WEBVTT 00:00:00.000 --> 00:00:02.830 NARRATOR: The following content is provided under a Creative 00:00:02.830 --> 00:00:04.370 Commons license. 00:00:04.370 --> 00:00:06.670 Your support will help MIT Open Courseware 00:00:06.670 --> 00:00:11.030 continue to offer high quality educational resources for free. 00:00:11.030 --> 00:00:13.660 To make a donation or view additional materials 00:00:13.660 --> 00:00:17.130 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.130 --> 00:00:18.140 at ocw.mit.edu. 00:00:25.282 --> 00:00:26.740 JOANNE STUBBE: So what I want to do 00:00:26.740 --> 00:00:30.190 is finish up purines today and talk 00:00:30.190 --> 00:00:33.598 about some interesting aspects of purine metabolism. 00:00:33.598 --> 00:00:35.390 I hope I'm going to be able to get through. 00:00:35.390 --> 00:00:40.240 I've given you handouts for pyrimidines and deoxynucleotide 00:00:40.240 --> 00:00:41.200 biosynthesis as well. 00:00:41.200 --> 00:00:44.620 The pyrimidines are pretty straightforward, much simpler 00:00:44.620 --> 00:00:45.490 than the purines. 00:00:45.490 --> 00:00:47.260 And so I think if I have time today, 00:00:47.260 --> 00:00:49.360 depending on when I get finished, 00:00:49.360 --> 00:00:52.780 I might talk a little bit about deoxynucleotide metabolism, 00:00:52.780 --> 00:00:57.380 since both Drennan's lab and my lab, both work in that area. 00:00:57.380 --> 00:00:59.470 So it would be good for you guys to know what's 00:00:59.470 --> 00:01:01.150 going on in the department and it's 00:01:01.150 --> 00:01:04.480 central to nucleotide metabolism. 00:01:08.380 --> 00:01:10.000 We started out-- we were drawing this. 00:01:10.000 --> 00:01:16.630 This is my notes that I tried to reproduce for you to look at. 00:01:16.630 --> 00:01:18.160 And I'm not going to read. 00:01:18.160 --> 00:01:21.370 So this was a big overview slide where we're going. 00:01:21.370 --> 00:01:25.030 And so central to everything. 00:01:25.030 --> 00:01:28.030 This wasn't in the original packet, but I will put this up. 00:01:28.030 --> 00:01:30.350 I'll try to get a better version of that. 00:01:30.350 --> 00:01:33.100 But PRPP is central. 00:01:33.100 --> 00:01:38.200 And we are talking about de novo purine bio-synthesis, 00:01:38.200 --> 00:01:43.930 but again, not only is de novo important, so is salvage. 00:01:43.930 --> 00:01:45.912 It depends on the cell type. 00:01:45.912 --> 00:01:48.370 You know, if you have cancer cells that are rapidly growing 00:01:48.370 --> 00:01:51.640 or B cells and T cells, de novo becomes really important. 00:01:51.640 --> 00:01:55.150 In other types of cells almost everything is salvage. 00:01:55.150 --> 00:02:00.358 And so I have that PRPP, at least in the purine case-- 00:02:00.358 --> 00:02:02.650 and I'll show you an example of that in a few minutes-- 00:02:02.650 --> 00:02:06.680 goes directly-- can make your nucleotide directly. 00:02:06.680 --> 00:02:08.949 That's a salvage pathway. 00:02:08.949 --> 00:02:11.920 And we'll see that the de novo pathway, 00:02:11.920 --> 00:02:15.190 which is what I was describing at the end-- 00:02:15.190 --> 00:02:19.300 and you've already seen this in recitation from last week-- 00:02:19.300 --> 00:02:23.470 is 10 steps to get to IMP. 00:02:23.470 --> 00:02:27.880 But then you need to get to GMP and AMP. 00:02:27.880 --> 00:02:35.880 And I showed you how all of this branches off with the cofactor 00:02:35.880 --> 00:02:39.790 folate between purines and pyrimidines. 00:02:42.660 --> 00:02:48.330 And in the end, we need both purines and pyrimidines. 00:02:48.330 --> 00:02:50.520 We need it in the nucleotide levels. 00:02:50.520 --> 00:02:54.240 So two hydroxyls, the two prime three prime cis hydroxyls, 00:02:54.240 --> 00:02:58.050 which in the diphosphate stage, that's also unusual. 00:02:58.050 --> 00:03:00.660 Most of the time you don't see high levels of diphosphates 00:03:00.660 --> 00:03:02.850 inside the cell, either they're monophosphates 00:03:02.850 --> 00:03:04.350 or triphosphates. 00:03:04.350 --> 00:03:09.030 So part of the complexity I think of nucleotide metabolism 00:03:09.030 --> 00:03:10.770 is figuring out where the kinases are 00:03:10.770 --> 00:03:12.360 and the phosotases are. 00:03:12.360 --> 00:03:14.340 And you'll notice that I've avoided that. 00:03:14.340 --> 00:03:16.770 And that's because every organism is different 00:03:16.770 --> 00:03:19.320 and every cell type is different, 00:03:19.320 --> 00:03:21.810 and the regulation is a little bit different. 00:03:21.810 --> 00:03:23.820 But I think it's important to realize 00:03:23.820 --> 00:03:27.240 that to make deoxynucleotides, which are required 00:03:27.240 --> 00:03:29.310 for DNA replication and repair, is 00:03:29.310 --> 00:03:31.200 done at the diphosphate level. 00:03:31.200 --> 00:03:35.010 So you make deoxynucleotides, but they still 00:03:35.010 --> 00:03:42.870 have to be converted to deoxy NTPs for DNA. 00:03:42.870 --> 00:03:52.440 And over here you need to again, make NTPs for RNA. 00:03:52.440 --> 00:03:54.480 So that's sort of the big picture. 00:03:54.480 --> 00:04:00.402 We have a purine pathway de novo. 00:04:00.402 --> 00:04:02.610 We're not going to be able to talk about pyrimidines, 00:04:02.610 --> 00:04:08.760 but the salvage pathway with pyrimidines 00:04:08.760 --> 00:04:11.020 is extremely important. 