1 00:00:00,000 --> 00:00:02,830 NARRATOR: The following content is provided under a Creative 2 00:00:02,830 --> 00:00:04,370 Commons license. 3 00:00:04,370 --> 00:00:06,670 Your support will help MIT Open Courseware 4 00:00:06,670 --> 00:00:11,030 continue to offer high quality educational resources for free. 5 00:00:11,030 --> 00:00:13,660 To make a donation or view additional materials 6 00:00:13,660 --> 00:00:17,130 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,130 --> 00:00:18,140 at ocw.mit.edu. 8 00:00:25,282 --> 00:00:26,740 JOANNE STUBBE: So what I want to do 9 00:00:26,740 --> 00:00:30,190 is finish up purines today and talk 10 00:00:30,190 --> 00:00:33,598 about some interesting aspects of purine metabolism. 11 00:00:33,598 --> 00:00:35,390 I hope I'm going to be able to get through. 12 00:00:35,390 --> 00:00:40,240 I've given you handouts for pyrimidines and deoxynucleotide 13 00:00:40,240 --> 00:00:41,200 biosynthesis as well. 14 00:00:41,200 --> 00:00:44,620 The pyrimidines are pretty straightforward, much simpler 15 00:00:44,620 --> 00:00:45,490 than the purines. 16 00:00:45,490 --> 00:00:47,260 And so I think if I have time today, 17 00:00:47,260 --> 00:00:49,360 depending on when I get finished, 18 00:00:49,360 --> 00:00:52,780 I might talk a little bit about deoxynucleotide metabolism, 19 00:00:52,780 --> 00:00:57,380 since both Drennan's lab and my lab, both work in that area. 20 00:00:57,380 --> 00:00:59,470 So it would be good for you guys to know what's 21 00:00:59,470 --> 00:01:01,150 going on in the department and it's 22 00:01:01,150 --> 00:01:04,480 central to nucleotide metabolism. 23 00:01:08,380 --> 00:01:10,000 We started out-- we were drawing this. 24 00:01:10,000 --> 00:01:16,630 This is my notes that I tried to reproduce for you to look at. 25 00:01:16,630 --> 00:01:18,160 And I'm not going to read. 26 00:01:18,160 --> 00:01:21,370 So this was a big overview slide where we're going. 27 00:01:21,370 --> 00:01:25,030 And so central to everything. 28 00:01:25,030 --> 00:01:28,030 This wasn't in the original packet, but I will put this up. 29 00:01:28,030 --> 00:01:30,350 I'll try to get a better version of that. 30 00:01:30,350 --> 00:01:33,100 But PRPP is central. 31 00:01:33,100 --> 00:01:38,200 And we are talking about de novo purine bio-synthesis, 32 00:01:38,200 --> 00:01:43,930 but again, not only is de novo important, so is salvage. 33 00:01:43,930 --> 00:01:45,912 It depends on the cell type. 34 00:01:45,912 --> 00:01:48,370 You know, if you have cancer cells that are rapidly growing 35 00:01:48,370 --> 00:01:51,640 or B cells and T cells, de novo becomes really important. 36 00:01:51,640 --> 00:01:55,150 In other types of cells almost everything is salvage. 37 00:01:55,150 --> 00:02:00,358 And so I have that PRPP, at least in the purine case-- 38 00:02:00,358 --> 00:02:02,650 and I'll show you an example of that in a few minutes-- 39 00:02:02,650 --> 00:02:06,680 goes directly-- can make your nucleotide directly. 40 00:02:06,680 --> 00:02:08,949 That's a salvage pathway. 41 00:02:08,949 --> 00:02:11,920 And we'll see that the de novo pathway, 42 00:02:11,920 --> 00:02:15,190 which is what I was describing at the end-- 43 00:02:15,190 --> 00:02:19,300 and you've already seen this in recitation from last week-- 44 00:02:19,300 --> 00:02:23,470 is 10 steps to get to IMP. 45 00:02:23,470 --> 00:02:27,880 But then you need to get to GMP and AMP. 46 00:02:27,880 --> 00:02:35,880 And I showed you how all of this branches off with the cofactor 47 00:02:35,880 --> 00:02:39,790 folate between purines and pyrimidines. 48 00:02:42,660 --> 00:02:48,330 And in the end, we need both purines and pyrimidines. 49 00:02:48,330 --> 00:02:50,520 We need it in the nucleotide levels. 50 00:02:50,520 --> 00:02:54,240 So two hydroxyls, the two prime three prime cis hydroxyls, 51 00:02:54,240 --> 00:02:58,050 which in the diphosphate stage, that's also unusual. 52 00:02:58,050 --> 00:03:00,660 Most of the time you don't see high levels of diphosphates 53 00:03:00,660 --> 00:03:02,850 inside the cell, either they're monophosphates 54 00:03:02,850 --> 00:03:04,350 or triphosphates. 55 00:03:04,350 --> 00:03:09,030 So part of the complexity I think of nucleotide metabolism 56 00:03:09,030 --> 00:03:10,770 is figuring out where the kinases are 57 00:03:10,770 --> 00:03:12,360 and the phosotases are. 58 00:03:12,360 --> 00:03:14,340 And you'll notice that I've avoided that. 59 00:03:14,340 --> 00:03:16,770 And that's because every organism is different 60 00:03:16,770 --> 00:03:19,320 and every cell type is different, 61 00:03:19,320 --> 00:03:21,810 and the regulation is a little bit different. 62 00:03:21,810 --> 00:03:23,820 But I think it's important to realize 63 00:03:23,820 --> 00:03:27,240 that to make deoxynucleotides, which are required 64 00:03:27,240 --> 00:03:29,310 for DNA replication and repair, is 65 00:03:29,310 --> 00:03:31,200 done at the diphosphate level. 66 00:03:31,200 --> 00:03:35,010 So you make deoxynucleotides, but they still 67 00:03:35,010 --> 00:03:42,870 have to be converted to deoxy NTPs for DNA. 68 00:03:42,870 --> 00:03:52,440 And over here you need to again, make NTPs for RNA. 69 00:03:52,440 --> 00:03:54,480 So that's sort of the big picture. 70 00:03:54,480 --> 00:04:00,402 We have a purine pathway de novo. 71 00:04:00,402 --> 00:04:02,610 We're not going to be able to talk about pyrimidines, 72 00:04:02,610 --> 00:04:08,760 but the salvage pathway with pyrimidines 73 00:04:08,760 --> 00:04:11,020 is extremely important. 74 00:04:11,020 --> 00:04:14,310 It's a major target of cancer therapeutics now. 75 00:04:14,310 --> 00:04:15,990 I think only in the last few years 76 00:04:15,990 --> 00:04:18,990 has it been realized that in many cancers 77 00:04:18,990 --> 00:04:21,640 you have both pathways going on. 78 00:04:21,640 --> 00:04:24,090 And it turns out now with isotopic labeling 79 00:04:24,090 --> 00:04:27,780 and mass spec, metabolomics is coming into its own. 80 00:04:27,780 --> 00:04:31,000 So you can tell actually by feeding the cells this 81 00:04:31,000 --> 00:04:32,250 is all done in tissue culture. 82 00:04:32,250 --> 00:04:34,170 But you can tell by feeding the cells 83 00:04:34,170 --> 00:04:38,610 whether the deoxynucleotides came from de novo 84 00:04:38,610 --> 00:04:40,500 or whether they came from salvage. 85 00:04:40,500 --> 00:04:43,020 And so we're getting a really different view 86 00:04:43,020 --> 00:04:44,460 of nucleotide metabolism. 87 00:04:44,460 --> 00:04:45,960 And as I said in the very beginning, 88 00:04:45,960 --> 00:04:47,660 I think the next decade we're going 89 00:04:47,660 --> 00:04:52,560 to understand a lot more about how all these things interact 90 00:04:52,560 --> 00:04:54,810 and the kinases and phosatases that 91 00:04:54,810 --> 00:04:58,860 put the nucleotides into the correct phosphorylation state. 92 00:04:58,860 --> 00:05:03,180 So that's key to everything and it's complicated. 93 00:05:03,180 --> 00:05:08,070 So what I want to do now is briefly 94 00:05:08,070 --> 00:05:10,560 talk about the purine pathway. 95 00:05:14,700 --> 00:05:17,430 So we can look at the biology. 96 00:05:17,430 --> 00:05:20,090 I'm not going to write this down, because most of you 97 00:05:20,090 --> 00:05:20,840 already know this. 98 00:05:20,840 --> 00:05:23,250 So we'll just go through it again. 99 00:05:23,250 --> 00:05:26,280 Purine nucleotides are central to everything. 100 00:05:26,280 --> 00:05:28,980 So knowing where they come from and how you control them 101 00:05:28,980 --> 00:05:30,780 is really pretty important. 102 00:05:30,780 --> 00:05:33,030 And we don't understand that much. 103 00:05:33,030 --> 00:05:36,660 So I mean, NTPs and dNTPs are central 104 00:05:36,660 --> 00:05:38,430 to our genetic material. 105 00:05:38,430 --> 00:05:41,520 So we need to get them and we need to control them. 106 00:05:41,520 --> 00:05:44,580 If these levels become imbalanced, 107 00:05:44,580 --> 00:05:49,230 you have mutater phenotypes in DNA replication. 108 00:05:49,230 --> 00:05:55,950 And so fidelity of deoxynucleotide DNA replication 109 00:05:55,950 --> 00:05:58,860 is really important and regulated 110 00:05:58,860 --> 00:06:01,650 by ribonucelotide reductases. 111 00:06:01,650 --> 00:06:04,470 Building blocks for cofactors. 112 00:06:04,470 --> 00:06:05,460 We've seen flavins. 