990 --> 00:07:03,810 want the enzymes to assemble, because they 134 00:07:03,810 --> 00:07:07,020 need to make purines because you've blocked the pathway. 135 00:07:07,020 --> 00:07:11,910 And the inhibitor they used is a molecule that looks like this. 136 00:07:11,910 --> 00:07:15,480 They used azaserine, but it has another methylene in it. 137 00:07:15,480 --> 00:07:18,000 This is DON. 138 00:07:18,000 --> 00:07:22,110 And this is a diazoketone. 139 00:07:22,110 --> 00:07:24,030 This is a natural product, and it 140 00:07:24,030 --> 00:07:26,910 was discovered by Buchanan's lab at MIT, 141 00:07:26,910 --> 00:07:30,930 and it was the first diazo compound that people had seen. 142 00:07:30,930 --> 00:07:32,693 And it inhibits all-- 143 00:07:32,693 --> 00:07:34,110 this is something that's important 144 00:07:34,110 --> 00:07:36,060 when thinking about what's happening 145 00:07:36,060 --> 00:07:40,080 when you're treating cells with it to stop purine metabolism-- 146 00:07:40,080 --> 00:07:42,750 it inhibits all glutamine-requiring enzymes, 147 00:07:42,750 --> 00:07:44,832 because the mechanisms are similar. 148 00:07:44,832 --> 00:07:47,040 So the mechanism, if you sit down and think about it, 149 00:07:47,040 --> 00:07:48,490 is pretty simple. 150 00:07:48,490 --> 00:07:52,050 You have a diazo group, and now the proposal 151 00:07:52,050 --> 00:07:54,600 is that this needs to be protonated 152 00:07:54,600 --> 00:07:56,910 by the cysteine in the active site. 153 00:07:56,910 --> 00:08:03,270 And now you have an N2 to that's dying to leave, N2+, 154 00:08:03,270 --> 00:08:06,120 and so you just do an SN2 reaction forming a covalent 155 00:08:06,120 --> 00:08:07,320 bond. 156 00:08:07,320 --> 00:08:11,380 That's the basis for how azaserine in the Bankovic paper 157 00:08:11,380 --> 00:08:11,880 works. 158 00:08:11,880 --> 00:08:13,422 There was another way that they block 159 00:08:13,422 --> 00:08:15,510 the pathway, which hopefully we'll have time 160 00:08:15,510 --> 00:08:17,220 to come back to in the end. 161 00:08:17,220 --> 00:08:20,220 So, again, this idea of coming together and going apart-- how 162 00:08:20,220 --> 00:08:22,020 do you perturb this? 163 00:08:22,020 --> 00:08:25,320 One way they perturbed it was depletion of purines. 164 00:08:25,320 --> 00:08:26,550 We discussed that. 165 00:08:26,550 --> 00:08:29,850 We didn't really discuss this particular step. 166 00:08:32,549 --> 00:08:33,870 The next step in this pathway. 167 00:08:37,950 --> 00:08:42,000 Now we have R, which is ribose 5-phosphate. 168 00:08:42,000 --> 00:08:45,840 I'm not going to write that out, because every single step now 169 00:08:45,840 --> 00:08:49,020 has ribose 5-phosphate as a scaffold. 170 00:08:49,020 --> 00:08:50,460 And what we added was glycine. 171 00:08:57,140 --> 00:09:06,865 Again, here's the first time that we need to use ATP and Pi. 172 00:09:09,152 --> 00:09:11,360 Lots of times, you don't know, when you look at this, 173 00:09:11,360 --> 00:09:14,845 whether you're going to transfer pyrophosphate 174 00:09:14,845 --> 00:09:16,220 or you're going to phosphorylate, 175 00:09:16,220 --> 00:09:20,210 so where you have attack on your ATP. 176 00:09:20,210 --> 00:09:22,820 Almost all the enzymes, but not all of them, in the period 177 00:09:22,820 --> 00:09:26,180 pathway have ATP going to ADP, so that tells you 178 00:09:26,180 --> 00:09:30,710 the attack has to be on the gamma position. 179 00:09:30,710 --> 00:09:38,300 This is an ATP grasp superfamily member, 180 00:09:38,300 --> 00:09:41,180 and they all go through the same mechanism, which I briefly 181 00:09:41,180 --> 00:09:44,510 talked about last time, so I'm not going to write this out 182 00:09:44,510 --> 00:09:47,420 again, but basically, you're going 183 00:09:47,420 --> 00:09:55,740 to go through a phosphoanhydride, which is then 184 00:09:55,740 --> 00:09:59,940 attacked by a nucleophile. 185 00:09:59,940 --> 00:10:04,740 We're converting the hydroxyl group of the carboxylic acid 186 00:10:04,740 --> 00:10:07,320 into a good leaving group. 187 00:10:07,320 --> 00:10:09,390 You've seen this used over and over again 188 00:10:09,390 --> 00:10:10,810 over the course of the semester. 189 00:10:10,810 --> 00:10:15,600 But over here, this is all written out for you. 190 00:10:15,600 --> 00:10:17,220 Here we have glycine. 191 00:10:17,220 --> 00:10:19,860 R is CH2NH2. 192 00:10:19,860 --> 00:10:22,830 You phosphorylate to form the anhydride. 193 00:10:22,830 --> 00:10:26,520 You still need to neutralize this to make it 194 00:10:26,520 --> 00:10:31,080 into a good leaving group, which is done in the active site, 195 00:10:31,080 --> 00:10:34,230 and then you can have a variety of nucleophiles 196 00:10:34,230 --> 00:10:38,820 that could come in and attack to form the covalent linkage. 197 00:10:38,820 --> 00:10:42,960 In this case, the nucleophile is not the NH3+. 198 00:10:42,960 --> 00:10:44,560 It needs to be converted to the NH2-- 199 00:10:48,280 --> 00:10:48,780 Sorry. 200 00:10:48,780 --> 00:10:50,480 The nucleophile is over here. 201 00:10:50,480 --> 00:10:51,990 It's phosphoribosylamine. 202 00:10:51,990 --> 00:10:55,830 So it's the NH2 of the phosphoribosylamine that's 203 00:10:55,830 --> 00:10:57,720 attacking. 204 00:10:57,720 --> 00:11:00,450 Again, to be a nucleophile, it's got to be deprotonated. 205 00:11:00,450 --> 00:11:02,550 Hopefully, you all know that at this stage. 206 00:11:05,700 --> 00:11:07,590 So what do these enzymes look like? 207 00:11:07,590 --> 00:11:09,570 They all look the same. 208 00:11:09,570 --> 00:11:12,690 It turns out that if you look at, globally, 209 00:11:12,690 --> 00:11:16,230 purine biosynthesis, not just focus on mammalian systems, 210 00:11:16,230 --> 00:11:18,330 there are four or five enzymes that 211 00:11:18,330 --> 00:11:20,370 actually are ATP grasp superfamily 212 00:11:20,370 --> 00:11:23,120 members in the purine pathway. 213 00:11:23,120 --> 00:11:25,200 And they all look like this. 214 00:11:25,200 --> 00:11:27,860 They have a little domain with a lid, 215 00:11:27,860 --> 00:11:29,850 and all the chemistry happens in between, 216 00:11:29,850 --> 00:11:32,220 and the lid opens and closes. 217 00:11:32,220 --> 00:11:36,360 You can pick these out by bioinformatics. 218 00:11:36,360 --> 00:11:39,810 That's the second step in the pathway. 219 00:11:39,810 --> 00:11:44,150 And this just shows what all of the products can be, 220 00:11:44,150 --> 00:11:48,180 so if you go back and you pull out the pathway, 221 00:11:48,180 --> 00:11:50,802 there are ATP grasp superfamily members, 222 00:11:50,802 --> 00:11:52,260 and these are the products that are 223 00:11:52,260 --> 00:11:55,170 formed by this common type of mechanism 224 00:11:55,170 --> 00:11:56,950 through a phosphoanhydride. 225 00:11:59,940 --> 00:12:01,590 The next step in the pathway. 226 00:12:01,590 --> 00:12:02,940 So now we formed-- 227 00:12:10,480 --> 00:12:13,120 The next step in this pathway, let's see if I put this. 228 00:12:18,880 --> 00:12:19,460 All right. 