00:04:11.020 --> 00:04:14.310 It's a major target of cancer therapeutics now. 00:04:14.310 --> 00:04:15.990 I think only in the last few years 00:04:15.990 --> 00:04:18.990 has it been realized that in many cancers 00:04:18.990 --> 00:04:21.640 you have both pathways going on. 00:04:21.640 --> 00:04:24.090 And it turns out now with isotopic labeling 00:04:24.090 --> 00:04:27.780 and mass spec, metabolomics is coming into its own. 00:04:27.780 --> 00:04:31.000 So you can tell actually by feeding the cells this 00:04:31.000 --> 00:04:32.250 is all done in tissue culture. 00:04:32.250 --> 00:04:34.170 But you can tell by feeding the cells 00:04:34.170 --> 00:04:38.610 whether the deoxynucleotides came from de novo 00:04:38.610 --> 00:04:40.500 or whether they came from salvage. 00:04:40.500 --> 00:04:43.020 And so we're getting a really different view 00:04:43.020 --> 00:04:44.460 of nucleotide metabolism. 00:04:44.460 --> 00:04:45.960 And as I said in the very beginning, 00:04:45.960 --> 00:04:47.660 I think the next decade we're going 00:04:47.660 --> 00:04:52.560 to understand a lot more about how all these things interact 00:04:52.560 --> 00:04:54.810 and the kinases and phosatases that 00:04:54.810 --> 00:04:58.860 put the nucleotides into the correct phosphorylation state. 00:04:58.860 --> 00:05:03.180 So that's key to everything and it's complicated. 00:05:03.180 --> 00:05:08.070 So what I want to do now is briefly 00:05:08.070 --> 00:05:10.560 talk about the purine pathway. 00:05:14.700 --> 00:05:17.430 So we can look at the biology. 00:05:17.430 --> 00:05:20.090 I'm not going to write this down, because most of you 00:05:20.090 --> 00:05:20.840 already know this. 00:05:20.840 --> 00:05:23.250 So we'll just go through it again. 00:05:23.250 --> 00:05:26.280 Purine nucleotides are central to everything. 00:05:26.280 --> 00:05:28.980 So knowing where they come from and how you control them 00:05:28.980 --> 00:05:30.780 is really pretty important. 00:05:30.780 --> 00:05:33.030 And we don't understand that much. 00:05:33.030 --> 00:05:36.660 So I mean, NTPs and dNTPs are central 00:05:36.660 --> 00:05:38.430 to our genetic material. 00:05:38.430 --> 00:05:41.520 So we need to get them and we need to control them. 00:05:41.520 --> 00:05:44.580 If these levels become imbalanced, 00:05:44.580 --> 00:05:49.230 you have mutater phenotypes in DNA replication. 00:05:49.230 --> 00:05:55.950 And so fidelity of deoxynucleotide DNA replication 00:05:55.950 --> 00:05:58.860 is really important and regulated 00:05:58.860 --> 00:06:01.650 by ribonucelotide reductases. 00:06:01.650 --> 00:06:04.470 Building blocks for cofactors. 00:06:04.470 --> 00:06:05.460 We've seen flavins. 00:06:05.460 --> 00:06:06.450 We've seen NAD. 00:06:06.450 --> 00:06:07.830 We've seen CoA. 00:06:07.830 --> 00:06:09.190 None of this is an accident. 00:06:09.190 --> 00:06:13.530 Adenine can self-assemble from cyanide formate 00:06:13.530 --> 00:06:15.360 in the prebiotic world. 00:06:15.360 --> 00:06:19.110 And so that's why they're central to everything. 00:06:19.110 --> 00:06:22.350 So they're in a lot of the cofactors we've already 00:06:22.350 --> 00:06:25.590 talked about is they're not necessarily the business end, 00:06:25.590 --> 00:06:28.680 but they've got the phosphates and the adenines stuck on 00:06:28.680 --> 00:06:33.600 to end, which presumably helps in some way for binding. 00:06:33.600 --> 00:06:36.570 We're using GDP and ATP everywhere 00:06:36.570 --> 00:06:38.250 in the course of this semester. 00:06:38.250 --> 00:06:41.910 You've seen it in your macromolecular machines 00:06:41.910 --> 00:06:43.440 that you've talked about, especially 00:06:43.440 --> 00:06:47.520 in the first part of the course with the translational 00:06:47.520 --> 00:06:51.030 and protein folding and protein degradation all 00:06:51.030 --> 00:06:55.410 require energy ATP. 00:06:55.410 --> 00:06:58.490 We will see in today's pathway and today's lecture 00:06:58.490 --> 00:07:01.260 on purine biosynthesis de novo, it 00:07:01.260 --> 00:07:05.610 turns out 5 out of the 10 enzymes use ATP. 00:07:05.610 --> 00:07:06.380 So we'll see. 00:07:06.380 --> 00:07:11.352 And what you will hopefully now will know is what ATP does. 00:07:11.352 --> 00:07:12.810 I'm going to show you two examples. 00:07:12.810 --> 00:07:15.255 But you see the same thing really over and over 00:07:15.255 --> 00:07:15.880 and over again. 00:07:15.880 --> 00:07:17.820 So this should be sort of-- you might not 00:07:17.820 --> 00:07:22.710 know whether it uses ATP to get at the gamma position-- 00:07:22.710 --> 00:07:25.410 chemistry at the gamma position, or at the alpha position 00:07:25.410 --> 00:07:28.440 but the chemistry is the same over and over again. 00:07:28.440 --> 00:07:32.340 And so that part hopefully is part of your repertoire now, 00:07:32.340 --> 00:07:34.440 about thinking about the role of the ATP 00:07:34.440 --> 00:07:36.540 on primary metabolic pathways. 00:07:36.540 --> 00:07:39.300 And we've also seen in the last-- 00:07:39.300 --> 00:07:43.080 in the reactive oxygen species section, 00:07:43.080 --> 00:07:45.150 we are signaling by many mechanisms, 00:07:45.150 --> 00:07:47.690 signaling by phosphorylation is all over the place. 00:07:47.690 --> 00:07:50.220 And a lot of people are trying to understand. 00:07:50.220 --> 00:07:51.690 And I think one of the futures is 00:07:51.690 --> 00:07:56.520 how do you integrate signaling and primary metabolic pathways? 