113 00:06:05,460 --> 00:06:06,450 We've seen NAD. 114 00:06:06,450 --> 00:06:07,830 We've seen CoA. 115 00:06:07,830 --> 00:06:09,190 None of this is an accident. 116 00:06:09,190 --> 00:06:13,530 Adenine can self-assemble from cyanide formate 117 00:06:13,530 --> 00:06:15,360 in the prebiotic world. 118 00:06:15,360 --> 00:06:19,110 And so that's why they're central to everything. 119 00:06:19,110 --> 00:06:22,350 So they're in a lot of the cofactors we've already 120 00:06:22,350 --> 00:06:25,590 talked about is they're not necessarily the business end, 121 00:06:25,590 --> 00:06:28,680 but they've got the phosphates and the adenines stuck on 122 00:06:28,680 --> 00:06:33,600 to end, which presumably helps in some way for binding. 123 00:06:33,600 --> 00:06:36,570 We're using GDP and ATP everywhere 124 00:06:36,570 --> 00:06:38,250 in the course of this semester. 125 00:06:38,250 --> 00:06:41,910 You've seen it in your macromolecular machines 126 00:06:41,910 --> 00:06:43,440 that you've talked about, especially 127 00:06:43,440 --> 00:06:47,520 in the first part of the course with the translational 128 00:06:47,520 --> 00:06:51,030 and protein folding and protein degradation all 129 00:06:51,030 --> 00:06:55,410 require energy ATP. 130 00:06:55,410 --> 00:06:58,490 We will see in today's pathway and today's lecture 131 00:06:58,490 --> 00:07:01,260 on purine biosynthesis de novo, it 132 00:07:01,260 --> 00:07:05,610 turns out 5 out of the 10 enzymes use ATP. 133 00:07:05,610 --> 00:07:06,380 So we'll see. 134 00:07:06,380 --> 00:07:11,352 And what you will hopefully now will know is what ATP does. 135 00:07:11,352 --> 00:07:12,810 I'm going to show you two examples. 136 00:07:12,810 --> 00:07:15,255 But you see the same thing really over and over 137 00:07:15,255 --> 00:07:15,880 and over again. 138 00:07:15,880 --> 00:07:17,820 So this should be sort of-- you might not 139 00:07:17,820 --> 00:07:22,710 know whether it uses ATP to get at the gamma position-- 140 00:07:22,710 --> 00:07:25,410 chemistry at the gamma position, or at the alpha position 141 00:07:25,410 --> 00:07:28,440 but the chemistry is the same over and over again. 142 00:07:28,440 --> 00:07:32,340 And so that part hopefully is part of your repertoire now, 143 00:07:32,340 --> 00:07:34,440 about thinking about the role of the ATP 144 00:07:34,440 --> 00:07:36,540 on primary metabolic pathways. 145 00:07:36,540 --> 00:07:39,300 And we've also seen in the last-- 146 00:07:39,300 --> 00:07:43,080 in the reactive oxygen species section, 147 00:07:43,080 --> 00:07:45,150 we are signaling by many mechanisms, 148 00:07:45,150 --> 00:07:47,690 signaling by phosphorylation is all over the place. 149 00:07:47,690 --> 00:07:50,220 And a lot of people are trying to understand. 150 00:07:50,220 --> 00:07:51,690 And I think one of the futures is 151 00:07:51,690 --> 00:07:56,520 how do you integrate signaling and primary metabolic pathways? 152 00:07:56,520 --> 00:07:57,960 And we're almost there. 153 00:07:57,960 --> 00:08:01,740 I decided-- I wrote a lecture on this and decided-- 154 00:08:01,740 --> 00:08:04,950 it's really still very phenomenological. 155 00:08:04,950 --> 00:08:08,550 But all of these key regulators and signaling 156 00:08:08,550 --> 00:08:11,160 linked to purines and pyrimidines in some way. 157 00:08:11,160 --> 00:08:16,080 I think the linkages aren't totally clear, in my opinion. 158 00:08:16,080 --> 00:08:20,460 So why else do we care about purines? 159 00:08:20,460 --> 00:08:24,840 When I was your age, purine and nucleotide metabolism 160 00:08:24,840 --> 00:08:26,560 was front and center. 161 00:08:26,560 --> 00:08:27,060 Why? 162 00:08:27,060 --> 00:08:30,360 Because people were successful at making drugs 163 00:08:30,360 --> 00:08:32,730 based on these molecules. 164 00:08:32,730 --> 00:08:36,690 The central role it plays in replication and repair 165 00:08:36,690 --> 00:08:41,309 has made them successful targets at many different levels. 166 00:08:41,309 --> 00:08:44,002 Here, this has both purines and pyramidines, 167 00:08:44,002 --> 00:08:45,210 but I'll just pick out a few. 168 00:08:45,210 --> 00:08:51,720 This guy acyclovir is what we use as an anti-herpes medicine. 169 00:08:51,720 --> 00:08:54,260 In fact, I think I've taken it. 170 00:08:54,260 --> 00:08:58,610 Here, mercaptopurine cures childhood leukemia. 171 00:08:58,610 --> 00:09:02,190 Clofarabine is something that's been studied my lab. 172 00:09:02,190 --> 00:09:03,872 It's a drug that-- 173 00:09:03,872 --> 00:09:05,580 it's not particularly effective, but it's 174 00:09:05,580 --> 00:09:11,820 used clinically against certain hematological cancers. 175 00:09:11,820 --> 00:09:14,720 And so these are all anti-metabolites not 176 00:09:14,720 --> 00:09:17,700 focused on signaling, which is what everybody is focused on. 177 00:09:17,700 --> 00:09:19,590 In reality, I think the success-- 178 00:09:19,590 --> 00:09:21,810 if there is success against cancer-- 179 00:09:21,810 --> 00:09:23,550 is going to be mixing the two. 180 00:09:23,550 --> 00:09:28,470 I think you need combinations of metabolic inhibitors. 181 00:09:28,470 --> 00:09:29,370 They're toxic. 182 00:09:29,370 --> 00:09:30,180 So is everything. 183 00:09:30,180 --> 00:09:34,590 But somehow figuring out how to use multiple approaches 184 00:09:34,590 --> 00:09:36,510 to avoid the resistance problem, which 185 00:09:36,510 --> 00:09:39,180 is a really important problem. 186 00:09:39,180 --> 00:09:42,690 And to combine the two once we understand the interconnections 187 00:09:42,690 --> 00:09:44,970 better, I think is where it will be to get 188 00:09:44,970 --> 00:09:48,030 successful, more successful. 189 00:09:48,030 --> 00:09:50,880 Therapeutics, but ultimately what we would like to do 190 00:09:50,880 --> 00:09:54,360 is catch it in the bud, rather than waiting 191 00:09:54,360 --> 00:09:59,340 to try to treat something where it's completely out of control. 192 00:09:59,340 --> 00:10:02,550 So I'll just show you one of my favorite ladies, Gertrude 193 00:10:02,550 --> 00:10:03,630 Elion. 194 00:10:03,630 --> 00:10:07,740 She worked at Burroughs Wellcome for many, many years. 195 00:10:07,740 --> 00:10:10,140 She went to Hunter College, as did many-- 196 00:10:10,140 --> 00:10:14,610 in New York City-- as did many outstanding women scientists. 197 00:10:14,610 --> 00:10:18,560 And she was involved at Burroughs Wellcome 198 00:10:18,560 --> 00:10:24,180 in discovery of mercaptopurines, acyclovir treating 199 00:10:24,180 --> 00:10:27,640 herpes, and AZT. 200 00:10:27,640 --> 00:10:28,950 She made several contributions. 201 00:10:28,950 --> 00:10:30,990 Never had a PhD. 202 00:10:30,990 --> 00:10:35,160 So anyhow-- 203 00:10:35,160 --> 00:10:36,750 So what I also wanted to show you, 204 00:10:36,750 --> 00:10:40,470 we're going to talk about de novo pathways. 205 00:10:40,470 --> 00:10:43,560 I just want to show you this is a typical-- 206 00:10:43,560 --> 00:10:44,960 in the case of the purines-- 207 00:10:44,960 --> 00:10:46,660 salvage pathway. 208 00:10:46,660 --> 00:10:47,970 So what does that mean? 209 00:10:47,970 --> 00:10:52,980 You get the bases, the nucleic acid bases from your diet. 210 00:10:52,980 --> 00:10:55,200 Or you're breaking down your DNA and your RNA. 211 00:10:55,200 --> 00:10:56,790 You have nucleic acid bases. 212 00:10:56,790 --> 00:10:58,590 Or you have nucleosides. 213 00:10:58,590 --> 00:11:03,180 So can you take those and make them into the right components 214 00:11:03,180 --> 00:11:07,170 to do RNA biosynthesis and DNA replication, 215 00:11:07,170 --> 00:11:08,990 make ATP, et cetera. 216 00:11:08,990 --> 00:11:13,890 And so here's an example of hypoxanthine reacting 217 00:11:13,890 --> 00:11:16,740 with our central phosphoribosyl pyrophosphate, which 218 00:11:16,740 --> 00:11:19,440 I had in the original slide that I 219 00:11:19,440 --> 00:11:24,450 talked about last time to make, in this case, the nucleotide. 220 00:11:24,450 --> 00:11:27,630 And why is this interesting? 221 00:11:27,630 --> 00:11:31,110 It's interesting because it turns out 222 00:11:31,110 --> 00:11:36,420 that many parasites like in malaria don't have any purines. 223 00:11:36,420 --> 00:11:38,430 So where do they get their purines from 224 00:11:38,430 --> 00:11:40,020 to replicate the DNA? 225 00:11:40,020 --> 00:11:42,870 They have to use salvage. 