229 00:12:19,460 --> 00:12:20,610 Sorry. 230 00:12:20,610 --> 00:12:22,340 I thought I put another copy of this in. 231 00:12:22,340 --> 00:12:26,630 The next step in the pathway is we need to formylate. 232 00:12:26,630 --> 00:12:28,370 What do we use as formylation? 233 00:12:28,370 --> 00:12:30,620 That's why we spent the introductory part 234 00:12:30,620 --> 00:12:32,780 of this course talking about folates, 235 00:12:32,780 --> 00:12:36,440 which can transfer carbon at three different oxidation 236 00:12:36,440 --> 00:12:37,520 levels. 237 00:12:37,520 --> 00:12:40,820 What you have here is, and I'm not 238 00:12:40,820 --> 00:12:43,310 going to draw the whole thing out, 239 00:12:43,310 --> 00:12:46,100 this is the part I told you was the business end. 240 00:12:51,730 --> 00:12:53,830 This is N10-formyltetrahydrofolate. 241 00:12:59,500 --> 00:13:02,980 Theoretically, this could be either here or here, 242 00:13:02,980 --> 00:13:06,100 and chemically they can actually interconvert 243 00:13:06,100 --> 00:13:08,210 under certain kinds of conditions. 244 00:13:08,210 --> 00:13:11,020 But we know, for all purine pathways 245 00:13:11,020 --> 00:13:14,470 that people have looked at, it's always the N10. 246 00:13:14,470 --> 00:13:16,270 That's distinct from methylation, 247 00:13:16,270 --> 00:13:17,860 where it's always from the N5. 248 00:13:17,860 --> 00:13:19,840 I don't know how things evolved, but that's 249 00:13:19,840 --> 00:13:21,880 what the results are. 250 00:13:21,880 --> 00:13:23,260 How does this happen? 251 00:13:23,260 --> 00:13:25,780 Hopefully, you all know this without me 252 00:13:25,780 --> 00:13:29,170 having to write this down, but this needs to be a nucleophile. 253 00:13:29,170 --> 00:13:31,780 It needs to be deprotonated. 254 00:13:31,780 --> 00:13:36,580 You need a base to remove a proton, 255 00:13:36,580 --> 00:13:39,250 and then you form a tetrahedral adduct, and then 256 00:13:39,250 --> 00:13:43,960 the tetrahedral adduct high energy intermediate collapses, 257 00:13:43,960 --> 00:13:51,040 and the formyl group gets transferred from here to here. 258 00:13:51,040 --> 00:14:02,770 This then becomes a molecule that looks like that. 259 00:14:02,770 --> 00:14:06,680 I've just transferred the formyl group, which is called FGAR. 260 00:14:11,650 --> 00:14:14,020 Formylglycinamide ribonucleotide. 261 00:14:14,020 --> 00:14:15,070 Horrible names. 262 00:14:15,070 --> 00:14:16,450 This molecule is unstable. 263 00:14:16,450 --> 00:14:19,750 It loses its formyl group actually quite rapidly. 264 00:14:19,750 --> 00:14:22,000 It took them a long time to figure this out. 265 00:14:22,000 --> 00:14:24,160 One of the premises is a purine pathway, 266 00:14:24,160 --> 00:14:27,280 because people were interested in it, is that it falls apart. 267 00:14:27,280 --> 00:14:29,230 When you're trying to look at metabolomics, 268 00:14:29,230 --> 00:14:30,970 which is the next decade-- 269 00:14:30,970 --> 00:14:33,730 hundreds of people are using mass spec, which you guys have 270 00:14:33,730 --> 00:14:36,190 thought about, to look for metabolites-- 271 00:14:36,190 --> 00:14:39,490 you need to know something about the stability of the molecules 272 00:14:39,490 --> 00:14:41,410 you're looking for, and how you separate them 273 00:14:41,410 --> 00:14:43,370 from everything else. 274 00:14:43,370 --> 00:14:45,550 So this is going to be a major focus, 275 00:14:45,550 --> 00:14:48,420 and most people haven't found very many intermediates 276 00:14:48,420 --> 00:14:50,140 in this pathway, and I guarantee you 277 00:14:50,140 --> 00:14:53,680 it's because they break down. 278 00:14:53,680 --> 00:14:58,180 I think that was clear from Buchanan's work really early 279 00:14:58,180 --> 00:14:59,470 on. 280 00:14:59,470 --> 00:15:02,800 The next enzyme in the pathway. 281 00:15:02,800 --> 00:15:06,310 We've seen this, again, before. 282 00:15:06,310 --> 00:15:11,560 Now we're going from an amide to an amidine. 283 00:15:11,560 --> 00:15:13,330 That's all we're doing, so an oxygen 284 00:15:13,330 --> 00:15:15,010 is being replaced by ammonia. 285 00:15:15,010 --> 00:15:16,660 So what are we going to use? 286 00:15:16,660 --> 00:15:18,730 We use glutamine. 287 00:15:18,730 --> 00:15:25,690 The next enzyme in the pathway uses glutamine to glutamate, 288 00:15:25,690 --> 00:15:30,830 and again, this is the source of ammonia. 289 00:15:30,830 --> 00:15:35,950 As I showed you before, there's a channel where this happens. 290 00:15:35,950 --> 00:15:44,170 This is another way you can use ATP going to ADP and Pi. 291 00:15:44,170 --> 00:15:48,340 This is the second kind of mechanism. 292 00:15:48,340 --> 00:15:54,250 This enzyme is called PurL. 293 00:15:54,250 --> 00:15:57,310 Anyhow, we're using ATP again. 294 00:15:57,310 --> 00:15:59,800 Why are we using ATP in this case? 295 00:15:59,800 --> 00:16:02,770 What we're trying to do is convert this amide 296 00:16:02,770 --> 00:16:03,880 into an amidine. 297 00:16:16,670 --> 00:16:19,460 We're converting this into this. 298 00:16:19,460 --> 00:16:21,530 So we need a source of ammonia. 299 00:16:21,530 --> 00:16:23,720 That's the source of ammonia. 300 00:16:23,720 --> 00:16:27,270 What we have is, we're using ATP to facilitate a dehydration 301 00:16:27,270 --> 00:16:27,770 reaction. 302 00:16:27,770 --> 00:16:30,560 Again, you've seen this before with a carboxylic acid. 303 00:16:30,560 --> 00:16:34,370 Now we're doing it with the oxygen of the amide. 304 00:16:34,370 --> 00:16:42,650 The ATP is used to remove oxygen of the amide. 305 00:16:47,230 --> 00:16:48,980 What I'm going to show you, and then we'll 306 00:16:48,980 --> 00:16:55,730 come back to this again, is the generic mechanism for this. 307 00:16:55,730 --> 00:16:58,190 Let me show you now, before we move on, 308 00:16:58,190 --> 00:16:59,870 the next enzyme in the pathway. 309 00:16:59,870 --> 00:17:02,720 Here is using glutamine, and we use 310 00:17:02,720 --> 00:17:07,970 ATP to help us attach the glutamine to the carbonyl. 311 00:17:07,970 --> 00:17:11,037 The next enzyme in the pathway. 312 00:17:11,037 --> 00:17:13,579 What you're doing, basically, I'll show you this in a second, 313 00:17:13,579 --> 00:17:15,890 but you're just cyclizing. 314 00:17:15,890 --> 00:17:18,950 This amino group becomes this amino group, 315 00:17:18,950 --> 00:17:23,780 and this guy has to attack that position. 316 00:17:23,780 --> 00:17:25,910 That position, again, is an amide, 317 00:17:25,910 --> 00:17:29,450 and the mechanism, again, uses ATP, just 318 00:17:29,450 --> 00:17:34,010 like this enzyme, PurL, and I'm going to show you how it works. 319 00:17:34,010 --> 00:17:36,620 These two enzymes in the pathway are structurally 320 00:17:36,620 --> 00:17:38,840 homologous to each other. 321 00:17:38,840 --> 00:17:42,800 The product of one enzyme is the substrate 322 00:17:42,800 --> 00:17:45,303 for the next enzyme in the pathway, 323 00:17:45,303 --> 00:17:46,970 and they clearly evolve from each other. 