00:07:56.520 --> 00:07:57.960 And we're almost there. 00:07:57.960 --> 00:08:01.740 I decided-- I wrote a lecture on this and decided-- 00:08:01.740 --> 00:08:04.950 it's really still very phenomenological. 00:08:04.950 --> 00:08:08.550 But all of these key regulators and signaling 00:08:08.550 --> 00:08:11.160 linked to purines and pyrimidines in some way. 00:08:11.160 --> 00:08:16.080 I think the linkages aren't totally clear, in my opinion. 00:08:16.080 --> 00:08:20.460 So why else do we care about purines? 00:08:20.460 --> 00:08:24.840 When I was your age, purine and nucleotide metabolism 00:08:24.840 --> 00:08:26.560 was front and center. 00:08:26.560 --> 00:08:27.060 Why? 00:08:27.060 --> 00:08:30.360 Because people were successful at making drugs 00:08:30.360 --> 00:08:32.730 based on these molecules. 00:08:32.730 --> 00:08:36.690 The central role it plays in replication and repair 00:08:36.690 --> 00:08:41.309 has made them successful targets at many different levels. 00:08:41.309 --> 00:08:44.002 Here, this has both purines and pyramidines, 00:08:44.002 --> 00:08:45.210 but I'll just pick out a few. 00:08:45.210 --> 00:08:51.720 This guy acyclovir is what we use as an anti-herpes medicine. 00:08:51.720 --> 00:08:54.260 In fact, I think I've taken it. 00:08:54.260 --> 00:08:58.610 Here, mercaptopurine cures childhood leukemia. 00:08:58.610 --> 00:09:02.190 Clofarabine is something that's been studied my lab. 00:09:02.190 --> 00:09:03.872 It's a drug that-- 00:09:03.872 --> 00:09:05.580 it's not particularly effective, but it's 00:09:05.580 --> 00:09:11.820 used clinically against certain hematological cancers. 00:09:11.820 --> 00:09:14.720 And so these are all anti-metabolites not 00:09:14.720 --> 00:09:17.700 focused on signaling, which is what everybody is focused on. 00:09:17.700 --> 00:09:19.590 In reality, I think the success-- 00:09:19.590 --> 00:09:21.810 if there is success against cancer-- 00:09:21.810 --> 00:09:23.550 is going to be mixing the two. 00:09:23.550 --> 00:09:28.470 I think you need combinations of metabolic inhibitors. 00:09:28.470 --> 00:09:29.370 They're toxic. 00:09:29.370 --> 00:09:30.180 So is everything. 00:09:30.180 --> 00:09:34.590 But somehow figuring out how to use multiple approaches 00:09:34.590 --> 00:09:36.510 to avoid the resistance problem, which 00:09:36.510 --> 00:09:39.180 is a really important problem. 00:09:39.180 --> 00:09:42.690 And to combine the two once we understand the interconnections 00:09:42.690 --> 00:09:44.970 better, I think is where it will be to get 00:09:44.970 --> 00:09:48.030 successful, more successful. 00:09:48.030 --> 00:09:50.880 Therapeutics, but ultimately what we would like to do 00:09:50.880 --> 00:09:54.360 is catch it in the bud, rather than waiting 00:09:54.360 --> 00:09:59.340 to try to treat something where it's completely out of control. 00:09:59.340 --> 00:10:02.550 So I'll just show you one of my favorite ladies, Gertrude 00:10:02.550 --> 00:10:03.630 Elion. 00:10:03.630 --> 00:10:07.740 She worked at Burroughs Wellcome for many, many years. 00:10:07.740 --> 00:10:10.140 She went to Hunter College, as did many-- 00:10:10.140 --> 00:10:14.610 in New York City-- as did many outstanding women scientists. 00:10:14.610 --> 00:10:18.560 And she was involved at Burroughs Wellcome 00:10:18.560 --> 00:10:24.180 in discovery of mercaptopurines, acyclovir treating 00:10:24.180 --> 00:10:27.640 herpes, and AZT. 00:10:27.640 --> 00:10:28.950 She made several contributions. 00:10:28.950 --> 00:10:30.990 Never had a PhD. 00:10:30.990 --> 00:10:35.160 So anyhow-- 00:10:35.160 --> 00:10:36.750 So what I also wanted to show you, 00:10:36.750 --> 00:10:40.470 we're going to talk about de novo pathways. 00:10:40.470 --> 00:10:43.560 I just want to show you this is a typical-- 00:10:43.560 --> 00:10:44.960 in the case of the purines-- 00:10:44.960 --> 00:10:46.660 salvage pathway. 00:10:46.660 --> 00:10:47.970 So what does that mean? 00:10:47.970 --> 00:10:52.980 You get the bases, the nucleic acid bases from your diet. 00:10:52.980 --> 00:10:55.200 Or you're breaking down your DNA and your RNA. 00:10:55.200 --> 00:10:56.790 You have nucleic acid bases. 00:10:56.790 --> 00:10:58.590 Or you have nucleosides. 00:10:58.590 --> 00:11:03.180 So can you take those and make them into the right components 00:11:03.180 --> 00:11:07.170 to do RNA biosynthesis and DNA replication, 00:11:07.170 --> 00:11:08.990 make ATP, et cetera. 00:11:08.990 --> 00:11:13.890 And so here's an example of hypoxanthine reacting 00:11:13.890 --> 00:11:16.740 with our central phosphoribosyl pyrophosphate, which 00:11:16.740 --> 00:11:19.440 I had in the original slide that I 00:11:19.440 --> 00:11:24.450 talked about last time to make, in this case, the nucleotide. 00:11:24.450 --> 00:11:27.630 And why is this interesting? 00:11:27.630 --> 00:11:31.110 It's interesting because it turns out 00:11:31.110 --> 00:11:36.420 that many parasites like in malaria don't have any purines. 00:11:36.420 --> 00:11:38.430 So where do they get their purines from 00:11:38.430 --> 00:11:40.020 to replicate the DNA? 00:11:40.020 --> 00:11:42.870 They have to use salvage. 00:11:42.870 --> 00:11:46.170 So the salvage pathways have-- for treatment of those things-- 00:11:46.170 --> 00:11:47.640 have become front and center. 