226 00:11:42,870 --> 00:11:46,170 So the salvage pathways have-- for treatment of those things-- 227 00:11:46,170 --> 00:11:47,640 have become front and center. 228 00:11:47,640 --> 00:11:52,170 Can you make specific inhibitors of phosphoribosyl pyrophosphate 229 00:11:52,170 --> 00:11:55,170 reaction with the bases? 230 00:11:55,170 --> 00:11:58,070 And we're pretty good at that actually. 231 00:11:58,070 --> 00:12:00,300 Vern Schramm's lab has done some beautiful work. 232 00:12:00,300 --> 00:12:05,310 And there's a lot of things in clinical trial targeting 233 00:12:05,310 --> 00:12:07,560 salvage pathways. 234 00:12:07,560 --> 00:12:11,010 So again, there's something different about the metabolism 235 00:12:11,010 --> 00:12:15,990 of us and whatever is invading us. 236 00:12:15,990 --> 00:12:17,820 That's not true in cancer. 237 00:12:17,820 --> 00:12:19,380 So cancer is a much tougher problem, 238 00:12:19,380 --> 00:12:22,470 because you get normal cells as well. 239 00:12:22,470 --> 00:12:25,590 It's a question of what the therapeutic index is. 240 00:12:29,250 --> 00:12:31,260 So that's all I want to say in the introduction 241 00:12:31,260 --> 00:12:33,270 to the biology. 242 00:12:33,270 --> 00:12:35,520 And then I want to talk about one cofactor. 243 00:12:35,520 --> 00:12:38,160 And then I'm going to talk about the pathway itself. 244 00:12:38,160 --> 00:12:41,280 So there's one cofactor, which I sort of told you 245 00:12:41,280 --> 00:12:45,170 I was going there in the first place. 246 00:12:45,170 --> 00:12:49,190 Let me break this down over here. 247 00:12:49,190 --> 00:12:52,190 So the one cofactor that I wanted to talk about is folate. 248 00:12:55,400 --> 00:12:56,400 Let me also show you. 249 00:12:56,400 --> 00:12:59,902 You don't have to sit and look at this. 250 00:12:59,902 --> 00:13:01,860 But I'm going to show you it's all written out. 251 00:13:01,860 --> 00:13:04,220 So you don't have to bob up and down. 252 00:13:04,220 --> 00:13:06,050 It's all written out on the handout. 253 00:13:06,050 --> 00:13:08,210 So this is folate. 254 00:13:08,210 --> 00:13:11,280 And let me just point out a few things. 255 00:13:11,280 --> 00:13:13,590 This is going to be the business end of the molecule. 256 00:13:13,590 --> 00:13:16,280 So I want you to know where the business end of the molecule 257 00:13:16,280 --> 00:13:16,780 is. 258 00:13:16,780 --> 00:13:20,000 I don't expect you to remember the structures. 259 00:13:23,120 --> 00:13:25,540 But what does this sort of look like? 260 00:13:25,540 --> 00:13:26,478 Anybody? 261 00:13:26,478 --> 00:13:27,770 This is the kind of chemistry-- 262 00:13:27,770 --> 00:13:31,670 I mean, I think there's a bunch of heterocyclic chemistry 263 00:13:31,670 --> 00:13:35,570 that you find in biology that most of you 264 00:13:35,570 --> 00:13:37,070 haven't been exposed to. 265 00:13:37,070 --> 00:13:40,280 And it's not intuitive what the most reactive positions are. 266 00:13:40,280 --> 00:13:44,840 This cofactor is much simpler than flavins, which we very 267 00:13:44,840 --> 00:13:47,780 briefly talked about before. 268 00:13:52,990 --> 00:13:56,400 So this has a polyglutamate on the end. 269 00:13:56,400 --> 00:13:57,460 So this is folate. 270 00:13:57,460 --> 00:14:04,540 And what you really need to know is that this is 5, 6, 7, 8. 271 00:14:04,540 --> 00:14:06,110 And this is 10. 272 00:14:06,110 --> 00:14:11,320 So the active part of this cofactor is here. 273 00:14:11,320 --> 00:14:15,550 So everything's going to happen at either N5-- 274 00:14:15,550 --> 00:14:22,180 if you have a copy of this, you can just circle N5, N10 and N5. 275 00:14:22,180 --> 00:14:24,590 That's where all the chemistry is going to happen. 276 00:14:24,590 --> 00:14:27,220 And it turns out the way this cofactor 277 00:14:27,220 --> 00:14:33,320 works-- so this is 1, 2, 3, 4. 278 00:14:33,320 --> 00:14:38,020 And so this is 4a, and this is 10a, 8a. 279 00:14:42,640 --> 00:14:44,800 It sort of looks like flavins. 280 00:14:44,800 --> 00:14:46,660 And it sort of looks like pterins. 281 00:14:46,660 --> 00:14:49,120 And pterins actually can undergo redox chemistry 282 00:14:49,120 --> 00:14:51,520 under certain sets of conditions. 283 00:14:51,520 --> 00:14:53,440 These molecules are only involved 284 00:14:53,440 --> 00:14:55,720 in one carbon transfer. 285 00:14:55,720 --> 00:15:04,630 So the major focus is one carbon transfers. 286 00:15:04,630 --> 00:15:13,730 And it can do it in the methyl state, in the aldehyde state, 287 00:15:13,730 --> 00:15:17,430 or it can do it in the acid state. 288 00:15:17,430 --> 00:15:22,310 So all three oxidation states from one carbon transfers. 289 00:15:22,310 --> 00:15:24,200 And so then how does it do it? 290 00:15:24,200 --> 00:15:27,020 And the chemistry actually is fairly simple 291 00:15:27,020 --> 00:15:30,075 compared to the chemistry that we've looked at before. 292 00:15:30,075 --> 00:15:31,700 And we looked at a little bit at hemes. 293 00:15:31,700 --> 00:15:36,200 We looked at a little bit at flavins. 294 00:15:36,200 --> 00:15:38,390 This is much simpler. 295 00:15:38,390 --> 00:15:41,600 And so what we're after in the end-- 296 00:15:41,600 --> 00:15:46,980 and I'll show you how we get there-- so here's N5 methyl. 297 00:15:46,980 --> 00:15:49,380 And we'll see this is tetrahydrofolate. 298 00:15:49,380 --> 00:15:53,770 So this ring is completely reduced. 299 00:15:53,770 --> 00:15:56,034 And so this is tetrahydrofolate. 300 00:16:02,100 --> 00:16:04,320 And this can undergo oxidation and reduction. 301 00:16:04,320 --> 00:16:06,840 And that becomes very important in the pyrimidine pathway 302 00:16:06,840 --> 00:16:11,685 to form thymidine, which is a major target of fluorouracil, 303 00:16:11,685 --> 00:16:13,560 which is a drug that's still used clinically. 304 00:16:13,560 --> 00:16:15,870 Anyhow, this is the reduced state here. 305 00:16:15,870 --> 00:16:18,390 So this is where the tetrahydro is. 306 00:16:18,390 --> 00:16:21,930 So both of these can be oxidized. 307 00:16:21,930 --> 00:16:24,060 And that would be folate. 308 00:16:24,060 --> 00:16:28,800 So you can make dihydrofolate, folate, and tetrahydrofolate. 309 00:16:28,800 --> 00:16:32,190 And the oxidations occur here and here. 310 00:16:32,190 --> 00:16:33,690 And we're not going to look at that, 311 00:16:33,690 --> 00:16:34,860 because we're not going to have time to look 312 00:16:34,860 --> 00:16:36,470 at pyrimidine metabolism. 313 00:16:36,470 --> 00:16:39,630 But the dihydrofolate plays an important role. 314 00:16:39,630 --> 00:16:42,000 It's the target of methotrexate. 315 00:16:42,000 --> 00:16:43,920 If you have rheumatoid arthritis, 316 00:16:43,920 --> 00:16:46,740 you take methotrexate is one of the drugs 317 00:16:46,740 --> 00:16:49,110 that people take nowadays. 318 00:16:49,110 --> 00:16:51,240 So what's unusual about this-- 319 00:16:51,240 --> 00:16:53,190 and this is key to the purine pathway, 320 00:16:53,190 --> 00:16:55,200 it's also key to the pyrimidine pathway-- 321 00:16:55,200 --> 00:16:57,690 that's why folate have been central. 322 00:16:57,690 --> 00:16:59,190 People made folates for decades. 323 00:16:59,190 --> 00:17:02,220 Even when I was your age, people were making folates 324 00:17:02,220 --> 00:17:05,369 for treatment therapies in cancer. 325 00:17:05,369 --> 00:17:06,750 And it's been successful. 326 00:17:06,750 --> 00:17:09,630 In fact, and if you've gone to Princeton's chemistry 327 00:17:09,630 --> 00:17:12,869 department, the whole department was funded on an anti-folate 328 00:17:12,869 --> 00:17:17,369 that Ted Taylor made 25 or 30 years ago. 329 00:17:17,369 --> 00:17:19,650 And they've tried it again under different conditions, 330 00:17:19,650 --> 00:17:23,069 and it's now being used clinically. 331 00:17:23,069 --> 00:17:26,160 So how does this work? 332 00:17:26,160 --> 00:17:29,310 So we have this oxidation state. 333 00:17:29,310 --> 00:17:30,840 We have this oxidation state. 334 00:17:30,840 --> 00:17:33,240 And then we'll see that this can ring open. 335 00:17:33,240 --> 00:17:35,610 And so this would be the aldehyde state. 336 00:17:35,610 --> 00:17:37,020 And this can hydrolyze. 337 00:17:37,020 --> 00:17:39,168 And that would be the acid state. 