324 00:17:46,970 --> 00:17:49,220 This is something that everybody's been interested in. 325 00:17:49,220 --> 00:17:51,350 How can you tell something about the evolution 326 00:17:51,350 --> 00:17:54,887 of a biosynthetic pathway, and thinking 327 00:17:54,887 --> 00:17:55,970 about how to control this. 328 00:17:55,970 --> 00:17:56,180 Why? 329 00:17:56,180 --> 00:17:57,555 Because everybody and his brother 330 00:17:57,555 --> 00:18:00,590 now is focused on bioengineering of metabolic pathways. 331 00:18:00,590 --> 00:18:03,928 So the more you understand about the basic principles of how 332 00:18:03,928 --> 00:18:05,720 nature designed this, the better off you're 333 00:18:05,720 --> 00:18:08,870 going to be in trying to get this to happen robustly 334 00:18:08,870 --> 00:18:14,480 and control things by using an enzymatic system and enzymes 335 00:18:14,480 --> 00:18:16,670 from many different sources. 336 00:18:16,670 --> 00:18:20,720 So what's the generic mechanism? 337 00:18:20,720 --> 00:18:26,640 This is called-- this enzyme is part of the PurM-- 338 00:18:26,640 --> 00:18:30,290 the nomenclature is horrible-- superfamily. 339 00:18:30,290 --> 00:18:34,100 So I just told you this ATP was the ATP grasp superfamily. 340 00:18:34,100 --> 00:18:37,340 This is the PurM. 341 00:18:37,340 --> 00:18:39,590 Why is it called the PurM superfamily? 342 00:18:39,590 --> 00:18:43,140 Because it was the first structure of any molecule 343 00:18:43,140 --> 00:18:46,217 that looked like this, and it was the PurM enzyme. 344 00:18:46,217 --> 00:18:48,050 So that's where the horrible name came from. 345 00:18:50,570 --> 00:18:55,370 This enzyme is PurL, and this enzyme is PurN, 346 00:18:55,370 --> 00:18:57,740 and they're structurally homologous to each other. 347 00:18:57,740 --> 00:19:00,750 How do they work? 348 00:19:00,750 --> 00:19:05,300 Again, I think once you see it. 349 00:19:05,300 --> 00:19:07,610 Here's the general mechanism. 350 00:19:07,610 --> 00:19:12,560 Here we have our amide, and what we want to do 351 00:19:12,560 --> 00:19:19,010 is facilitate dehydration of the oxygen. What you're going to do 352 00:19:19,010 --> 00:19:24,340 is phosphorylate the oxygen of the amide. 353 00:19:24,340 --> 00:19:28,570 Now what you have is a system that 354 00:19:28,570 --> 00:19:33,010 is activated for nucleophilic attack by a nucleophile. 355 00:19:33,010 --> 00:19:35,380 That's the generic mechanism. 356 00:19:35,380 --> 00:19:45,130 There is a generic mechanism where you simply 357 00:19:45,130 --> 00:19:46,510 phosphorylate this. 358 00:19:53,890 --> 00:19:56,800 Now, if this is positively charged, 359 00:19:56,800 --> 00:20:00,020 this is activated for nucleophilic attack, 360 00:20:00,020 --> 00:20:02,110 and then you lose phosphate. 361 00:20:04,850 --> 00:20:08,270 People have studied this over the course of years, 362 00:20:08,270 --> 00:20:11,540 and the mechanism for this is understood. 363 00:20:11,540 --> 00:20:15,140 I don't have the structures but, again, this enzyme and then 364 00:20:15,140 --> 00:20:21,710 the next enzyme in the pathway use the same sort of approach. 365 00:20:21,710 --> 00:20:29,390 The next enzyme in the pathway takes the amidine. 366 00:20:34,590 --> 00:20:36,915 What it's going to form is a cyclized product. 367 00:20:50,400 --> 00:20:53,710 This is aminoimidazole ribonucleotide. 368 00:20:53,710 --> 00:20:55,020 So we finally found-- 369 00:20:55,020 --> 00:20:57,990 Remember, I told you, you form the imidazole ring, 370 00:20:57,990 --> 00:21:05,730 and then you're going to put on the pyrimidine ring afterwards. 371 00:21:05,730 --> 00:21:07,620 How does this happen? 372 00:21:07,620 --> 00:21:10,440 It looks sort of wonky. 373 00:21:10,440 --> 00:21:13,020 But what you can see is that this guy-- 374 00:21:13,020 --> 00:21:18,240 so let's just put a box around this guy-- becomes this guy. 375 00:21:21,780 --> 00:21:25,800 This guy is where we're doing the chemistry. 376 00:21:25,800 --> 00:21:27,890 That's the one we're going to attach, 377 00:21:27,890 --> 00:21:29,820 we're going to phosphorylate. 378 00:21:29,820 --> 00:21:34,290 What you have here, now, is an intramolecular attack. 379 00:21:34,290 --> 00:21:37,660 So, the nucleophile, instead of being ammonia, 380 00:21:37,660 --> 00:21:41,940 which is external, now happens intramolecularly. 381 00:21:41,940 --> 00:21:46,560 In the end, after you activate this, 382 00:21:46,560 --> 00:21:50,100 you get intramolecular chemistry. 383 00:21:50,100 --> 00:21:52,290 This was the site. 384 00:21:52,290 --> 00:21:57,450 This was the site that was activated in the beginning. 385 00:21:57,450 --> 00:21:59,460 The chemistry in these two systems 386 00:21:59,460 --> 00:22:03,330 is pretty much the same, and now we've got our imidazole ring, 387 00:22:03,330 --> 00:22:06,690 and now what we need to do is build up 388 00:22:06,690 --> 00:22:10,190 the rest of this system. 389 00:22:10,190 --> 00:22:12,180 Is everybody with me, or am I going too fast? 390 00:22:12,180 --> 00:22:13,380 I'm probably going too fast. 391 00:22:19,380 --> 00:22:22,410 Anyhow, that gives you the generic mechanism for this. 392 00:22:22,410 --> 00:22:24,810 I didn't draw the structures all out. 393 00:22:24,810 --> 00:22:26,830 The folates we've already talked about. 394 00:22:26,830 --> 00:22:29,523 So I'm not going to talk about that again. 395 00:22:29,523 --> 00:22:31,440 We're going to see the folate-requiring enzyme 396 00:22:31,440 --> 00:22:33,750 again later on in the pathway. 397 00:22:33,750 --> 00:22:36,900 Now the pathway just repeats itself. 398 00:22:36,900 --> 00:22:38,670 Really, I think what's most striking, 399 00:22:38,670 --> 00:22:42,570 this is really an ancient pathway. 400 00:22:42,570 --> 00:22:48,420 There are huge numbers of ATPs used in this pathway. 401 00:22:48,420 --> 00:22:53,640 I think, if any of you wind up thinking about cancer therapy 402 00:22:53,640 --> 00:22:56,130 and stuff, and whether you have de novo biosynthesis 403 00:22:56,130 --> 00:22:59,025 because you need a lot of purines fast, 404 00:22:59,025 --> 00:23:02,580 or whether you use salvage, this really 405 00:23:02,580 --> 00:23:06,810 requires a huge amount of energy to make this pathway actually 406 00:23:06,810 --> 00:23:09,030 work. 407 00:23:09,030 --> 00:23:10,980 Now we have this molecule, and then 408 00:23:10,980 --> 00:23:14,520 the next step in this pathway. 409 00:23:14,520 --> 00:23:21,640 In the human system, what you do in the human system-- 410 00:23:21,640 --> 00:23:23,020 this-- it's not right. 411 00:23:26,480 --> 00:23:29,150 This enzyme, cross this off. 412 00:23:29,150 --> 00:23:30,450 This is a Bankovic's lie. 413 00:23:30,450 --> 00:23:31,340 Cross that off. 414 00:23:31,340 --> 00:23:33,620 It doesn't use ATP. 415 00:23:33,620 --> 00:23:36,020 So you need to cross that off. 416 00:23:36,020 --> 00:23:37,940 It just picks up CO2. 417 00:23:37,940 --> 00:23:42,207 If you look at this, what do you have happening here? 418 00:23:42,207 --> 00:23:43,790 We're going to go from here, and we're 419 00:23:43,790 --> 00:23:46,040 going to pick up CO2 there. 