00:11:47.640 --> 00:11:52.170 Can you make specific inhibitors of phosphoribosyl pyrophosphate 00:11:52.170 --> 00:11:55.170 reaction with the bases? 00:11:55.170 --> 00:11:58.070 And we're pretty good at that actually. 00:11:58.070 --> 00:12:00.300 Vern Schramm's lab has done some beautiful work. 00:12:00.300 --> 00:12:05.310 And there's a lot of things in clinical trial targeting 00:12:05.310 --> 00:12:07.560 salvage pathways. 00:12:07.560 --> 00:12:11.010 So again, there's something different about the metabolism 00:12:11.010 --> 00:12:15.990 of us and whatever is invading us. 00:12:15.990 --> 00:12:17.820 That's not true in cancer. 00:12:17.820 --> 00:12:19.380 So cancer is a much tougher problem, 00:12:19.380 --> 00:12:22.470 because you get normal cells as well. 00:12:22.470 --> 00:12:25.590 It's a question of what the therapeutic index is. 00:12:29.250 --> 00:12:31.260 So that's all I want to say in the introduction 00:12:31.260 --> 00:12:33.270 to the biology. 00:12:33.270 --> 00:12:35.520 And then I want to talk about one cofactor. 00:12:35.520 --> 00:12:38.160 And then I'm going to talk about the pathway itself. 00:12:38.160 --> 00:12:41.280 So there's one cofactor, which I sort of told you 00:12:41.280 --> 00:12:45.170 I was going there in the first place. 00:12:45.170 --> 00:12:49.190 Let me break this down over here. 00:12:49.190 --> 00:12:52.190 So the one cofactor that I wanted to talk about is folate. 00:12:55.400 --> 00:12:56.400 Let me also show you. 00:12:56.400 --> 00:12:59.902 You don't have to sit and look at this. 00:12:59.902 --> 00:13:01.860 But I'm going to show you it's all written out. 00:13:01.860 --> 00:13:04.220 So you don't have to bob up and down. 00:13:04.220 --> 00:13:06.050 It's all written out on the handout. 00:13:06.050 --> 00:13:08.210 So this is folate. 00:13:08.210 --> 00:13:11.280 And let me just point out a few things. 00:13:11.280 --> 00:13:13.590 This is going to be the business end of the molecule. 00:13:13.590 --> 00:13:16.280 So I want you to know where the business end of the molecule 00:13:16.280 --> 00:13:16.780 is. 00:13:16.780 --> 00:13:20.000 I don't expect you to remember the structures. 00:13:23.120 --> 00:13:25.540 But what does this sort of look like? 00:13:25.540 --> 00:13:26.478 Anybody? 00:13:26.478 --> 00:13:27.770 This is the kind of chemistry-- 00:13:27.770 --> 00:13:31.670 I mean, I think there's a bunch of heterocyclic chemistry 00:13:31.670 --> 00:13:35.570 that you find in biology that most of you 00:13:35.570 --> 00:13:37.070 haven't been exposed to. 00:13:37.070 --> 00:13:40.280 And it's not intuitive what the most reactive positions are. 00:13:40.280 --> 00:13:44.840 This cofactor is much simpler than flavins, which we very 00:13:44.840 --> 00:13:47.780 briefly talked about before. 00:13:52.990 --> 00:13:56.400 So this has a polyglutamate on the end. 00:13:56.400 --> 00:13:57.460 So this is folate. 00:13:57.460 --> 00:14:04.540 And what you really need to know is that this is 5, 6, 7, 8. 00:14:04.540 --> 00:14:06.110 And this is 10. 00:14:06.110 --> 00:14:11.320 So the active part of this cofactor is here. 00:14:11.320 --> 00:14:15.550 So everything's going to happen at either N5-- 00:14:15.550 --> 00:14:22.180 if you have a copy of this, you can just circle N5, N10 and N5. 00:14:22.180 --> 00:14:24.590 That's where all the chemistry is going to happen. 00:14:24.590 --> 00:14:27.220 And it turns out the way this cofactor 00:14:27.220 --> 00:14:33.320 works-- so this is 1, 2, 3, 4. 00:14:33.320 --> 00:14:38.020 And so this is 4a, and this is 10a, 8a. 00:14:42.640 --> 00:14:44.800 It sort of looks like flavins. 00:14:44.800 --> 00:14:46.660 And it sort of looks like pterins. 00:14:46.660 --> 00:14:49.120 And pterins actually can undergo redox chemistry 00:14:49.120 --> 00:14:51.520 under certain sets of conditions. 00:14:51.520 --> 00:14:53.440 These molecules are only involved 00:14:53.440 --> 00:14:55.720 in one carbon transfer. 00:14:55.720 --> 00:15:04.630 So the major focus is one carbon transfers. 00:15:04.630 --> 00:15:13.730 And it can do it in the methyl state, in the aldehyde state, 00:15:13.730 --> 00:15:17.430 or it can do it in the acid state. 00:15:17.430 --> 00:15:22.310 So all three oxidation states from one carbon transfers. 00:15:22.310 --> 00:15:24.200 And so then how does it do it? 00:15:24.200 --> 00:15:27.020 And the chemistry actually is fairly simple 00:15:27.020 --> 00:15:30.075 compared to the chemistry that we've looked at before. 00:15:30.075 --> 00:15:31.700 And we looked at a little bit at hemes. 00:15:31.700 --> 00:15:36.200 We looked at a little bit at flavins. 00:15:36.200 --> 00:15:38.390 This is much simpler. 00:15:38.390 --> 00:15:41.600 And so what we're after in the end-- 00:15:41.600 --> 00:15:46.980 and I'll show you how we get there-- so here's N5 methyl. 00:15:46.980 --> 00:15:49.380 And we'll see this is tetrahydrofolate. 00:15:49.380 --> 00:15:53.770 So this ring is completely reduced. 00:15:53.770 --> 00:15:56.034 And so this is tetrahydrofolate. 00:16:02.100 --> 00:16:04.320 And this can undergo oxidation and reduction. 00:16:04.320 --> 00:16:06.840 And that becomes very important in the pyrimidine pathway 00:16:06.840 --> 00:16:11.685 to form thymidine, which is a major target of fluorouracil, 00:16:11.685 --> 00:16:13.560 which is a drug that's still used clinically. 