338 00:17:39,168 --> 00:17:40,710 So I'm going to show you in a second, 339 00:17:40,710 --> 00:17:43,740 I'm going to walk you through where those different states 340 00:17:43,740 --> 00:17:44,300 came from. 341 00:17:44,300 --> 00:17:48,780 So methyl state, aldehyde state, acid state. 342 00:17:51,410 --> 00:17:56,930 So there's the model, because I like to have the windows open, 343 00:17:56,930 --> 00:17:59,450 you probably can't see the model very well up there now. 344 00:17:59,450 --> 00:18:01,760 But you can pull it up on your computer if you want. 345 00:18:01,760 --> 00:18:03,820 I'm going to write out the model. 346 00:18:03,820 --> 00:18:09,410 So we start out over here with tetrahydrofolate. 347 00:18:09,410 --> 00:18:15,140 So this is tetrahydrofolate. 348 00:18:15,140 --> 00:18:16,910 And we have nothing here, which you 349 00:18:16,910 --> 00:18:18,710 notice was we could have something 350 00:18:18,710 --> 00:18:21,380 at N5, something at N10. 351 00:18:21,380 --> 00:18:23,720 We'll see the methyl group is always at N5. 352 00:18:23,720 --> 00:18:25,700 It could be at either, chemically, 353 00:18:25,700 --> 00:18:27,530 but it's always at N5. 354 00:18:27,530 --> 00:18:30,080 We'll see the aldehyde group is always at N10. 355 00:18:30,080 --> 00:18:33,050 It could be either chemically, but it's not. 356 00:18:33,050 --> 00:18:35,150 So these are going to be the key stages. 357 00:18:35,150 --> 00:18:37,040 And here we have no carbons. 358 00:18:37,040 --> 00:18:40,650 So somehow we have to get the carbons into the molecules. 359 00:18:40,650 --> 00:18:45,200 So we start out with this molecule, tetrahydrofolate. 360 00:18:45,200 --> 00:18:53,690 So what happens is you can start out here, and use formate. 361 00:18:53,690 --> 00:18:57,110 So formate is going to be the source of the one 362 00:18:57,110 --> 00:18:59,430 carbon in this case. 363 00:18:59,430 --> 00:19:03,620 So the names in this pathway are again, 364 00:19:03,620 --> 00:19:06,280 horrible, just like the purine pathway. 365 00:19:06,280 --> 00:19:09,980 And on the next slide I've written out the names. 366 00:19:09,980 --> 00:19:13,100 So it turns out that one enzyme can 367 00:19:13,100 --> 00:19:15,380 do three of these activities. 368 00:19:15,380 --> 00:19:17,970 So this is one of the enzymes. 369 00:19:17,970 --> 00:19:20,270 And so this is activity one. 370 00:19:20,270 --> 00:19:25,190 And it attaches a formate, so they call it a formate ligase. 371 00:19:25,190 --> 00:19:27,770 The names again, in my opinion, are horrible. 372 00:19:27,770 --> 00:19:33,430 But what it allows you to do is-- 373 00:19:33,430 --> 00:19:38,570 so what I'm going to draw out now is not the whole structure. 374 00:19:38,570 --> 00:19:41,000 I'm just going to focus on the business 375 00:19:41,000 --> 00:19:46,160 end of the molecule over here, and skip this ring over here. 376 00:19:46,160 --> 00:19:49,730 But that ring is there, and is key to making all of this work. 377 00:19:49,730 --> 00:19:54,140 So I'm just going to do this like that. 378 00:19:54,140 --> 00:20:04,690 And so what can happen is that you can formulate and form. 379 00:20:04,690 --> 00:20:13,160 And so this is now N10 formal tetrahydrofolate. 380 00:20:13,160 --> 00:20:19,970 So this is N10, and we'll call this R. 381 00:20:19,970 --> 00:20:21,830 So that's the first step. 382 00:20:21,830 --> 00:20:22,880 That's the enzyme. 383 00:20:22,880 --> 00:20:26,750 The same enzyme catalyzes the next step. 384 00:20:26,750 --> 00:20:28,970 And what you can picture happening here, 385 00:20:28,970 --> 00:20:32,840 if you watch me, is this nitrogen 386 00:20:32,840 --> 00:20:37,190 is juxtaposed to this imid. 387 00:20:37,190 --> 00:20:39,800 So can attack to form a tetrahedral intermediate 388 00:20:39,800 --> 00:20:42,350 and then lose a molecule of water. 389 00:20:42,350 --> 00:20:45,230 So that's called a cyclohydrolase. 390 00:20:45,230 --> 00:20:48,560 So this guy is attacking. 391 00:20:48,560 --> 00:20:52,330 And then you have loss of water. 392 00:20:52,330 --> 00:20:53,960 And this is a cyclohydrolase. 393 00:20:57,600 --> 00:20:59,010 So this is the same enzyme. 394 00:21:02,620 --> 00:21:03,780 So this is two. 395 00:21:03,780 --> 00:21:04,530 This is one. 396 00:21:04,530 --> 00:21:07,230 But they're both on the same polypeptide. 397 00:21:07,230 --> 00:21:09,390 So there are three of these on one polypeptide. 398 00:21:09,390 --> 00:21:13,110 You've seen that before in recitation last week. 399 00:21:13,110 --> 00:21:15,960 And so now what you've formed is-- 400 00:21:15,960 --> 00:21:18,030 and again, this is a cyclohydrolase-- 401 00:21:18,030 --> 00:21:19,740 now what you're formed is this structure. 402 00:21:25,950 --> 00:21:28,980 So we've lost a molecule of water. 403 00:21:28,980 --> 00:21:34,410 So you can draw NR. 404 00:21:34,410 --> 00:21:36,120 And so if you hydrolyze this, you 405 00:21:36,120 --> 00:21:39,960 can get back to the aldehyde stage. 406 00:21:39,960 --> 00:21:45,750 So if water adds here, this is an iminium system. 407 00:21:45,750 --> 00:21:48,660 Water can add, it can collapse, it can ring open, 408 00:21:48,660 --> 00:21:51,270 it can ring close. 409 00:21:51,270 --> 00:21:53,040 So the chemistry here-- we're going 410 00:21:53,040 --> 00:21:55,240 to see some really similar chemistry actually, 411 00:21:55,240 --> 00:21:58,770 because we can use N10 formal tetrahydrofolate in two 412 00:21:58,770 --> 00:22:00,430 steps in the purine pathway. 413 00:22:00,430 --> 00:22:02,880 So this chemistry I'm drawing right now 414 00:22:02,880 --> 00:22:06,960 is related to the pathway in general. 415 00:22:06,960 --> 00:22:16,890 And so this is called 5, 10 methylidine-- 416 00:22:16,890 --> 00:22:20,910 the names again, are horrible-- tetrahydrofolate. 417 00:22:20,910 --> 00:22:29,710 And then the third enzyme in this pathway 418 00:22:29,710 --> 00:22:33,100 is a dehydrogenase, so DH. 419 00:22:33,100 --> 00:22:36,640 And so what you can imagine you could do here 420 00:22:36,640 --> 00:22:38,490 is we have an iminium system. 421 00:22:38,490 --> 00:22:41,210 And NAD pH is the reductive. 422 00:22:41,210 --> 00:22:45,430 So you can reduce this down to methylene tetrahydrofolate. 423 00:22:45,430 --> 00:22:53,585 So this can be converted to an NADP. 424 00:22:56,340 --> 00:22:58,620 So this is the dehydrogenase. 425 00:22:58,620 --> 00:23:00,450 We've seen that used over and over again. 426 00:23:00,450 --> 00:23:02,340 This is the same enzyme. 427 00:23:02,340 --> 00:23:03,660 So this is also MTHFD. 428 00:23:06,340 --> 00:23:09,510 And I've given you the nomenclature on the next slide. 429 00:23:09,510 --> 00:23:10,960 So if you want to look at-- 430 00:23:10,960 --> 00:23:15,090 So this is tetrahydrofolate whatever. 431 00:23:15,090 --> 00:23:20,100 So it has of formal ligase, it has a cyclohydrolase, 432 00:23:20,100 --> 00:23:26,100 and it has a D hydrogenase all on one enzyme. 433 00:23:26,100 --> 00:23:27,630 And so what do you generate then? 434 00:23:27,630 --> 00:23:44,040 You generate-- so this is methylene tetrahydrofolate. 435 00:23:47,480 --> 00:23:51,290 And this is the key player in pyrimidine biosynthesis, which 436 00:23:51,290 --> 00:23:52,850 we are going to talk about. 437 00:23:52,850 --> 00:23:56,030 And it's an enzyme called thymidylate synthase, which 438 00:23:56,030 --> 00:24:01,690 makes thymidine, which is a major target for drugs 439 00:24:01,690 --> 00:24:04,220 in the treatment of cancer. 440 00:24:04,220 --> 00:24:06,170 So now you can even take this a step further 441 00:24:06,170 --> 00:24:07,970 and reduce this further. 442 00:24:07,970 --> 00:24:09,620 We're still now here. 443 00:24:09,620 --> 00:24:12,950 If you ring open this, you're at the aldehyde stage. 444 00:24:12,950 --> 00:24:15,920 You can reduce the aldehyde stage down to the methyl group. 445 00:24:15,920 --> 00:24:18,200 And that's then getting us into the methyl 446 00:24:18,200 --> 00:24:21,033 state, the aldehyde state, and the acid state. 447 00:24:21,033 --> 00:24:22,950 So I think when you sit down and look at this, 448 00:24:22,950 --> 00:24:24,200 it looks complicated at first. 449 00:24:24,200 --> 00:24:26,430 It's really not that complicated. 450 00:24:26,430 --> 00:24:28,880 So this can just ring open. 451 00:24:28,880 --> 00:24:31,700 And conceivably, it could ring open in either direction. 452 00:24:31,700 --> 00:24:34,070 It depends on the enzyme that's catalyzing it. 453 00:24:34,070 --> 00:24:36,980 But we always get N5 methyl tetrahydrofolate. 