420 00:23:46,040 --> 00:23:51,620 CO2 actually can react really rapidly at this position. 421 00:23:51,620 --> 00:23:57,020 So you need CO2, and let me write this down. 422 00:23:57,020 --> 00:23:58,010 No ATP. 423 00:24:01,130 --> 00:24:01,940 I don't know why. 424 00:24:01,940 --> 00:24:03,857 I probably didn't look at this very carefully, 425 00:24:03,857 --> 00:24:08,810 but there's no ATP required for this step. 426 00:24:08,810 --> 00:24:09,920 What's unusual? 427 00:24:09,920 --> 00:24:11,780 Do you think it's unusual to use CO2? 428 00:24:15,660 --> 00:24:16,500 This is called PurE. 429 00:24:19,260 --> 00:24:27,680 How much CO2 is there inside the cell 430 00:24:27,680 --> 00:24:36,530 at physiological concentrations? 431 00:24:36,530 --> 00:24:37,940 Think there's a lot or a little? 432 00:24:41,910 --> 00:24:46,230 Where have you seen CO2 used before? 433 00:24:46,230 --> 00:24:48,390 Remember fatty acid biosynthesis? 434 00:24:48,390 --> 00:24:52,900 Do you use CO2 in fatty acid biosynthesis? 435 00:24:52,900 --> 00:24:54,682 Anybody remember? 436 00:24:54,682 --> 00:24:56,446 AUDIENCE: [INAUDIBLE] 437 00:24:56,446 --> 00:24:59,360 JOANNE STUBBE: The what? 438 00:24:59,360 --> 00:25:00,830 Anybody know how you-- 439 00:25:00,830 --> 00:25:02,510 do you use CO2 directly? 440 00:25:02,510 --> 00:25:04,510 AUDIENCE: [INAUDIBLE] 441 00:25:04,510 --> 00:25:06,810 JOANNE STUBBE: We'll use bicarbonate? 442 00:25:06,810 --> 00:25:09,410 OK, why do you use bicarbonate? 443 00:25:09,410 --> 00:25:11,870 That's where the equilibrium is at pH 7. 444 00:25:11,870 --> 00:25:16,370 There is almost no CO2 unless you go down to acidic pHs, 445 00:25:16,370 --> 00:25:18,173 so almost no enzymes use CO2. 446 00:25:18,173 --> 00:25:18,965 So this is unusual. 447 00:25:22,160 --> 00:25:25,100 That's also true of biotin. 448 00:25:25,100 --> 00:25:28,430 And, in fact, so this is the human enzyme, 449 00:25:28,430 --> 00:25:31,410 and it generates that product. 450 00:25:31,410 --> 00:25:36,710 In bacterial systems, it turns out 451 00:25:36,710 --> 00:25:42,530 that it does use bicarbonate and ATP, 452 00:25:42,530 --> 00:25:51,180 and generates a carbomate of the same molecule. 453 00:25:56,588 --> 00:25:58,630 What do we know about the stability of carbonate? 454 00:25:58,630 --> 00:26:02,520 So, number one, why are we using bicarbonate and ATP? 455 00:26:02,520 --> 00:26:05,640 Have you seen that before? 456 00:26:05,640 --> 00:26:08,313 What does ATP due to the bicarbonate? 457 00:26:14,240 --> 00:26:16,290 We just saw this reaction two seconds ago. 458 00:26:20,030 --> 00:26:25,049 What does ATP do to bicarbonate? 459 00:26:25,049 --> 00:26:26,216 AUDIENCE: Phosphorylates it. 460 00:26:26,216 --> 00:26:27,841 JOANNE STUBBE: Yeah, phosphorylates it. 461 00:26:35,390 --> 00:26:37,280 You need to neutralize your charges. 462 00:26:37,280 --> 00:26:40,190 These are all magnesium ATP and you 463 00:26:40,190 --> 00:26:42,500 form carboxyphosphate, which has a lifetime 464 00:26:42,500 --> 00:26:44,090 on the order of a millisecond. 465 00:26:44,090 --> 00:26:47,360 That's the way biotin is made inside the cell. 466 00:26:47,360 --> 00:26:51,530 Almost all organisms do not use CO2, they use bicarbonate. 467 00:26:51,530 --> 00:26:53,630 And to activate bicarbonate, the other reason-- 468 00:26:53,630 --> 00:26:55,070 What's wrong with CO2? 469 00:26:55,070 --> 00:26:57,020 How do you hold on CO2? 470 00:26:57,020 --> 00:26:59,773 You think that's easy to bind in the active site? 471 00:26:59,773 --> 00:27:01,190 No, there's nothing to hold on to. 472 00:27:01,190 --> 00:27:02,780 There's no charge. 473 00:27:02,780 --> 00:27:04,550 It's symmetrical. 474 00:27:04,550 --> 00:27:07,220 So what nature does is put bicarbonate, 475 00:27:07,220 --> 00:27:09,620 which is charged, into the active site, 476 00:27:09,620 --> 00:27:13,340 and uses ATP to phosphorylate it, 477 00:27:13,340 --> 00:27:17,900 to form carboxyphosphate, which then 478 00:27:17,900 --> 00:27:20,730 reacts with the nucleophile, in this case the amino group. 479 00:27:20,730 --> 00:27:22,168 Yeah. 480 00:27:22,168 --> 00:27:23,210 Did I screw something up? 481 00:27:23,210 --> 00:27:24,970 AUDIENCE: So you do need ATP? 482 00:27:24,970 --> 00:27:28,100 JOANNE STUBBE: You do need ATP for the bicarbonate-dependent 483 00:27:28,100 --> 00:27:29,120 reaction. 484 00:27:29,120 --> 00:27:31,100 So, there are two different reactions. 485 00:27:31,100 --> 00:27:38,170 This is eucaryotes, and this is bacteria. 486 00:27:38,170 --> 00:27:41,560 They have two different pathways. 487 00:27:41,560 --> 00:27:46,210 I think this is sort of amazing, because what happens now 488 00:27:46,210 --> 00:27:51,060 is the bacterial enzyme then takes this, and generates this. 489 00:27:51,060 --> 00:27:53,665 So nobody even knew that this intermediate-- my lab 490 00:27:53,665 --> 00:27:56,040 discovered this a long time ago-- existed on the pathway. 491 00:27:56,040 --> 00:27:56,540 Why? 492 00:27:56,540 --> 00:27:58,920 Because its half-life is on the order of 15 seconds. 493 00:27:58,920 --> 00:28:00,210 Carbomates. 494 00:28:00,210 --> 00:28:04,800 That's how you carry CO2 from the tissues back to the lungs. 495 00:28:04,800 --> 00:28:10,170 It's carried on the surface with lysines forming carbomates, 496 00:28:10,170 --> 00:28:14,190 and what's striking is that these enzymes-- 497 00:28:14,190 --> 00:28:22,440 one enzyme uses this substrate, one uses this substrate. 498 00:28:22,440 --> 00:28:25,860 The proteins are structurally homologous to each other. 499 00:28:25,860 --> 00:28:27,990 Nobody really understands that. 500 00:28:27,990 --> 00:28:31,710 Nature has done a shift on what normally happens 501 00:28:31,710 --> 00:28:33,570 in the eukaryotic system. 502 00:28:33,570 --> 00:28:37,050 CO2 is added in the procaryotic system. 503 00:28:37,050 --> 00:28:40,440 You need bicarbonate, and you need CO2, 504 00:28:40,440 --> 00:28:43,200 and I think that tells you something about where 505 00:28:43,200 --> 00:28:45,300 these things evolved. 506 00:28:45,300 --> 00:28:46,770 What's the pH? 507 00:28:46,770 --> 00:28:48,960 And was there enough CO2 to be able to do 508 00:28:48,960 --> 00:28:53,380 these kinds of experiments over the evolution of these systems? 509 00:28:53,380 --> 00:28:57,270 I think things change. 510 00:28:57,270 --> 00:29:05,400 You've now produced this molecule, which is called CAIR. 511 00:29:05,400 --> 00:29:10,680 So, that's carboxyaminoimidazole ribonucleotide. 512 00:29:13,270 --> 00:29:16,220 Then the next step in the pathway. 513 00:29:16,220 --> 00:29:20,500 Now, we only need this and another carbon 514 00:29:20,500 --> 00:29:23,560 to complete the pyrimidine ring. 515 00:29:23,560 --> 00:29:24,910 It turns out aspartic acid-- 516 00:29:24,910 --> 00:29:32,200 which is also a major player in pyrimidine biosynthesis-- 517 00:29:32,200 --> 00:29:37,840 this nitrogen is going to come from aspartic acid. 518 00:29:37,840 --> 00:29:38,920 What are we going to do? 519 00:29:38,920 --> 00:29:42,790 We need to activate this carboxylate 520 00:29:42,790 --> 00:29:45,940 to attach the amino group of aspartic acid. 521 00:29:45,940 --> 00:29:48,160 How do we do that? 522 00:29:48,160 --> 00:29:49,390 With ATP. 523 00:29:49,390 --> 00:29:54,250 We phosphorylate it, and then we have nucleophilic attack. 