00:16:13.560 --> 00:16:15.870 Anyhow, this is the reduced state here. 00:16:15.870 --> 00:16:18.390 So this is where the tetrahydro is. 00:16:18.390 --> 00:16:21.930 So both of these can be oxidized. 00:16:21.930 --> 00:16:24.060 And that would be folate. 00:16:24.060 --> 00:16:28.800 So you can make dihydrofolate, folate, and tetrahydrofolate. 00:16:28.800 --> 00:16:32.190 And the oxidations occur here and here. 00:16:32.190 --> 00:16:33.690 And we're not going to look at that, 00:16:33.690 --> 00:16:34.860 because we're not going to have time to look 00:16:34.860 --> 00:16:36.470 at pyrimidine metabolism. 00:16:36.470 --> 00:16:39.630 But the dihydrofolate plays an important role. 00:16:39.630 --> 00:16:42.000 It's the target of methotrexate. 00:16:42.000 --> 00:16:43.920 If you have rheumatoid arthritis, 00:16:43.920 --> 00:16:46.740 you take methotrexate is one of the drugs 00:16:46.740 --> 00:16:49.110 that people take nowadays. 00:16:49.110 --> 00:16:51.240 So what's unusual about this-- 00:16:51.240 --> 00:16:53.190 and this is key to the purine pathway, 00:16:53.190 --> 00:16:55.200 it's also key to the pyrimidine pathway-- 00:16:55.200 --> 00:16:57.690 that's why folate have been central. 00:16:57.690 --> 00:16:59.190 People made folates for decades. 00:16:59.190 --> 00:17:02.220 Even when I was your age, people were making folates 00:17:02.220 --> 00:17:05.369 for treatment therapies in cancer. 00:17:05.369 --> 00:17:06.750 And it's been successful. 00:17:06.750 --> 00:17:09.630 In fact, and if you've gone to Princeton's chemistry 00:17:09.630 --> 00:17:12.869 department, the whole department was funded on an anti-folate 00:17:12.869 --> 00:17:17.369 that Ted Taylor made 25 or 30 years ago. 00:17:17.369 --> 00:17:19.650 And they've tried it again under different conditions, 00:17:19.650 --> 00:17:23.069 and it's now being used clinically. 00:17:23.069 --> 00:17:26.160 So how does this work? 00:17:26.160 --> 00:17:29.310 So we have this oxidation state. 00:17:29.310 --> 00:17:30.840 We have this oxidation state. 00:17:30.840 --> 00:17:33.240 And then we'll see that this can ring open. 00:17:33.240 --> 00:17:35.610 And so this would be the aldehyde state. 00:17:35.610 --> 00:17:37.020 And this can hydrolyze. 00:17:37.020 --> 00:17:39.168 And that would be the acid state. 00:17:39.168 --> 00:17:40.710 So I'm going to show you in a second, 00:17:40.710 --> 00:17:43.740 I'm going to walk you through where those different states 00:17:43.740 --> 00:17:44.300 came from. 00:17:44.300 --> 00:17:48.780 So methyl state, aldehyde state, acid state. 00:17:51.410 --> 00:17:56.930 So there's the model, because I like to have the windows open, 00:17:56.930 --> 00:17:59.450 you probably can't see the model very well up there now. 00:17:59.450 --> 00:18:01.760 But you can pull it up on your computer if you want. 00:18:01.760 --> 00:18:03.820 I'm going to write out the model. 00:18:03.820 --> 00:18:09.410 So we start out over here with tetrahydrofolate. 00:18:09.410 --> 00:18:15.140 So this is tetrahydrofolate. 00:18:15.140 --> 00:18:16.910 And we have nothing here, which you 00:18:16.910 --> 00:18:18.710 notice was we could have something 00:18:18.710 --> 00:18:21.380 at N5, something at N10. 00:18:21.380 --> 00:18:23.720 We'll see the methyl group is always at N5. 00:18:23.720 --> 00:18:25.700 It could be at either, chemically, 00:18:25.700 --> 00:18:27.530 but it's always at N5. 00:18:27.530 --> 00:18:30.080 We'll see the aldehyde group is always at N10. 00:18:30.080 --> 00:18:33.050 It could be either chemically, but it's not. 00:18:33.050 --> 00:18:35.150 So these are going to be the key stages. 00:18:35.150 --> 00:18:37.040 And here we have no carbons. 00:18:37.040 --> 00:18:40.650 So somehow we have to get the carbons into the molecules. 00:18:40.650 --> 00:18:45.200 So we start out with this molecule, tetrahydrofolate. 00:18:45.200 --> 00:18:53.690 So what happens is you can start out here, and use formate. 00:18:53.690 --> 00:18:57.110 So formate is going to be the source of the one 00:18:57.110 --> 00:18:59.430 carbon in this case. 00:18:59.430 --> 00:19:03.620 So the names in this pathway are again, 00:19:03.620 --> 00:19:06.280 horrible, just like the purine pathway. 00:19:06.280 --> 00:19:09.980 And on the next slide I've written out the names. 00:19:09.980 --> 00:19:13.100 So it turns out that one enzyme can 00:19:13.100 --> 00:19:15.380 do three of these activities. 00:19:15.380 --> 00:19:17.970 So this is one of the enzymes. 00:19:17.970 --> 00:19:20.270 And so this is activity one. 00:19:20.270 --> 00:19:25.190 And it attaches a formate, so they call it a formate ligase. 00:19:25.190 --> 00:19:27.770 The names again, in my opinion, are horrible. 00:19:27.770 --> 00:19:33.430 But what it allows you to do is-- 00:19:33.430 --> 00:19:38.570 so what I'm going to draw out now is not the whole structure. 00:19:38.570 --> 00:19:41.000 I'm just going to focus on the business 00:19:41.000 --> 00:19:46.160 end of the molecule over here, and skip this ring over here. 00:19:46.160 --> 00:19:49.730 But that ring is there, and is key to making all of this work. 00:19:49.730 --> 00:19:54.140 So I'm just going to do this like that. 00:19:54.140 --> 00:20:04.690 And so what can happen is that you can formulate and form. 00:20:04.690 --> 00:20:13.160 And so this is now N10 formal tetrahydrofolate. 00:20:13.160 --> 00:20:19.970 So this is N10, and we'll call this R. 00:20:19.970 --> 00:20:21.830 So that's the first step. 00:20:21.830 --> 00:20:22.880 That's the enzyme. 