454 00:24:36,980 --> 00:24:38,990 That's what's used inside the cell. 455 00:24:38,990 --> 00:24:42,290 People don't find N10 methyl tetrahydrofolate, 456 00:24:42,290 --> 00:24:44,930 but chemically, that could happen. 457 00:24:44,930 --> 00:24:45,950 So what happens? 458 00:24:45,950 --> 00:24:47,570 This is now a new enzyme. 459 00:24:47,570 --> 00:24:50,880 And again, it's a dehydrogenase. 460 00:24:50,880 --> 00:24:55,580 So NADPH is going to NADP. 461 00:24:55,580 --> 00:24:57,920 So this is a new enzyme. 462 00:24:57,920 --> 00:24:59,990 I'm not going to write out the name. 463 00:24:59,990 --> 00:25:14,560 But this then reduces this to N5 methyl tetrahydrofolate. 464 00:25:14,560 --> 00:25:18,730 So what we've done then is, in the pathway 465 00:25:18,730 --> 00:25:21,670 I've drawn out here is, where do we get the one carbon from? 466 00:25:21,670 --> 00:25:25,300 Here, we got it from the formate. 467 00:25:25,300 --> 00:25:31,900 And we can change the oxidation states to get all three 468 00:25:31,900 --> 00:25:33,670 of these oxidation states, depending 469 00:25:33,670 --> 00:25:35,380 on what we need to do with it. 470 00:25:35,380 --> 00:25:38,950 You have to have the right enzymes and the right complexes 471 00:25:38,950 --> 00:25:41,680 to be able to make this all work. 472 00:25:41,680 --> 00:25:45,760 Now many of you might not recall this, but in the Benkovic paper 473 00:25:45,760 --> 00:25:49,900 you read for recitation last week, one of the controls 474 00:25:49,900 --> 00:25:52,580 with this tri-functional protein. 475 00:25:52,580 --> 00:25:56,980 And it does not exist in the purinosome. 476 00:25:56,980 --> 00:25:59,230 Benkovic's been interested never in these enzymes, 477 00:25:59,230 --> 00:26:02,200 and channeling of reactive intermediates in these systems. 478 00:26:02,200 --> 00:26:06,430 This does not exist in the purinosome. 479 00:26:06,430 --> 00:26:08,440 So then the question is how do you get back? 480 00:26:11,350 --> 00:26:15,310 And so there are three methylating agents 481 00:26:15,310 --> 00:26:17,140 inside the cell in a biology. 482 00:26:17,140 --> 00:26:19,990 Does anybody know what the other two are? 483 00:26:19,990 --> 00:26:22,600 So this is unusual, N5 methyl. 484 00:26:22,600 --> 00:26:25,140 So this is N5, this is again, N10. 485 00:26:25,140 --> 00:26:26,630 STUDENT: [INAUDIBLE]. 486 00:26:26,630 --> 00:26:28,570 JOANNE STUBBE: So S-adenosyl methionine 487 00:26:28,570 --> 00:26:29,930 is probably the most prevalent. 488 00:26:29,930 --> 00:26:31,580 What's another one? 489 00:26:31,580 --> 00:26:33,287 STUDENT: Methylcobalamin. 490 00:26:33,287 --> 00:26:34,120 JOANNE STUBBE: Yeah. 491 00:26:34,120 --> 00:26:35,290 So methylcobalamin. 492 00:26:35,290 --> 00:26:38,072 So S-adenosyl methionine 493 00:26:40,330 --> 00:26:45,410 is the universal methylating agent inside the cell. 494 00:26:45,410 --> 00:26:47,453 And then you also have-- 495 00:26:47,453 --> 00:26:49,120 I'm not going to draw the structure out. 496 00:26:49,120 --> 00:26:52,800 We're not going to talk about it, but methylcobalamin. 497 00:26:52,800 --> 00:26:56,620 And there's a single enzyme that uses all three 498 00:26:56,620 --> 00:26:58,270 of these methyl groups. 499 00:26:58,270 --> 00:27:00,188 And if I had another five lectures, 500 00:27:00,188 --> 00:27:01,480 I would talk about this enzyme. 501 00:27:01,480 --> 00:27:05,360 This was studied extensively by Rowena Matthews' lab, who 502 00:27:05,360 --> 00:27:07,152 was one of Cathy's mentors. 503 00:27:07,152 --> 00:27:09,610 And then Cathy was involved in getting the first structures 504 00:27:09,610 --> 00:27:11,412 many years ago with the little pieces. 505 00:27:11,412 --> 00:27:12,620 So it's one of these enzymes. 506 00:27:12,620 --> 00:27:13,720 It's huge. 507 00:27:13,720 --> 00:27:15,700 And it's got to juggle these three methyl 508 00:27:15,700 --> 00:27:16,930 groups to do the chemistry. 509 00:27:16,930 --> 00:27:19,300 It's really sort of fascinating. 510 00:27:19,300 --> 00:27:22,540 And so what it does is it takes homocysteine-- 511 00:27:28,280 --> 00:27:29,990 so this is homocysteine-- 512 00:27:29,990 --> 00:27:31,460 and converts it to methionine. 513 00:27:31,460 --> 00:27:32,600 I'm not going to draw the structure. 514 00:27:32,600 --> 00:27:33,530 So you methylate it. 515 00:27:33,530 --> 00:27:37,610 So you're going to methylate that cysteine. 516 00:27:37,610 --> 00:27:40,790 And then you're back to tetrahydrofolate. 517 00:27:40,790 --> 00:27:43,370 So there's another important reaction 518 00:27:43,370 --> 00:27:48,260 that I just want to put in here is that there's another way 519 00:27:48,260 --> 00:27:53,390 to go from tetrahydrofolate to this one, which is methylene 520 00:27:53,390 --> 00:27:55,490 tetrahydrofolate. 521 00:27:55,490 --> 00:27:58,880 And so in addition to being able to put on the one carbon 522 00:27:58,880 --> 00:28:02,540 with formate, does anybody have any idea what 523 00:28:02,540 --> 00:28:04,040 another major way-- it's probably 524 00:28:04,040 --> 00:28:06,710 the major way of doing one carbon 525 00:28:06,710 --> 00:28:11,060 transfers from metabolic labeling experiments? 526 00:28:11,060 --> 00:28:12,450 It comes from an amino acid. 527 00:28:12,450 --> 00:28:14,010 What amino acid could you use? 528 00:28:16,850 --> 00:28:21,500 So somehow we want to get from here to here. 529 00:28:21,500 --> 00:28:25,010 This is also a major target of therapeutics. 530 00:28:28,140 --> 00:28:29,610 Anybody got any ideas? 531 00:28:29,610 --> 00:28:34,642 We need to get one carbon out of an amino acid. 532 00:28:34,642 --> 00:28:35,350 What did you say? 533 00:28:35,350 --> 00:28:36,056 STUDENT: Thymine? 534 00:28:36,056 --> 00:28:37,000 JOANNE STUBBE: Thymine? 535 00:28:37,000 --> 00:28:38,050 That's not an amino acid. 536 00:28:38,050 --> 00:28:38,380 STUDENT: Thiamine. 537 00:28:38,380 --> 00:28:39,250 JOANNE STUBBE: Oh, thiamine. 538 00:28:39,250 --> 00:28:40,083 STUDENT: Methionine. 539 00:28:40,083 --> 00:28:42,350 JOANNE STUBBE: Oh, methionine. 540 00:28:42,350 --> 00:28:42,850 No. 541 00:28:42,850 --> 00:28:44,093 See, I guess I'm deaf. 542 00:28:44,093 --> 00:28:45,010 OK, I didn't hear you. 543 00:28:45,010 --> 00:28:48,010 No, that's, not it. 544 00:28:48,010 --> 00:28:52,515 So I'm not going to spend a lot of time, but serine-- 545 00:28:55,090 --> 00:28:59,890 so this is ours-- 546 00:28:59,890 --> 00:29:03,640 I'll draw this out, because I think this is really important. 547 00:29:03,640 --> 00:29:08,212 This can be converted into formaldehyde. 548 00:29:08,212 --> 00:29:09,670 Does anybody know what the cofactor 549 00:29:09,670 --> 00:29:10,837 would be that would do that? 550 00:29:14,930 --> 00:29:16,895 And then what you end up with is glycine. 551 00:29:20,170 --> 00:29:22,240 So this is the major way-- 552 00:29:22,240 --> 00:29:25,900 serine is a major one carbon donor. 553 00:29:25,900 --> 00:29:37,360 So seramine is going to generate the formaldehyde equivalent, 554 00:29:37,360 --> 00:29:38,940 which then can get picked up here 555 00:29:38,940 --> 00:29:43,740 and make methylene tetrahydrofolate. 556 00:29:43,740 --> 00:29:46,860 Anybody have any idea of how you would convert serine 557 00:29:46,860 --> 00:29:49,860 into glycine? 558 00:29:49,860 --> 00:29:53,190 You do learn about this cofactor. 559 00:29:53,190 --> 00:29:55,470 What is the cofactor that works on all amino acids, 560 00:29:55,470 --> 00:29:56,940 if you want to do something to it? 561 00:29:56,940 --> 00:29:58,740 There's only one. 562 00:29:58,740 --> 00:29:59,730 STUDENT: PLP. 563 00:29:59,730 --> 00:30:01,710 JOANNE STUBBE: PLP, yeah. 564 00:30:01,710 --> 00:30:03,300 So this isn't unusual-- 565 00:30:03,300 --> 00:30:05,880 PLP is sort of an amazing cofactor. 566 00:30:05,880 --> 00:30:09,330 It can do alpha decarboxylations, 567 00:30:09,330 --> 00:30:11,760 racemizations. 568 00:30:11,760 --> 00:30:14,760 It can do aldol reactions. 569 00:30:14,760 --> 00:30:16,850 And then it activates the beta positions 570 00:30:16,850 --> 00:30:18,990 so you can do beta eliminations replacements. 571 00:30:18,990 --> 00:30:21,750 It can do probably 10 or 15 different reactions. 572 00:30:21,750 --> 00:30:24,480 This one is unusual in that what you're doing 573 00:30:24,480 --> 00:30:28,320 is you're doing an aldol reaction. 