524 00:29:54,250 --> 00:29:57,670 I'm going to go up onto the board up there, 525 00:29:57,670 --> 00:30:00,340 so you can still see what's happening. 526 00:30:00,340 --> 00:30:01,255 This next reaction. 527 00:30:07,340 --> 00:30:11,900 CAIR now reacts with aspartic acid. 528 00:30:21,560 --> 00:30:28,610 And we need, again, ATP, and we go to ADP. 529 00:30:28,610 --> 00:30:32,165 Now what we have, aspartic acid, we form an amide linkage. 530 00:30:45,360 --> 00:30:49,530 R is ribose 5-phosphate. 531 00:30:49,530 --> 00:30:53,130 What we've done now is attached-- 532 00:30:53,130 --> 00:30:55,410 we're deviating, but we're going to see it 533 00:30:55,410 --> 00:30:57,030 near the end of the purine pathway. 534 00:30:57,030 --> 00:31:00,570 We use this strategy again. 535 00:31:00,570 --> 00:31:04,600 Almost all the time, if you have to guess at this, 536 00:31:04,600 --> 00:31:07,278 the source of ammonia or nitrogen 537 00:31:07,278 --> 00:31:08,320 is going to be glutamine. 538 00:31:08,320 --> 00:31:10,528 So if you don't know and you're seeing a new pathway, 539 00:31:10,528 --> 00:31:12,030 use glutamine. 540 00:31:12,030 --> 00:31:13,552 But here's an example where nature 541 00:31:13,552 --> 00:31:14,760 has used something different. 542 00:31:14,760 --> 00:31:17,250 She's used aspartic acid. 543 00:31:17,250 --> 00:31:25,350 And the ATP is, again, activating the carboxylate. 544 00:31:25,350 --> 00:31:30,000 So we're using the same strategy over and over and over again. 545 00:31:30,000 --> 00:31:33,620 Then the next enzyme in this pathway. 546 00:31:33,620 --> 00:31:34,620 What are we going to do? 547 00:31:34,620 --> 00:31:37,560 We convert this intermediate called SAICAR. 548 00:31:37,560 --> 00:31:39,540 We now lose fumarate. 549 00:31:39,540 --> 00:31:41,070 Where have you seen fumarate before? 550 00:31:41,070 --> 00:31:43,920 Does everybody know what fumarate is? 551 00:31:43,920 --> 00:31:46,200 That's an intermediate in the TCA cycle. 552 00:31:46,200 --> 00:31:48,000 This is an anaplerotic pathway, and you've 553 00:31:48,000 --> 00:31:50,970 got to feed the fumarate back in. 554 00:31:50,970 --> 00:31:52,120 What are we doing here? 555 00:31:52,120 --> 00:31:54,180 We're going to lose fumarate, which 556 00:31:54,180 --> 00:31:59,400 has the two carboxylates transfer the double bond. 557 00:31:59,400 --> 00:32:03,010 We're going to do an elimination reaction. 558 00:32:03,010 --> 00:32:09,130 The next step in this pathway is catalyzed by PurB, 559 00:32:09,130 --> 00:32:12,190 and we'll see that nature uses the same strategy 560 00:32:12,190 --> 00:32:15,420 to convert IMP into AMP at the end of the pathway. 561 00:32:15,420 --> 00:32:17,980 Uses the same enzyme, actually. 562 00:32:17,980 --> 00:32:20,920 So you lose fumarate. 563 00:32:20,920 --> 00:32:24,130 So what we're doing now is we're going to do-- 564 00:32:24,130 --> 00:32:26,390 actually, the enzymes have been very well studied. 565 00:32:26,390 --> 00:32:29,100 We have structures of all these things. 566 00:32:29,100 --> 00:32:30,520 You use fumarate. 567 00:32:36,920 --> 00:32:41,270 Now what we have is this guy. 568 00:32:49,070 --> 00:32:52,400 And this guy actually has now been found 569 00:32:52,400 --> 00:32:55,710 as a regulator of glycolysis. 570 00:32:55,710 --> 00:32:57,267 So we're linking now. 571 00:32:57,267 --> 00:32:59,100 You're going to see this, and I think you're 572 00:32:59,100 --> 00:32:59,970 going to see more of this. 573 00:32:59,970 --> 00:33:01,720 The only reason these guys have been found 574 00:33:01,720 --> 00:33:03,510 is this guy's pretty stable, so people 575 00:33:03,510 --> 00:33:06,390 can find it using metabolomics. 576 00:33:06,390 --> 00:33:09,390 But this molecule is a regulator of glycolysis, 577 00:33:09,390 --> 00:33:11,370 and I think the more we look, the more we're 578 00:33:11,370 --> 00:33:13,470 going to find basic intermediates 579 00:33:13,470 --> 00:33:16,470 and metabolic pathways controlling fluxes 580 00:33:16,470 --> 00:33:17,430 through other things. 581 00:33:17,430 --> 00:33:21,750 We need glycolysis to ultimately generate energy, 582 00:33:21,750 --> 00:33:24,240 because we need a lot of ATP to synthesize things, 583 00:33:24,240 --> 00:33:26,520 but the connections between all these things, I think, 584 00:33:26,520 --> 00:33:28,970 remains to be established. 585 00:33:28,970 --> 00:33:36,185 So this is involved in regulation of glycolysis. 586 00:33:36,185 --> 00:33:38,250 If I'd had another couple of lectures, 587 00:33:38,250 --> 00:33:42,100 I would have showed you how that fit in. 588 00:33:42,100 --> 00:33:43,230 And then, where are we? 589 00:33:43,230 --> 00:33:44,280 We're not very far away. 590 00:33:44,280 --> 00:33:46,950 We only need one carbon left. 591 00:33:46,950 --> 00:33:49,628 Where do we get the one carbon from? 592 00:33:49,628 --> 00:33:50,516 AUDIENCE: Folate. 593 00:33:50,516 --> 00:33:51,990 JOANNE STUBBE: Yeah, from folate. 594 00:33:51,990 --> 00:33:54,270 So here we have it again. 595 00:33:54,270 --> 00:34:00,360 Now we have N10-formyltetrahydrofolate. 596 00:34:00,360 --> 00:34:02,638 That's why I spent the time in the beginning. 597 00:34:05,680 --> 00:34:09,219 And this guy, through the same kind of a mechanism, 598 00:34:09,219 --> 00:34:13,389 is going to be attached to that guy. 599 00:34:13,389 --> 00:34:17,770 Once we have the one carbon there, then you can cyclize. 600 00:34:17,770 --> 00:34:18,760 You attach that. 601 00:34:38,550 --> 00:34:44,190 Now we're ready to cyclize and lose a molecule of water. 602 00:34:44,190 --> 00:34:52,290 So, the last step is cyclization and loss of water 603 00:34:52,290 --> 00:34:55,096 to form inosine monophosphate. 604 00:34:57,720 --> 00:35:00,510 Inosine monophosphate is the end goal. 605 00:35:00,510 --> 00:35:02,710 That's the first time we now have it purined. 606 00:35:02,710 --> 00:35:06,150 So we have both the imidazole ring and the pyrimidine ring, 607 00:35:06,150 --> 00:35:12,180 generating this purine, which then is the branch point 608 00:35:12,180 --> 00:35:15,830 to form GMP and AMP. 609 00:35:20,310 --> 00:35:24,820 Both of these are going to involve two steps. 610 00:35:24,820 --> 00:35:27,600 And this tells us something about the overall regulation 611 00:35:27,600 --> 00:35:29,310 of the pathway. 612 00:35:29,310 --> 00:35:34,800 Pathways are often regulated by feedback inhibition. 613 00:35:34,800 --> 00:35:38,610 The M-products can come back and inhibit the first step, 614 00:35:38,610 --> 00:35:41,520 so things don't build up. 615 00:35:41,520 --> 00:35:48,690 If we come over here, If we look at PurF, This is a stop. 616 00:35:48,690 --> 00:35:54,670 These are inhibitors-- our AMP and GMP. 617 00:35:58,170 --> 00:36:02,150 We're going to see, in this pathway, 618 00:36:02,150 --> 00:36:09,820 AMP inhibits its own biosynthesis, 619 00:36:09,820 --> 00:36:17,230 and we're also going to see GMP inhibits its own biosynthesis. 