00:20:22.880 --> 00:20:26.750 The same enzyme catalyzes the next step. 00:20:26.750 --> 00:20:28.970 And what you can picture happening here, 00:20:28.970 --> 00:20:32.840 if you watch me, is this nitrogen 00:20:32.840 --> 00:20:37.190 is juxtaposed to this imid. 00:20:37.190 --> 00:20:39.800 So can attack to form a tetrahedral intermediate 00:20:39.800 --> 00:20:42.350 and then lose a molecule of water. 00:20:42.350 --> 00:20:45.230 So that's called a cyclohydrolase. 00:20:45.230 --> 00:20:48.560 So this guy is attacking. 00:20:48.560 --> 00:20:52.330 And then you have loss of water. 00:20:52.330 --> 00:20:53.960 And this is a cyclohydrolase. 00:20:57.600 --> 00:20:59.010 So this is the same enzyme. 00:21:02.620 --> 00:21:03.780 So this is two. 00:21:03.780 --> 00:21:04.530 This is one. 00:21:04.530 --> 00:21:07.230 But they're both on the same polypeptide. 00:21:07.230 --> 00:21:09.390 So there are three of these on one polypeptide. 00:21:09.390 --> 00:21:13.110 You've seen that before in recitation last week. 00:21:13.110 --> 00:21:15.960 And so now what you've formed is-- 00:21:15.960 --> 00:21:18.030 and again, this is a cyclohydrolase-- 00:21:18.030 --> 00:21:19.740 now what you're formed is this structure. 00:21:25.950 --> 00:21:28.980 So we've lost a molecule of water. 00:21:28.980 --> 00:21:34.410 So you can draw NR. 00:21:34.410 --> 00:21:36.120 And so if you hydrolyze this, you 00:21:36.120 --> 00:21:39.960 can get back to the aldehyde stage. 00:21:39.960 --> 00:21:45.750 So if water adds here, this is an iminium system. 00:21:45.750 --> 00:21:48.660 Water can add, it can collapse, it can ring open, 00:21:48.660 --> 00:21:51.270 it can ring close. 00:21:51.270 --> 00:21:53.040 So the chemistry here-- we're going 00:21:53.040 --> 00:21:55.240 to see some really similar chemistry actually, 00:21:55.240 --> 00:21:58.770 because we can use N10 formal tetrahydrofolate in two 00:21:58.770 --> 00:22:00.430 steps in the purine pathway. 00:22:00.430 --> 00:22:02.880 So this chemistry I'm drawing right now 00:22:02.880 --> 00:22:06.960 is related to the pathway in general. 00:22:06.960 --> 00:22:16.890 And so this is called 5, 10 methylidine-- 00:22:16.890 --> 00:22:20.910 the names again, are horrible-- tetrahydrofolate. 00:22:20.910 --> 00:22:29.710 And then the third enzyme in this pathway 00:22:29.710 --> 00:22:33.100 is a dehydrogenase, so DH. 00:22:33.100 --> 00:22:36.640 And so what you can imagine you could do here 00:22:36.640 --> 00:22:38.490 is we have an iminium system. 00:22:38.490 --> 00:22:41.210 And NAD pH is the reductive. 00:22:41.210 --> 00:22:45.430 So you can reduce this down to methylene tetrahydrofolate. 00:22:45.430 --> 00:22:53.585 So this can be converted to an NADP. 00:22:56.340 --> 00:22:58.620 So this is the dehydrogenase. 00:22:58.620 --> 00:23:00.450 We've seen that used over and over again. 00:23:00.450 --> 00:23:02.340 This is the same enzyme. 00:23:02.340 --> 00:23:03.660 So this is also MTHFD. 00:23:06.340 --> 00:23:09.510 And I've given you the nomenclature on the next slide. 00:23:09.510 --> 00:23:10.960 So if you want to look at-- 00:23:10.960 --> 00:23:15.090 So this is tetrahydrofolate whatever. 00:23:15.090 --> 00:23:20.100 So it has of formal ligase, it has a cyclohydrolase, 00:23:20.100 --> 00:23:26.100 and it has a D hydrogenase all on one enzyme. 00:23:26.100 --> 00:23:27.630 And so what do you generate then? 00:23:27.630 --> 00:23:44.040 You generate-- so this is methylene tetrahydrofolate. 00:23:47.480 --> 00:23:51.290 And this is the key player in pyrimidine biosynthesis, which 00:23:51.290 --> 00:23:52.850 we are going to talk about. 00:23:52.850 --> 00:23:56.030 And it's an enzyme called thymidylate synthase, which 00:23:56.030 --> 00:24:01.690 makes thymidine, which is a major target for drugs 00:24:01.690 --> 00:24:04.220 in the treatment of cancer. 00:24:04.220 --> 00:24:06.170 So now you can even take this a step further 00:24:06.170 --> 00:24:07.970 and reduce this further. 00:24:07.970 --> 00:24:09.620 We're still now here. 00:24:09.620 --> 00:24:12.950 If you ring open this, you're at the aldehyde stage. 00:24:12.950 --> 00:24:15.920 You can reduce the aldehyde stage down to the methyl group. 00:24:15.920 --> 00:24:18.200 And that's then getting us into the methyl 00:24:18.200 --> 00:24:21.033 state, the aldehyde state, and the acid state. 00:24:21.033 --> 00:24:22.950 So I think when you sit down and look at this, 00:24:22.950 --> 00:24:24.200 it looks complicated at first. 00:24:24.200 --> 00:24:26.430 It's really not that complicated. 00:24:26.430 --> 00:24:28.880 So this can just ring open. 00:24:28.880 --> 00:24:31.700 And conceivably, it could ring open in either direction. 00:24:31.700 --> 00:24:34.070 It depends on the enzyme that's catalyzing it. 00:24:34.070 --> 00:24:36.980 But we always get N5 methyl tetrahydrofolate. 00:24:36.980 --> 00:24:38.990 That's what's used inside the cell. 00:24:38.990 --> 00:24:42.290 People don't find N10 methyl tetrahydrofolate, 00:24:42.290 --> 00:24:44.930 but chemically, that could happen. 00:24:44.930 --> 00:24:45.950 So what happens? 00:24:45.950 --> 00:24:47.570 This is now a new enzyme. 00:24:47.570 --> 00:24:50.880 And again, it's a dehydrogenase. 00:24:50.880 --> 00:24:55.580 So NADPH is going to NADP. 00:24:55.580 --> 00:24:57.920 So this is a new enzyme. 