574 00:30:28,320 --> 00:30:32,100 So you're cleaving that bond, and a reverse aldol reaction 575 00:30:32,100 --> 00:30:34,080 in this case. 576 00:30:34,080 --> 00:30:35,610 And then the other thing is if you 577 00:30:35,610 --> 00:30:38,190 want to link this into pyrimidines, 578 00:30:38,190 --> 00:30:39,660 you have dihydrofolate. 579 00:30:49,030 --> 00:30:51,880 So this is dihydrofolate. 580 00:30:51,880 --> 00:30:55,540 And that's a major player in pyrimidine metabolism 581 00:30:55,540 --> 00:30:57,010 to make thymidine. 582 00:30:57,010 --> 00:30:58,990 I'm not going to have time to talk about this. 583 00:30:58,990 --> 00:31:02,880 But folate is a central player in both purine and pyrimidine 584 00:31:02,880 --> 00:31:04,570 metabolism. 585 00:31:04,570 --> 00:31:07,813 And people have spent a lot of time thinking about it. 586 00:31:07,813 --> 00:31:09,730 And I think the chemistry of interconversions, 587 00:31:09,730 --> 00:31:12,910 once you sit and walk through this yourself, 588 00:31:12,910 --> 00:31:15,917 start over here and see if you can draw out the mechanisms. 589 00:31:15,917 --> 00:31:18,250 It's the same mechanisms we've seen over and over again, 590 00:31:18,250 --> 00:31:20,950 in addition to a carbonyl and loss of water. 591 00:31:26,560 --> 00:31:28,630 So that was the introductory part. 592 00:31:28,630 --> 00:31:30,395 And really what I want to do now is-- 593 00:31:30,395 --> 00:31:31,770 we can put that up here for those 594 00:31:31,770 --> 00:31:33,490 who still want to stare at it-- 595 00:31:33,490 --> 00:31:37,120 what I want to do now is talk about the pathway. 596 00:31:37,120 --> 00:31:40,270 And what I want to do is write out the pathway, 597 00:31:40,270 --> 00:31:42,160 and then use a PowerPoint to talk 598 00:31:42,160 --> 00:31:45,220 about a few features of the pathway 599 00:31:45,220 --> 00:31:47,740 that I think are the most interesting, 600 00:31:47,740 --> 00:31:52,040 and that you can make generalizations 601 00:31:52,040 --> 00:31:55,300 to other pathways, like, what is the role of glutamine? 602 00:31:55,300 --> 00:31:58,750 That's universally conserved. 603 00:31:58,750 --> 00:32:01,730 What is the role of ATP? 604 00:32:01,730 --> 00:32:04,630 And we're going to see the roles you 605 00:32:04,630 --> 00:32:06,700 see in the purine pathway are used 606 00:32:06,700 --> 00:32:08,320 in many metabolic pathways. 607 00:32:08,320 --> 00:32:12,280 So those are the ones I decided to focus on. 608 00:32:12,280 --> 00:32:15,880 So what I want to do is go step by step 609 00:32:15,880 --> 00:32:19,420 and just make a few comments. 610 00:32:19,420 --> 00:32:22,450 And then I'm going to use a PowerPoint over here so you can 611 00:32:22,450 --> 00:32:23,710 see what I have written down. 612 00:32:23,710 --> 00:32:26,530 I'm going to write down a few things. 613 00:32:26,530 --> 00:32:28,750 So that's the nomenclature. 614 00:32:28,750 --> 00:32:29,710 There's the pathway. 615 00:32:29,710 --> 00:32:31,270 We will start there. 616 00:32:31,270 --> 00:32:35,500 So I told you that the first step in this pathway 617 00:32:35,500 --> 00:32:39,610 is we start with phosphoribosyl pyrophosphate. 618 00:32:39,610 --> 00:32:42,100 That's central to a lot of things. 619 00:32:42,100 --> 00:32:44,110 It's chemically very unstable. 620 00:32:44,110 --> 00:32:44,860 It falls apart. 621 00:32:44,860 --> 00:32:46,900 It's hard to isolate. 622 00:32:46,900 --> 00:32:49,320 And the first step in this pathway-- 623 00:32:49,320 --> 00:32:52,630 we talked about this briefly in recitation-- 624 00:32:52,630 --> 00:32:57,130 is to make phosphoribosylamine. 625 00:32:57,130 --> 00:32:59,830 So the interesting thing about this pathway-- 626 00:32:59,830 --> 00:33:02,110 so is you start out-- 627 00:33:05,270 --> 00:33:09,880 and again, the nomenclature, I've written out. 628 00:33:09,880 --> 00:33:14,580 On the exam, you probably will have something about purines 629 00:33:14,580 --> 00:33:15,080 there. 630 00:33:15,080 --> 00:33:17,860 I will give you the pathway, and I will give you 631 00:33:17,860 --> 00:33:19,390 all the names and the enzymes. 632 00:33:19,390 --> 00:33:22,180 So you don't need to memorize that. 633 00:33:22,180 --> 00:33:25,030 I'm probably the only one that knows the names, 634 00:33:25,030 --> 00:33:26,500 because I've worked on it. 635 00:33:26,500 --> 00:33:27,400 Very confusing. 636 00:33:31,470 --> 00:33:35,220 So what's unique, again, and we've already mentioned this, 637 00:33:35,220 --> 00:33:39,812 is you start out with ribosyl phosphate. 638 00:33:39,812 --> 00:33:41,020 And what you're going to do-- 639 00:33:41,020 --> 00:33:43,470 and this is what we're going to walk through-- 640 00:33:43,470 --> 00:33:45,390 is that the first thing you do is 641 00:33:45,390 --> 00:33:49,530 you build up the imidazole moeity of your purine. 642 00:33:49,530 --> 00:33:53,520 So using sort of basic metabolites in ATP-- 643 00:33:53,520 --> 00:33:57,750 there are five steps out of the 10 that use ATP-- 644 00:33:57,750 --> 00:34:02,580 you make this amino imidazole ribonucelotide. 645 00:34:02,580 --> 00:34:05,940 And then what you do again, step by step, 646 00:34:05,940 --> 00:34:09,630 is convert this into the pyrimidine moiety. 647 00:34:09,630 --> 00:34:11,610 So you make your purine. 648 00:34:11,610 --> 00:34:14,460 So that's a step, one step at a time. 649 00:34:14,460 --> 00:34:20,219 And this was unraveled using metabolic labeling experiments. 650 00:34:20,219 --> 00:34:23,489 So the first enzyme I'll spend a little bit of time 651 00:34:23,489 --> 00:34:27,030 on, because I think it's a paradigm for many enzymes 652 00:34:27,030 --> 00:34:31,739 in metabolism in general, where do you get ammonia 653 00:34:31,739 --> 00:34:32,909 from most of the time? 654 00:34:32,909 --> 00:34:36,420 The major source of ammonia is glutamine. 655 00:34:36,420 --> 00:34:42,000 So that's something that you see in this pathway. 656 00:34:42,000 --> 00:34:48,530 So glutamine-- you all know glutamine has this part in this 657 00:34:48,530 --> 00:34:50,520 side chain-- 658 00:34:50,520 --> 00:34:53,190 is going to glutamate. 659 00:34:53,190 --> 00:34:56,639 And so you form glutamic acid. 660 00:34:56,639 --> 00:34:59,280 And the ammonia from the amid is going 661 00:34:59,280 --> 00:35:03,150 to interact with phosphoribosyl pyrophosphate, which 662 00:35:03,150 --> 00:35:11,010 is always bound to magnesium to form phosphoribosylamine. 663 00:35:11,010 --> 00:35:20,073 And so I'm now going to start being sloppier. 664 00:35:20,073 --> 00:35:21,490 Instead of writing phosphate here, 665 00:35:21,490 --> 00:35:23,782 I'm going to have a phosphorus with a circle around it. 666 00:35:23,782 --> 00:35:26,340 That means we always have the five prime phosphate. 667 00:35:26,340 --> 00:35:29,700 And furthermore, what I'm going to do 668 00:35:29,700 --> 00:35:32,220 is replace all of this with an R group, 669 00:35:32,220 --> 00:35:36,180 ribosylphosphate is present at every single step 670 00:35:36,180 --> 00:35:37,410 in the pathway. 671 00:35:37,410 --> 00:35:39,870 And in fact, one of the reasons I thought this pathway was 672 00:35:39,870 --> 00:35:42,690 interesting, every enzyme in the pathway 673 00:35:42,690 --> 00:35:45,323 has to have a binding site for ribosylphosphate. 674 00:35:45,323 --> 00:35:46,740 Well, have any of you ever thought 675 00:35:46,740 --> 00:35:51,340 about how metabolic pathways evolved? 676 00:35:51,340 --> 00:35:52,430 Where does it come from? 677 00:35:52,430 --> 00:35:54,610 You have these really complicated pathways. 678 00:35:54,610 --> 00:35:55,820 Where do you start? 679 00:35:55,820 --> 00:35:57,070 How do you think about that? 680 00:35:57,070 --> 00:35:59,830 Well, this might be a fantastic place to look at that. 681 00:35:59,830 --> 00:36:00,490 Why? 682 00:36:00,490 --> 00:36:03,610 Because you might have a ribosyl binding site for everything. 683 00:36:03,610 --> 00:36:05,020 So maybe it starts with something 684 00:36:05,020 --> 00:36:07,030 that binds ribosylphosphate. 685 00:36:07,030 --> 00:36:08,945 Anyhow, this is an unusual pathway 686 00:36:08,945 --> 00:36:10,070 in that you have something. 