620 00:36:17,230 --> 00:36:19,270 So what you see is, ultimately, we 621 00:36:19,270 --> 00:36:22,840 want to control the relative ratios of purines 622 00:36:22,840 --> 00:36:26,080 and pyrimidines, which we're not going to get to, 623 00:36:26,080 --> 00:36:29,650 and these are examples of simple allosteric effectors. 624 00:36:29,650 --> 00:36:33,490 They bind outside the active site and shut things down. 625 00:36:33,490 --> 00:36:35,740 And we actually understand a lot about how that works, 626 00:36:35,740 --> 00:36:36,940 we just don't-- 627 00:36:36,940 --> 00:36:40,190 we're not going to have time to discuss that. 628 00:36:40,190 --> 00:36:43,270 So what we've gotten in to through all of this 629 00:36:43,270 --> 00:36:45,135 is inosine monophosphate. 630 00:36:47,710 --> 00:36:49,730 If you look at the next step in this path. 631 00:36:49,730 --> 00:36:55,950 If we go back here, here is IMP, and we want to go to AMP, 632 00:36:55,950 --> 00:36:58,120 and we want to go to GMP. 633 00:36:58,120 --> 00:37:02,680 If we look at AMP, what do we see? 634 00:37:02,680 --> 00:37:05,470 Have you seen this before? 635 00:37:05,470 --> 00:37:07,840 We're attaching aspartic acid. 636 00:37:07,840 --> 00:37:09,370 Where have we just seen that? 637 00:37:09,370 --> 00:37:12,840 We've just seen aspartic acid attachment. 638 00:37:15,510 --> 00:37:20,760 And what's interesting about this is, instead of using ATP, 639 00:37:20,760 --> 00:37:22,910 it's using GDP. 640 00:37:22,910 --> 00:37:23,960 Is that an accident? 641 00:37:23,960 --> 00:37:24,980 I don't know. 642 00:37:24,980 --> 00:37:29,930 GTP is regulating the flux to form AMP. 643 00:37:29,930 --> 00:37:34,640 So again, AMP, ATP, GTP, you've seen this over and over 644 00:37:34,640 --> 00:37:37,250 again over the course of the semester. 645 00:37:37,250 --> 00:37:39,560 You saw, with translation, it was all GTP. 646 00:37:39,560 --> 00:37:42,680 In other cases, you saw, with folding, with the proteasome, 647 00:37:42,680 --> 00:37:43,790 it's all ATP. 648 00:37:43,790 --> 00:37:47,930 You've got to control all of these ratios. 649 00:37:47,930 --> 00:37:51,470 Here is a place where the ratios are controlled. 650 00:37:51,470 --> 00:37:54,410 So how does this happen? 651 00:37:54,410 --> 00:37:58,900 What are we going to do with the GTP in that molecule? 652 00:37:58,900 --> 00:38:02,400 We want to go from here to here. 653 00:38:02,400 --> 00:38:08,040 This carbonyl is replaced with the nitrogen of aspartic acid. 654 00:38:08,040 --> 00:38:10,705 What are we going to do to that oxygen? 655 00:38:10,705 --> 00:38:11,830 AUDIENCE: Phosphorylate it. 656 00:38:11,830 --> 00:38:13,660 JOANNE STUBBE: Phosphorylate it. 657 00:38:13,660 --> 00:38:16,780 And that's done by GTP rather than ATP. 658 00:38:16,780 --> 00:38:19,180 So what you do is you phosphorylate 659 00:38:19,180 --> 00:38:21,670 through the mechanism that we just went through, 660 00:38:21,670 --> 00:38:25,985 that I wrote over here somewhere. 661 00:38:25,985 --> 00:38:26,860 Where did I write it? 662 00:38:30,850 --> 00:38:31,437 All right. 663 00:38:31,437 --> 00:38:33,645 I can't see where I wrote it, but it's in your notes. 664 00:38:37,260 --> 00:38:40,350 You then have your amino group of aspartic acid, 665 00:38:40,350 --> 00:38:44,280 displaces this, and then what happens in the last step? 666 00:38:44,280 --> 00:38:47,850 This is exactly what we saw over here. 667 00:38:47,850 --> 00:38:49,420 We're kicking out fumarate. 668 00:38:49,420 --> 00:38:50,760 So this is the same enzyme. 669 00:38:50,760 --> 00:38:55,200 So PurB also happens here. 670 00:38:55,200 --> 00:38:57,710 So it's kicking out fumarate. 671 00:38:57,710 --> 00:38:59,390 Now, what about this pathway? 672 00:38:59,390 --> 00:39:00,912 This pathway is of great interest, 673 00:39:00,912 --> 00:39:02,120 because it's a major target-- 674 00:39:05,210 --> 00:39:09,860 when you have a transplant, to prevent rejection-- 675 00:39:09,860 --> 00:39:11,360 of mycophenolic acids. 676 00:39:11,360 --> 00:39:14,000 There are many compounds that inhibit 677 00:39:14,000 --> 00:39:16,280 this step in the pathway, and it's widely 678 00:39:16,280 --> 00:39:21,560 used for organ transplant, subsequent to the transplant. 679 00:39:21,560 --> 00:39:24,950 This is called IMP dehydrogenase. 680 00:39:24,950 --> 00:39:26,540 How do you get from here to here? 681 00:39:26,540 --> 00:39:29,490 This is not so trivial. 682 00:39:29,490 --> 00:39:32,430 What you see, and this is the unusual thing about this, 683 00:39:32,430 --> 00:39:35,490 hopefully now you could actually think about this, 684 00:39:35,490 --> 00:39:37,330 but we're adding an oxygen here. 685 00:39:37,330 --> 00:39:41,700 So, somehow, we have to add water, 686 00:39:41,700 --> 00:39:45,990 and then we're using NAD and ADP, 687 00:39:45,990 --> 00:39:51,570 so we're going to have to do an oxidation and NAD gets reduced. 688 00:39:51,570 --> 00:39:53,550 If you look at this, what happens 689 00:39:53,550 --> 00:39:57,630 is this molecule is activated for nucleophilic attack 690 00:39:57,630 --> 00:40:01,140 at this position to add an OH here. 691 00:40:01,140 --> 00:40:03,180 So what you generate is-- 692 00:40:09,200 --> 00:40:14,150 then this guy needs to get oxidized by NAD. 693 00:40:14,150 --> 00:40:15,440 That's an unusual step. 694 00:40:15,440 --> 00:40:18,380 You should go back and you should think about that. 695 00:40:18,380 --> 00:40:21,650 It took people quite a while to figure this out. 696 00:40:21,650 --> 00:40:22,790 What about the last step? 697 00:40:22,790 --> 00:40:24,110 How does this work? 698 00:40:24,110 --> 00:40:26,230 Where have we seen this before? 699 00:40:26,230 --> 00:40:28,280 Glutamine. 700 00:40:28,280 --> 00:40:34,340 What we're doing is converting this oxygen to an amino group. 701 00:40:34,340 --> 00:40:35,090 What's doing that? 702 00:40:35,090 --> 00:40:38,000 I told you there were five glutamine-requiring enzymes 703 00:40:38,000 --> 00:40:38,630 in the pathway. 704 00:40:38,630 --> 00:40:40,700 This is one of them. 705 00:40:40,700 --> 00:40:43,280 What do we need to do to this oxygen to make it 706 00:40:43,280 --> 00:40:45,610 into a good leaving group? 707 00:40:45,610 --> 00:40:47,380 We need to phosphorylate it. 708 00:40:47,380 --> 00:40:51,320 Use ATP to phosphorylate this, and then 709 00:40:51,320 --> 00:40:56,740 glutamine supplies the ammonia, and that's how you get GMP. 710 00:40:56,740 --> 00:40:58,750 As an exercise, you should go back and think 711 00:40:58,750 --> 00:41:00,190 about these interconversions. 712 00:41:00,190 --> 00:41:04,920 If you have trouble, you can come back and talk to me. 713 00:41:04,920 --> 00:41:11,470 I put on the Stellar site a new version 714 00:41:11,470 --> 00:41:15,730 of a chapter on purines from a book by Appling 715 00:41:15,730 --> 00:41:18,160 that has come out last year. 716 00:41:18,160 --> 00:41:20,530 Within this section, it's by far and away much better 717 00:41:20,530 --> 00:41:22,010 than any of the others. 