00:24:57.920 --> 00:24:59.990 I'm not going to write out the name. 00:24:59.990 --> 00:25:14.560 But this then reduces this to N5 methyl tetrahydrofolate. 00:25:14.560 --> 00:25:18.730 So what we've done then is, in the pathway 00:25:18.730 --> 00:25:21.670 I've drawn out here is, where do we get the one carbon from? 00:25:21.670 --> 00:25:25.300 Here, we got it from the formate. 00:25:25.300 --> 00:25:31.900 And we can change the oxidation states to get all three 00:25:31.900 --> 00:25:33.670 of these oxidation states, depending 00:25:33.670 --> 00:25:35.380 on what we need to do with it. 00:25:35.380 --> 00:25:38.950 You have to have the right enzymes and the right complexes 00:25:38.950 --> 00:25:41.680 to be able to make this all work. 00:25:41.680 --> 00:25:45.760 Now many of you might not recall this, but in the Benkovic paper 00:25:45.760 --> 00:25:49.900 you read for recitation last week, one of the controls 00:25:49.900 --> 00:25:52.580 with this tri-functional protein. 00:25:52.580 --> 00:25:56.980 And it does not exist in the purinosome. 00:25:56.980 --> 00:25:59.230 Benkovic's been interested never in these enzymes, 00:25:59.230 --> 00:26:02.200 and channeling of reactive intermediates in these systems. 00:26:02.200 --> 00:26:06.430 This does not exist in the purinosome. 00:26:06.430 --> 00:26:08.440 So then the question is how do you get back? 00:26:11.350 --> 00:26:15.310 And so there are three methylating agents 00:26:15.310 --> 00:26:17.140 inside the cell in a biology. 00:26:17.140 --> 00:26:19.990 Does anybody know what the other two are? 00:26:19.990 --> 00:26:22.600 So this is unusual, N5 methyl. 00:26:22.600 --> 00:26:25.140 So this is N5, this is again, N10. 00:26:25.140 --> 00:26:26.630 STUDENT: [INAUDIBLE]. 00:26:26.630 --> 00:26:28.570 JOANNE STUBBE: So S-adenosyl methionine 00:26:28.570 --> 00:26:29.930 is probably the most prevalent. 00:26:29.930 --> 00:26:31.580 What's another one? 00:26:31.580 --> 00:26:33.287 STUDENT: Methylcobalamin. 00:26:33.287 --> 00:26:34.120 JOANNE STUBBE: Yeah. 00:26:34.120 --> 00:26:35.290 So methylcobalamin. 00:26:35.290 --> 00:26:38.072 So S-adenosyl methionine 00:26:40.330 --> 00:26:45.410 is the universal methylating agent inside the cell. 00:26:45.410 --> 00:26:47.453 And then you also have-- 00:26:47.453 --> 00:26:49.120 I'm not going to draw the structure out. 00:26:49.120 --> 00:26:52.800 We're not going to talk about it, but methylcobalamin. 00:26:52.800 --> 00:26:56.620 And there's a single enzyme that uses all three 00:26:56.620 --> 00:26:58.270 of these methyl groups. 00:26:58.270 --> 00:27:00.188 And if I had another five lectures, 00:27:00.188 --> 00:27:01.480 I would talk about this enzyme. 00:27:01.480 --> 00:27:05.360 This was studied extensively by Rowena Matthews' lab, who 00:27:05.360 --> 00:27:07.152 was one of Cathy's mentors. 00:27:07.152 --> 00:27:09.610 And then Cathy was involved in getting the first structures 00:27:09.610 --> 00:27:11.412 many years ago with the little pieces. 00:27:11.412 --> 00:27:12.620 So it's one of these enzymes. 00:27:12.620 --> 00:27:13.720 It's huge. 00:27:13.720 --> 00:27:15.700 And it's got to juggle these three methyl 00:27:15.700 --> 00:27:16.930 groups to do the chemistry. 00:27:16.930 --> 00:27:19.300 It's really sort of fascinating. 00:27:19.300 --> 00:27:22.540 And so what it does is it takes homocysteine-- 00:27:28.280 --> 00:27:29.990 so this is homocysteine-- 00:27:29.990 --> 00:27:31.460 and converts it to methionine. 00:27:31.460 --> 00:27:32.600 I'm not going to draw the structure. 00:27:32.600 --> 00:27:33.530 So you methylate it. 00:27:33.530 --> 00:27:37.610 So you're going to methylate that cysteine. 00:27:37.610 --> 00:27:40.790 And then you're back to tetrahydrofolate. 00:27:40.790 --> 00:27:43.370 So there's another important reaction 00:27:43.370 --> 00:27:48.260 that I just want to put in here is that there's another way 00:27:48.260 --> 00:27:53.390 to go from tetrahydrofolate to this one, which is methylene 00:27:53.390 --> 00:27:55.490 tetrahydrofolate. 00:27:55.490 --> 00:27:58.880 And so in addition to being able to put on the one carbon 00:27:58.880 --> 00:28:02.540 with formate, does anybody have any idea what 00:28:02.540 --> 00:28:04.040 another major way-- it's probably 00:28:04.040 --> 00:28:06.710 the major way of doing one carbon 00:28:06.710 --> 00:28:11.060 transfers from metabolic labeling experiments? 00:28:11.060 --> 00:28:12.450 It comes from an amino acid. 00:28:12.450 --> 00:28:14.010 What amino acid could you use? 00:28:16.850 --> 00:28:21.500 So somehow we want to get from here to here. 00:28:21.500 --> 00:28:25.010 This is also a major target of therapeutics. 00:28:28.140 --> 00:28:29.610 Anybody got any ideas? 00:28:29.610 --> 00:28:34.642 We need to get one carbon out of an amino acid. 00:28:34.642 --> 00:28:35.350 What did you say? 00:28:35.350 --> 00:28:36.056 STUDENT: Thymine? 00:28:36.056 --> 00:28:37.000 JOANNE STUBBE: Thymine? 00:28:37.000 --> 00:28:38.050 That's not an amino acid. 00:28:38.050 --> 00:28:38.380 STUDENT: Thiamine. 00:28:38.380 --> 00:28:39.250 JOANNE STUBBE: Oh, thiamine. 00:28:39.250 --> 00:28:40.083 STUDENT: Methionine. 00:28:40.083 --> 00:28:42.350 JOANNE STUBBE: Oh, methionine. 