687 00:36:10,070 --> 00:36:13,482 You have a really good handle on to hang on to. 688 00:36:13,482 --> 00:36:15,190 And as we already talked about-- so this, 689 00:36:15,190 --> 00:36:17,410 we're going to call R-- 690 00:36:17,410 --> 00:36:19,188 what's unusual, there are a couple things 691 00:36:19,188 --> 00:36:20,230 I want to say about this. 692 00:36:20,230 --> 00:36:24,250 But we already talked about this a little in terms of channeling 693 00:36:24,250 --> 00:36:26,200 and this question of why you would ever want 694 00:36:26,200 --> 00:36:29,680 to have clustering enzymes. 695 00:36:29,680 --> 00:36:33,370 And that's because the half-life of this 696 00:36:33,370 --> 00:36:37,320 is about 10 seconds at 37 degrees. 697 00:36:37,320 --> 00:36:39,943 So it took a lot of effort to see this thing. 698 00:36:39,943 --> 00:36:41,860 I mean, you couldn't see it by normal methods. 699 00:36:41,860 --> 00:36:45,190 People inferred its presence because Buchanan actually 700 00:36:45,190 --> 00:36:48,730 was able to see the next intermediate in the pathway 701 00:36:48,730 --> 00:36:51,100 and inferred the existence of this. 702 00:36:51,100 --> 00:36:53,260 And many of these intermediates in the pathway, 703 00:36:53,260 --> 00:36:58,060 which is why Benkovic focused on this, are chemically unstable. 704 00:36:58,060 --> 00:36:59,727 Let's see if I have one of these. 705 00:36:59,727 --> 00:37:01,060 I don't have it in this pathway. 706 00:37:01,060 --> 00:37:02,410 Anyhow, I'll show you another one, which 707 00:37:02,410 --> 00:37:05,170 has a half-life of five seconds or something like that, that 708 00:37:05,170 --> 00:37:07,010 took forever for people to identify it, 709 00:37:07,010 --> 00:37:09,010 because when you try to work it up as a chemist, 710 00:37:09,010 --> 00:37:10,720 it falls apart. 711 00:37:10,720 --> 00:37:13,510 And I would say this is something any of you 712 00:37:13,510 --> 00:37:17,840 get into metabolomics, people are looking for metabolites 713 00:37:17,840 --> 00:37:18,710 now. 714 00:37:18,710 --> 00:37:20,530 There's one metabolite that people 715 00:37:20,530 --> 00:37:22,330 have found quite frequently, and it 716 00:37:22,330 --> 00:37:26,290 seems to be involved in regulation of glycolysis. 717 00:37:26,290 --> 00:37:29,390 It's this one. 718 00:37:29,390 --> 00:37:30,650 See, where am I? 719 00:37:33,390 --> 00:37:37,980 Aminoimidazole ribo-- this one. 720 00:37:37,980 --> 00:37:40,260 And that's because it's stable. 721 00:37:40,260 --> 00:37:42,550 And the lot of the other ones are not very stable. 722 00:37:42,550 --> 00:37:45,060 So I wouldn't be surprised if you ended up 723 00:37:45,060 --> 00:37:46,800 finding a lot more metabolites that 724 00:37:46,800 --> 00:37:50,430 are playing a central role in regulating enzymes 725 00:37:50,430 --> 00:37:52,920 in primary metabolism, because where 726 00:37:52,920 --> 00:37:54,660 does the serine come from? 727 00:37:54,660 --> 00:37:56,160 Does anybody know where the serine 728 00:37:56,160 --> 00:38:01,440 comes from that plays a key role in making this folate analog? 729 00:38:01,440 --> 00:38:02,790 Anybody have any idea? 730 00:38:06,980 --> 00:38:11,170 So serine is three phosphoglyceric acid 731 00:38:11,170 --> 00:38:12,740 in the glycolysis pathway. 732 00:38:12,740 --> 00:38:14,360 It's actually very straightforward 733 00:38:14,360 --> 00:38:17,180 to write a mechanism of how you get there. 734 00:38:17,180 --> 00:38:22,130 Intimately links the glycolysis pathway to purine metabolism. 735 00:38:22,130 --> 00:38:25,310 And we'll also see here of course, this is folate, 736 00:38:25,310 --> 00:38:26,690 but we also need glycine. 737 00:38:26,690 --> 00:38:30,242 That's the next step in small molecule in this pathway. 738 00:38:30,242 --> 00:38:30,950 It needs glycine. 739 00:38:30,950 --> 00:38:32,290 So everything is integrated. 740 00:38:32,290 --> 00:38:34,490 Once you see-- you sort of see the big picture 741 00:38:34,490 --> 00:38:37,280 and have central pictures of primary metabolism, 742 00:38:37,280 --> 00:38:41,420 everything becomes much more integrated. 743 00:38:41,420 --> 00:38:43,520 So how does this happen? 744 00:38:43,520 --> 00:38:46,130 So what I want to do is I want to talk 745 00:38:46,130 --> 00:38:47,510 a little bit about this enzyme. 746 00:38:50,890 --> 00:38:53,810 So here, let me just talk about this this. 747 00:38:53,810 --> 00:38:59,030 So if we call this Pur F, just so we have a name, 748 00:38:59,030 --> 00:39:03,875 Pur F is called an amidotransferase. 749 00:39:09,550 --> 00:39:15,535 And what it's going to do is it's going to take glutamine-- 750 00:39:24,420 --> 00:39:29,100 and it turns out these enzymes have a domain. 751 00:39:31,650 --> 00:39:33,960 They always have multiple domains. 752 00:39:33,960 --> 00:39:37,830 And the domain that uses the glutamine can be the same. 753 00:39:37,830 --> 00:39:40,860 There are actually two different convergent evolutions 754 00:39:40,860 --> 00:39:45,480 of glutamine binding domains that do the same chemistry. 755 00:39:45,480 --> 00:39:49,905 So what you do is-- we've seen this again many, many times-- 756 00:39:53,490 --> 00:40:00,570 so you form a covalent intermediate, 757 00:40:00,570 --> 00:40:04,370 which then hydrolyzes to glutamate regenerating ESH. 758 00:40:07,620 --> 00:40:14,560 And what happened during this reaction, you generate ammonia. 759 00:40:14,560 --> 00:40:18,130 So the goal of these amidotransferases in general, 760 00:40:18,130 --> 00:40:21,910 in many, many metabolic pathways, 761 00:40:21,910 --> 00:40:23,030 is to generate ammonia. 762 00:40:25,990 --> 00:40:27,970 And so to me, what's striking about 763 00:40:27,970 --> 00:40:33,310 this is the way nature evolved these metabolic enzymes 764 00:40:33,310 --> 00:40:34,780 that generate ammonia. 765 00:40:34,780 --> 00:40:36,970 And so what you see in a cartoon view-- 766 00:40:36,970 --> 00:40:41,950 so we are always going to have all of these enzymes. 767 00:40:41,950 --> 00:40:43,720 They may be a single polypeptide. 768 00:40:43,720 --> 00:40:46,780 They may be two polypeptides, but they all 769 00:40:46,780 --> 00:40:49,465 have a glutaminase domain. 770 00:40:55,470 --> 00:40:59,730 So the glutaminase is just generating the ammonia. 771 00:40:59,730 --> 00:41:01,360 But what do we have? 772 00:41:01,360 --> 00:41:03,600 We start out with phosphoribosyl pyrophosphate. 773 00:41:03,600 --> 00:41:06,450 So once we generate the ammonia, what can happen? 774 00:41:06,450 --> 00:41:10,990 You can now by-- it turns out by dissociated mechanism-- 775 00:41:10,990 --> 00:41:14,790 displace a pyrophosphate to form phosphoribosylamine. 776 00:41:14,790 --> 00:41:17,910 So all of these kinds of reactions 777 00:41:17,910 --> 00:41:21,850 involve dissociative rather than associative transition states. 778 00:41:21,850 --> 00:41:24,110 That's not important. 779 00:41:24,110 --> 00:41:34,545 But what it what's amazing about this is that PRPP, 780 00:41:34,545 --> 00:41:37,950 in this case, binds to one domain, 781 00:41:37,950 --> 00:41:42,360 and the glutamine binds to this second domain. 782 00:41:42,360 --> 00:41:44,400 So ammonia, what would happen to ammonia 783 00:41:44,400 --> 00:41:46,698 if it went out into solution? 784 00:41:46,698 --> 00:41:47,607 STUDENT: Protonated. 785 00:41:47,607 --> 00:41:48,440 JOANNE STUBBE: Yeah. 786 00:41:48,440 --> 00:41:51,240 Gets protonated really rapidly, becomes unreactive. 787 00:41:51,240 --> 00:41:53,130 I don't know why nature designed this. 788 00:41:53,130 --> 00:41:57,540 But what you see with all these enzymes is she 789 00:41:57,540 --> 00:42:01,860 makes a tunnel across the domain interface that's 790 00:42:01,860 --> 00:42:04,830 about 25 to 40 angstroms long. 791 00:42:04,830 --> 00:42:07,790 So the ammonia this released never gets out into solutions. 792 00:42:07,790 --> 00:42:09,540 This is another example of channeling 793 00:42:09,540 --> 00:42:13,500 a reactive intermediate, which we talked about as potentially 794 00:42:13,500 --> 00:42:16,200 a reason for channeling in the purine pathway. 795 00:42:16,200 --> 00:42:20,250 So there's a tunnel. 796 00:42:20,250 --> 00:42:23,790 And the tunnel can be 25-- 797 00:42:23,790 --> 00:42:31,125 we have a number of structures in the ammonia channels. 798 00:42:34,770 --> 00:42:36,280 And I have no idea-- 799 00:42:36,280 --> 00:42:38,620 I mean, this surprised the heck out of me. 800 00:42:38,620 --> 00:42:40,750 I thought the way nature would hold 801 00:42:40,750 --> 00:42:45,380 on to this is by hanging on to not the covalent intermediate, 802 00:42:45,380 --> 00:42:47,500 but the preceding tetrahedral intermediate. 