718 00:41:22,010 --> 00:41:24,940 So those of you who want to look at the chemistry of this, 719 00:41:24,940 --> 00:41:27,430 they've written this all out in detail. 720 00:41:27,430 --> 00:41:29,420 So you can pull it out and flip to that page. 721 00:41:29,420 --> 00:41:30,970 You don't have to read the whole chapter. 722 00:41:30,970 --> 00:41:32,428 You can flip to the page where they 723 00:41:32,428 --> 00:41:34,850 describe all of these things. 724 00:41:34,850 --> 00:41:37,720 So that's the purine pathway. 725 00:41:37,720 --> 00:41:42,490 My goal was to try to show you that everything 726 00:41:42,490 --> 00:41:45,910 has made from ribose 5-phosphate as a scaffold. 727 00:41:45,910 --> 00:41:48,950 You build up the imidazole, you build up the purine, 728 00:41:48,950 --> 00:41:51,310 and you use three types of reactions 729 00:41:51,310 --> 00:41:54,790 that are used over and over and over again in metabolism. 730 00:41:57,865 --> 00:42:00,490 One of the reasons I picked this topic, besides the fact that I 731 00:42:00,490 --> 00:42:04,870 like deoxynucleotides, which I never get to talk about, 732 00:42:04,870 --> 00:42:09,310 is the discovery of what we talked about in recitation 733 00:42:09,310 --> 00:42:13,330 13, this purinosome. 734 00:42:13,330 --> 00:42:15,820 What's the purinosome? 735 00:42:15,820 --> 00:42:23,380 You all know what it is, And we talked about some 736 00:42:23,380 --> 00:42:25,600 of the experiments, but the idea is 737 00:42:25,600 --> 00:42:28,270 that you have proteins from all over the place that 738 00:42:28,270 --> 00:42:30,100 organize transiently. 739 00:42:30,100 --> 00:42:32,680 So you have transient protein-protein interaction 740 00:42:32,680 --> 00:42:34,120 that arise to the occasion. 741 00:42:34,120 --> 00:42:37,780 There's going to be some signaling mechanisms 742 00:42:37,780 --> 00:42:40,210 that they know they're depleted in purines. 743 00:42:40,210 --> 00:42:41,440 That's the model. 744 00:42:41,440 --> 00:42:43,850 They come together, they do their thing. 745 00:42:43,850 --> 00:42:45,280 Why would you want to do this? 746 00:42:48,520 --> 00:42:50,020 The choice that everybody has looked 747 00:42:50,020 --> 00:43:02,890 at has been the purine pathway for this idea of multi enzyme 748 00:43:02,890 --> 00:43:11,450 complexes that form transiently, and I've 749 00:43:11,450 --> 00:43:13,190 asked you this question recitation, 750 00:43:13,190 --> 00:43:15,500 why would you want to do this? 751 00:43:15,500 --> 00:43:19,310 One reason you might want to do this everybody agrees on, 752 00:43:19,310 --> 00:43:21,740 and that's because if you have unstable intermediates, 753 00:43:21,740 --> 00:43:24,740 and these intermediates go into solution, they can degrade. 754 00:43:24,740 --> 00:43:26,330 So that would be a waste. 755 00:43:26,330 --> 00:43:27,290 Is that true? 756 00:43:27,290 --> 00:43:28,470 We don't know. 757 00:43:28,470 --> 00:43:36,503 But one reason would be to protect unstable intermediates. 758 00:43:42,840 --> 00:43:46,020 A second reason that you might want to do this 759 00:43:46,020 --> 00:43:47,940 is if you have a long metabolic pathway-- 760 00:43:47,940 --> 00:43:50,832 this is tensed up, it's a long pathway-- 761 00:43:50,832 --> 00:43:53,040 oftentimes, in the middle, you can have branch points 762 00:43:53,040 --> 00:43:55,290 to other pathways. 763 00:43:55,290 --> 00:43:58,230 Say you want your intermediate to go this way and not 764 00:43:58,230 --> 00:43:59,160 that way. 765 00:43:59,160 --> 00:44:04,650 If you have this organized, you can control where it goes. 766 00:44:04,650 --> 00:44:11,400 If you have a pathway, and you have some intermediate X, 767 00:44:11,400 --> 00:44:17,900 and it can go another way, so this would be a branch point, 768 00:44:17,900 --> 00:44:23,120 you can prevent formation going into another pathway. 769 00:44:23,120 --> 00:44:26,850 And in the purine pathway, this feeds into histidine metabolism 770 00:44:26,850 --> 00:44:31,850 and thiamin biosynthesis, and tryptophan biosynthesis. 771 00:44:31,850 --> 00:44:33,862 So, there are intermediates in this pathway, 772 00:44:33,862 --> 00:44:35,570 and when you start looking at metabolism, 773 00:44:35,570 --> 00:44:38,570 you find these connections all the time. 774 00:44:38,570 --> 00:44:40,220 We know a lot of these connections. 775 00:44:40,220 --> 00:44:41,900 I don't have time to go through them, 776 00:44:41,900 --> 00:44:44,660 but that would be another reason that you would 777 00:44:44,660 --> 00:44:47,460 like to be able to do that. 778 00:44:47,460 --> 00:44:49,730 The reason Bankovic got into this, 779 00:44:49,730 --> 00:44:51,740 and that's whose work you've been reading, 780 00:44:51,740 --> 00:44:54,080 is he was interested in the question of whether 781 00:44:54,080 --> 00:44:56,360 N10-formyltetrahydrofolate-- 782 00:44:56,360 --> 00:44:58,970 remember we talked about all the interconversions-- 783 00:44:58,970 --> 00:45:03,110 whether all of those intermediates were sequestered. 784 00:45:03,110 --> 00:45:04,490 That's why he got into it. 785 00:45:04,490 --> 00:45:05,870 And what is the answer? 786 00:45:05,870 --> 00:45:08,370 He was interested in this question 787 00:45:08,370 --> 00:45:14,180 of tetrahydrofolate metabolism-- central 788 00:45:14,180 --> 00:45:17,628 to both purine and pyrimidine metabolism. 789 00:45:17,628 --> 00:45:18,920 And what do we know about that? 790 00:45:18,920 --> 00:45:21,110 In the control experiments in the paper 791 00:45:21,110 --> 00:45:24,880 you needed to read, what did he use as a control? 792 00:45:24,880 --> 00:45:26,420 He used-- remember we talked about 793 00:45:26,420 --> 00:45:30,350 this trifunctional protein that has three activities? 794 00:45:30,350 --> 00:45:33,080 It puts on the formate, it does a cyclohydrolase, 795 00:45:33,080 --> 00:45:34,970 it does a dehydratase. 796 00:45:34,970 --> 00:45:40,940 So if you go back and you look up the enzyme in his notes, 797 00:45:40,940 --> 00:45:47,270 this is not in purinosomes. 798 00:45:47,270 --> 00:45:49,370 And that's one of the first experiments he did. 799 00:45:49,370 --> 00:45:50,630 It's not there. 800 00:45:50,630 --> 00:45:52,880 So why isn't it there? 801 00:45:52,880 --> 00:45:54,080 I don't know. 802 00:45:54,080 --> 00:45:55,850 And maybe that means that we should 803 00:45:55,850 --> 00:46:00,830 be thinking about these things in other ways. 804 00:46:00,830 --> 00:46:03,470 In the last minute or so. 805 00:46:03,470 --> 00:46:06,440 So, that summarizes the key thing. 806 00:46:06,440 --> 00:46:09,530 Unstable intermediates and multiple 807 00:46:09,530 --> 00:46:11,390 pathways, and sequestration. 808 00:46:11,390 --> 00:46:14,690 I think there's no debate about that. 809 00:46:14,690 --> 00:46:18,530 If you have things sequestered, can you increase fluxes 810 00:46:18,530 --> 00:46:20,120 through pathways? 