803 00:42:47,500 --> 00:42:49,840 And then when the white substrate was there, 804 00:42:49,840 --> 00:42:53,320 release it and then bind it, sitting right next to it. 805 00:42:53,320 --> 00:42:56,620 But nature, in all designs, has done this thing 806 00:42:56,620 --> 00:42:58,390 where you have this channel. 807 00:42:58,390 --> 00:43:03,010 And here is an example of Pur F. 808 00:43:03,010 --> 00:43:05,630 This is the glutaminase domain up here. 809 00:43:05,630 --> 00:43:08,680 And here is where the phosphoribosyl pyrophosphate 810 00:43:08,680 --> 00:43:09,880 binds down here. 811 00:43:09,880 --> 00:43:12,100 You can't see the channel, but this 812 00:43:12,100 --> 00:43:14,170 is work of Jan Smith a number of years ago, 813 00:43:14,170 --> 00:43:18,850 was the first one that showed the channel in this pathway. 814 00:43:18,850 --> 00:43:20,350 So that's common. 815 00:43:20,350 --> 00:43:23,200 And we're going to look at another glutamine requiring 816 00:43:23,200 --> 00:43:24,370 enzyme in this pathway. 817 00:43:24,370 --> 00:43:26,200 It's the fourth enzyme in the pathway. 818 00:43:26,200 --> 00:43:27,430 Also is a channel. 819 00:43:27,430 --> 00:43:29,800 Again, it's distinct. 820 00:43:29,800 --> 00:43:34,000 It all does this glutaminase covalent intermediate, 821 00:43:34,000 --> 00:43:39,520 but the structure of the glutaminase domain is distinct. 822 00:43:39,520 --> 00:43:41,360 So what's the next enzyme in the pathway? 823 00:43:41,360 --> 00:43:43,900 So the next enzyme in the pathway again, 824 00:43:43,900 --> 00:43:47,050 is a paradigm for many, many unsungs 825 00:43:47,050 --> 00:43:49,630 and primary metabolic pathways. 826 00:43:49,630 --> 00:43:51,040 And if you look at the structure, 827 00:43:51,040 --> 00:43:57,370 let's just go back to the pathway. 828 00:43:57,370 --> 00:44:01,240 If you look at this pathway, what you now want to do-- 829 00:44:01,240 --> 00:44:03,940 so we keep the ribose 5-phosphate all the way 830 00:44:03,940 --> 00:44:05,920 through the whole thing. 831 00:44:05,920 --> 00:44:07,540 That's the scaffold. 832 00:44:07,540 --> 00:44:08,830 Now what are you going to add? 833 00:44:08,830 --> 00:44:11,230 You're going to add glycine. 834 00:44:11,230 --> 00:44:13,140 So here is your phosphoribosylamine. 835 00:44:13,140 --> 00:44:15,340 And you're going to add glycine. 836 00:44:15,340 --> 00:44:19,060 How do you inactivate an amino acid? 837 00:44:19,060 --> 00:44:21,790 You've seen activation of amino acids now many times. 838 00:44:21,790 --> 00:44:24,858 What are the two ways you can activate amino acids? 839 00:44:24,858 --> 00:44:26,142 STUDENT: [INAUDIBLE]. 840 00:44:26,142 --> 00:44:29,100 JOANNE STUBBE: So either adenylate or phosphorylate. 841 00:44:29,100 --> 00:44:31,750 So that's a paradigm that you see over and over again 842 00:44:31,750 --> 00:44:33,910 in nature. 843 00:44:33,910 --> 00:44:37,290 This enzyme uses ATP. 844 00:44:37,290 --> 00:44:40,300 This is one of the five enzymes. 845 00:44:40,300 --> 00:44:42,670 And it forms inorganic phosphate. 846 00:44:42,670 --> 00:44:46,510 So you're phosphorylating, not adenylating. 847 00:44:46,510 --> 00:44:51,202 And so I'll show you what the mechanism is up there. 848 00:44:51,202 --> 00:44:52,660 You've already seen this mechanism, 849 00:44:52,660 --> 00:44:54,490 but the idea is you phosphorylate this. 850 00:44:54,490 --> 00:44:57,400 You're going to form the phosphoanhydride. 851 00:44:57,400 --> 00:45:03,340 And then the phosphoanhydride can react with the amino group. 852 00:45:03,340 --> 00:45:06,198 And kinetically-- this is something that's one 853 00:45:06,198 --> 00:45:07,990 of my students working on a long time ago-- 854 00:45:07,990 --> 00:45:10,150 there was evidence that this intermediate, which 855 00:45:10,150 --> 00:45:12,910 is chemically unstable, could channel between the two 856 00:45:12,910 --> 00:45:13,430 proteins. 857 00:45:13,430 --> 00:45:15,100 So you don't generate this own solution 858 00:45:15,100 --> 00:45:17,230 where it can fall apart and it can anomerize. 859 00:45:17,230 --> 00:45:18,580 It gets transferred directly. 860 00:45:18,580 --> 00:45:22,310 In fact, in the early days when we 861 00:45:22,310 --> 00:45:25,290 invented the first biochemistry labs at MIT, 862 00:45:25,290 --> 00:45:27,363 they used this system. 863 00:45:27,363 --> 00:45:28,780 I really pushed them to the limit, 864 00:45:28,780 --> 00:45:30,760 because they were dealing with the substrate. 865 00:45:30,760 --> 00:45:32,770 They had a very short half-life Anyhow, 866 00:45:32,770 --> 00:45:35,440 they learned a lot from the exercise. 867 00:45:35,440 --> 00:45:38,500 So what you're going to have then is ribos-- 868 00:45:38,500 --> 00:45:44,910 I'm just going to call it R. And so here's our glycine. 869 00:45:44,910 --> 00:45:45,856 Whoops. 870 00:45:45,856 --> 00:45:47,880 Guess I'd better get the structure right. 871 00:45:52,820 --> 00:45:56,970 So this is from lysine. 872 00:45:56,970 --> 00:46:03,008 So what we will see is that this is another-- 873 00:46:03,008 --> 00:46:04,550 we're not getting very far-- but this 874 00:46:04,550 --> 00:46:14,540 is a member of the ATP grasp superfamily of enzymes. 875 00:46:14,540 --> 00:46:15,920 They all do the same chemistry. 876 00:46:15,920 --> 00:46:19,400 So let me just move forward a little bit. 877 00:46:19,400 --> 00:46:20,930 I'm not going to draw this out. 878 00:46:20,930 --> 00:46:24,710 You guys have seen this chemistry many times. 879 00:46:24,710 --> 00:46:27,590 So what's happening in this chemistry 880 00:46:27,590 --> 00:46:30,140 is you have a carboxylate. 881 00:46:30,140 --> 00:46:36,360 ATP phosphorylates it, and then you attack by a nucleophile, 882 00:46:36,360 --> 00:46:40,730 in this case, the nucleophile is the amino group 883 00:46:40,730 --> 00:46:43,340 of phosphoribosylamine. 884 00:46:43,340 --> 00:46:46,400 So what I just want you to see here if you look at this, 885 00:46:46,400 --> 00:46:49,790 there are four enzymes that are involved in purine metabolism 886 00:46:49,790 --> 00:46:51,860 that all have the same structure. 887 00:46:51,860 --> 00:46:56,540 They all have ATP grasp structures. 888 00:46:56,540 --> 00:46:59,480 They all go through phosoanhydride intermediates. 889 00:46:59,480 --> 00:47:01,820 And you can, from bioinformatics, 890 00:47:01,820 --> 00:47:04,160 pick these structures out. 891 00:47:04,160 --> 00:47:05,720 So this is again, an example. 892 00:47:05,720 --> 00:47:09,560 Once people defined-- there's almost a no sequence homology 893 00:47:09,560 --> 00:47:11,060 between these proteins-- 894 00:47:11,060 --> 00:47:14,540 but by knowing this chemistry, you 895 00:47:14,540 --> 00:47:17,400 can actually pick out that these are going to be family members. 896 00:47:17,400 --> 00:47:19,010 And if you know if they are organized 897 00:47:19,010 --> 00:47:21,830 in bacteria and operons, you can even guess at the substrate. 898 00:47:21,830 --> 00:47:24,590 And then you can test this model that they 899 00:47:24,590 --> 00:47:28,520 go through phosoanhydride intermediates. 900 00:47:28,520 --> 00:47:31,025 And I'm over, but the next step in this pathway-- 901 00:47:36,570 --> 00:47:38,300 the next step in this pathway-- 902 00:47:38,300 --> 00:47:40,490 we're going to come back. 903 00:47:40,490 --> 00:47:42,300 And what are we going to use? 904 00:47:42,300 --> 00:47:44,660 We're going to use N10 formal tetrahydrofolate. 905 00:47:44,660 --> 00:47:46,280 That's why I went through this. 906 00:47:46,280 --> 00:47:49,430 We're going to put a formal group here. 907 00:47:49,430 --> 00:47:51,290 And again, the chemistry is just the same. 908 00:47:51,290 --> 00:47:54,020 Go home and think about the chemistry 909 00:47:54,020 --> 00:47:56,120 of how you generate all the different oxidation 910 00:47:56,120 --> 00:47:57,453 states of the carbon. 911 00:47:57,453 --> 00:47:59,870 And then I think you can see the chemistry in this pathway 912 00:47:59,870 --> 00:48:03,890 actually is pretty simple, once a few basic reactions. 913 00:48:03,890 --> 00:48:07,370 So the ATP grasp family is interesting. 914 00:48:07,370 --> 00:48:10,160 The amidotransferase and the channel 915 00:48:10,160 --> 00:48:14,320 is interesting as being general in metabolism.