811 00:46:20,120 --> 00:46:22,160 A lot of bioengineers say you can, 812 00:46:22,160 --> 00:46:24,020 other people say you can't. 813 00:46:24,020 --> 00:46:26,010 This, to me, becomes really important 814 00:46:26,010 --> 00:46:28,190 to metabolic engineering. 815 00:46:28,190 --> 00:46:30,650 If you read metabolic engineering papers, 816 00:46:30,650 --> 00:46:32,660 people will take a polymer, and they'll 817 00:46:32,660 --> 00:46:35,160 stick all the enzymes in the pathway onto a polymer. 818 00:46:35,160 --> 00:46:35,780 Why? 819 00:46:35,780 --> 00:46:37,730 Because they think it's important to have 820 00:46:37,730 --> 00:46:40,780 these things in multi-enzyme complexes, 821 00:46:40,780 --> 00:46:43,040 where you increase the effect of molarity. 822 00:46:43,040 --> 00:46:46,400 That's something else we've talked about extensively 823 00:46:46,400 --> 00:46:49,580 over the course of the semester. 824 00:46:49,580 --> 00:46:51,380 Methods used to study this. 825 00:46:51,380 --> 00:46:55,040 OK, we've talked about that in recitation 13. 826 00:46:55,040 --> 00:46:58,820 We talked about what the issues are. 827 00:46:58,820 --> 00:47:01,700 In all cases, he used the enzyme fused 828 00:47:01,700 --> 00:47:03,472 to a green fluorescent protein. 829 00:47:03,472 --> 00:47:05,180 You could have problems with aggregation. 830 00:47:05,180 --> 00:47:07,220 You could have problems with altered activity. 831 00:47:07,220 --> 00:47:11,180 We talked about all of that last time. 832 00:47:11,180 --> 00:47:13,370 Looking at these-- punctate staining, 833 00:47:13,370 --> 00:47:14,870 if you look at the punctate staining 834 00:47:14,870 --> 00:47:19,190 with one protein and another, they're widely different. 835 00:47:19,190 --> 00:47:23,390 The shapes of the stains are widely different. 836 00:47:23,390 --> 00:47:27,340 Azaserine and hypoxanthine-- Azaserine we just talked about. 837 00:47:27,340 --> 00:47:32,310 Hypoxanthine-- hopefully, you now remember that that-- 838 00:47:32,310 --> 00:47:46,360 IMP, hypoxanthine, with PRPP, this is salvage. 839 00:47:50,050 --> 00:47:52,630 You should now be able to, thinking about this, 840 00:47:52,630 --> 00:47:54,970 go back and read that experiment he did. 841 00:47:54,970 --> 00:47:58,480 That experiment makes no sense to me. 842 00:47:58,480 --> 00:48:01,360 That was an experiment he did because he made a prediction, 843 00:48:01,360 --> 00:48:03,482 knowing how all these things fit together, 844 00:48:03,482 --> 00:48:04,940 and it didn't do what he predicted. 845 00:48:04,940 --> 00:48:06,795 So then he made up something else. 846 00:48:06,795 --> 00:48:08,170 These are the kinds of things you 847 00:48:08,170 --> 00:48:10,090 need to think about when you're trying 848 00:48:10,090 --> 00:48:11,350 to test a model like this. 849 00:48:11,350 --> 00:48:14,200 It's a very appealing model, but it's also 850 00:48:14,200 --> 00:48:17,250 a very controversial model. 851 00:48:17,250 --> 00:48:18,700 I'm sort of at the end of my time, 852 00:48:18,700 --> 00:48:20,380 so I think I'm going to go to the end. 853 00:48:20,380 --> 00:48:22,780 We've looked at all of these-- 854 00:48:22,780 --> 00:48:26,620 punctate staining with no purines, when we add purines, 855 00:48:26,620 --> 00:48:28,060 we lose it. 856 00:48:28,060 --> 00:48:30,790 And I just want to go to a paper that was recently published. 857 00:48:30,790 --> 00:48:33,460 This is probably hard to see, but this just 858 00:48:33,460 --> 00:48:36,670 shows this is an ongoing area of research. 859 00:48:36,670 --> 00:48:38,740 The latest is, now, instead of looking 860 00:48:38,740 --> 00:48:41,410 at this fluorescent stuff where a lot of you commented, 861 00:48:41,410 --> 00:48:43,840 you really can't see the green overlapping 862 00:48:43,840 --> 00:48:45,130 with the red to form yellow. 863 00:48:45,130 --> 00:48:47,047 The pictures were terrible, and if you go back 864 00:48:47,047 --> 00:48:49,360 and you look up there, I can't see it either. 865 00:48:49,360 --> 00:48:52,750 Fluorescence changes, and red and green 866 00:48:52,750 --> 00:48:54,430 on top of each other showing yellow 867 00:48:54,430 --> 00:48:56,800 showing they're sort of in the same general area 868 00:48:56,800 --> 00:48:58,420 are often hard to see. 869 00:48:58,420 --> 00:49:00,850 So now, they've turned to super-resolution, 870 00:49:00,850 --> 00:49:04,030 and if you look at when you turn off the lights, 871 00:49:04,030 --> 00:49:09,010 this is mitochondria, and these little purple things 872 00:49:09,010 --> 00:49:14,260 are the putative purinosome using green attached 873 00:49:14,260 --> 00:49:16,270 fluorescent proteins. 874 00:49:16,270 --> 00:49:18,250 And what you can see is there-- 875 00:49:18,250 --> 00:49:22,060 and again, you need to look at the statistics of all of this-- 876 00:49:22,060 --> 00:49:25,600 they appear to be associated with the mitochondria. 877 00:49:25,600 --> 00:49:28,058 Does that make sense? 878 00:49:28,058 --> 00:49:28,600 I don't know. 879 00:49:28,600 --> 00:49:34,780 That's where you need purines to make all of your ATP. 880 00:49:34,780 --> 00:49:38,740 Anyhow, it's linked to signaling pathways, 881 00:49:38,740 --> 00:49:40,420 and they do that in this paper. 882 00:49:40,420 --> 00:49:42,960 But again, to me, this is just another example. 883 00:49:42,960 --> 00:49:45,290 I don't think they expected to find this. 884 00:49:45,290 --> 00:49:50,110 And they found that, and so now we have more complex systems 885 00:49:50,110 --> 00:49:53,360 to really try to understand why these things-- do they 886 00:49:53,360 --> 00:49:57,220 sequester, number one, and if they do sequester, what 887 00:49:57,220 --> 00:50:00,070 is the advantage to biology? 888 00:50:00,070 --> 00:50:02,410 So, we end here, and the bottom line 889 00:50:02,410 --> 00:50:08,290 is, when you think about all the data, it's a moving target. 890 00:50:08,290 --> 00:50:11,320 You can't prove something. 891 00:50:11,320 --> 00:50:14,230 If you're a mechanism person, you can't prove a mechanism. 892 00:50:14,230 --> 00:50:15,160 It keeps changing. 893 00:50:15,160 --> 00:50:16,510 That's the way life is. 894 00:50:16,510 --> 00:50:17,380 So you have a model. 895 00:50:17,380 --> 00:50:21,100 You make it as simple as possible, you get some data, 896 00:50:21,100 --> 00:50:23,740 you find something that doesn't agree with your hypothesis. 897 00:50:23,740 --> 00:50:24,790 You've got to change it. 898 00:50:24,790 --> 00:50:27,790 That's why science is so much fun. 899 00:50:27,790 --> 00:50:29,410 That's the end of purines, and I'm 900 00:50:29,410 --> 00:50:32,080 sorry I didn't get to tell you about ribonucleotide 901 00:50:32,080 --> 00:50:33,580 reductases. 902 00:50:33,580 --> 00:50:36,520 It's much more chemically complex than anything 903 00:50:36,520 --> 00:50:38,830 you saw in purines, so I am sure you 904 00:50:38,830 --> 00:50:40,750 are delighted that you didn't have 905 00:50:40,750 --> 00:50:43,360 to look at all the radicals. 906 00:50:43,360 --> 00:50:47,040 So we'll see you on, I guess, Tuesday.