00:28:42.350 --> 00:28:42.850 No. 00:28:42.850 --> 00:28:44.093 See, I guess I'm deaf. 00:28:44.093 --> 00:28:45.010 OK, I didn't hear you. 00:28:45.010 --> 00:28:48.010 No, that's, not it. 00:28:48.010 --> 00:28:52.515 So I'm not going to spend a lot of time, but serine-- 00:28:55.090 --> 00:28:59.890 so this is ours-- 00:28:59.890 --> 00:29:03.640 I'll draw this out, because I think this is really important. 00:29:03.640 --> 00:29:08.212 This can be converted into formaldehyde. 00:29:08.212 --> 00:29:09.670 Does anybody know what the cofactor 00:29:09.670 --> 00:29:10.837 would be that would do that? 00:29:14.930 --> 00:29:16.895 And then what you end up with is glycine. 00:29:20.170 --> 00:29:22.240 So this is the major way-- 00:29:22.240 --> 00:29:25.900 serine is a major one carbon donor. 00:29:25.900 --> 00:29:37.360 So seramine is going to generate the formaldehyde equivalent, 00:29:37.360 --> 00:29:38.940 which then can get picked up here 00:29:38.940 --> 00:29:43.740 and make methylene tetrahydrofolate. 00:29:43.740 --> 00:29:46.860 Anybody have any idea of how you would convert serine 00:29:46.860 --> 00:29:49.860 into glycine? 00:29:49.860 --> 00:29:53.190 You do learn about this cofactor. 00:29:53.190 --> 00:29:55.470 What is the cofactor that works on all amino acids, 00:29:55.470 --> 00:29:56.940 if you want to do something to it? 00:29:56.940 --> 00:29:58.740 There's only one. 00:29:58.740 --> 00:29:59.730 STUDENT: PLP. 00:29:59.730 --> 00:30:01.710 JOANNE STUBBE: PLP, yeah. 00:30:01.710 --> 00:30:03.300 So this isn't unusual-- 00:30:03.300 --> 00:30:05.880 PLP is sort of an amazing cofactor. 00:30:05.880 --> 00:30:09.330 It can do alpha decarboxylations, 00:30:09.330 --> 00:30:11.760 racemizations. 00:30:11.760 --> 00:30:14.760 It can do aldol reactions. 00:30:14.760 --> 00:30:16.850 And then it activates the beta positions 00:30:16.850 --> 00:30:18.990 so you can do beta eliminations replacements. 00:30:18.990 --> 00:30:21.750 It can do probably 10 or 15 different reactions. 00:30:21.750 --> 00:30:24.480 This one is unusual in that what you're doing 00:30:24.480 --> 00:30:28.320 is you're doing an aldol reaction. 00:30:28.320 --> 00:30:32.100 So you're cleaving that bond, and a reverse aldol reaction 00:30:32.100 --> 00:30:34.080 in this case. 00:30:34.080 --> 00:30:35.610 And then the other thing is if you 00:30:35.610 --> 00:30:38.190 want to link this into pyrimidines, 00:30:38.190 --> 00:30:39.660 you have dihydrofolate. 00:30:49.030 --> 00:30:51.880 So this is dihydrofolate. 00:30:51.880 --> 00:30:55.540 And that's a major player in pyrimidine metabolism 00:30:55.540 --> 00:30:57.010 to make thymidine. 00:30:57.010 --> 00:30:58.990 I'm not going to have time to talk about this. 00:30:58.990 --> 00:31:02.880 But folate is a central player in both purine and pyrimidine 00:31:02.880 --> 00:31:04.570 metabolism. 00:31:04.570 --> 00:31:07.813 And people have spent a lot of time thinking about it. 00:31:07.813 --> 00:31:09.730 And I think the chemistry of interconversions, 00:31:09.730 --> 00:31:12.910 once you sit and walk through this yourself, 00:31:12.910 --> 00:31:15.917 start over here and see if you can draw out the mechanisms. 00:31:15.917 --> 00:31:18.250 It's the same mechanisms we've seen over and over again, 00:31:18.250 --> 00:31:20.950 in addition to a carbonyl and loss of water. 00:31:26.560 --> 00:31:28.630 So that was the introductory part. 00:31:28.630 --> 00:31:30.395 And really what I want to do now is-- 00:31:30.395 --> 00:31:31.770 we can put that up here for those 00:31:31.770 --> 00:31:33.490 who still want to stare at it-- 00:31:33.490 --> 00:31:37.120 what I want to do now is talk about the pathway. 00:31:37.120 --> 00:31:40.270 And what I want to do is write out the pathway, 00:31:40.270 --> 00:31:42.160 and then use a PowerPoint to talk 00:31:42.160 --> 00:31:45.220 about a few features of the pathway 00:31:45.220 --> 00:31:47.740 that I think are the most interesting, 00:31:47.740 --> 00:31:52.040 and that you can make generalizations 00:31:52.040 --> 00:31:55.300 to other pathways, like, what is the role of glutamine? 00:31:55.300 --> 00:31:58.750 That's universally conserved. 00:31:58.750 --> 00:32:01.730 What is the role of ATP? 00:32:01.730 --> 00:32:04.630 And we're going to see the roles you 00:32:04.630 --> 00:32:06.700 see in the purine pathway are used 00:32:06.700 --> 00:32:08.320 in many metabolic pathways. 00:32:08.320 --> 00:32:12.280 So those are the ones I decided to focus on. 00:32:12.280 --> 00:32:15.880 So what I want to do is go step by step 00:32:15.880 --> 00:32:19.420 and just make a few comments. 00:32:19.420 --> 00:32:22.450 And then I'm going to use a PowerPoint over here so you can 00:32:22.450 --> 00:32:23.710 see what I have written down. 00:32:23.710 --> 00:32:26.530 I'm going to write down a few things. 00:32:26.530 --> 00:32:28.750 So that's the nomenclature. 00:32:28.750 --> 00:32:29.710 There's the pathway. 00:32:29.710 --> 00:32:31.270 We will start there. 00:32:31.270 --> 00:32:35.500 So I told you that the first step in this pathway 00:32:35.500 --> 00:32:39.610 is we start with phosphoribosyl pyrophosphate. 00:32:39.610 --> 00:32:42.100 That's central to a lot of things. 00:32:42.100 --> 00:32:44.110 It's chemically very unstable. 00:32:44.110 --> 00:32:44.860 It falls apart. 00:32:44.860 --> 00:32:46.900 It's hard to isolate. 00:32:46.900 --> 00:32:49.320 And the first step in this pathway-- 00:32:49.320 --> 00:32:52.630 we talked about this briefly in recitation-- 00:32:52.630 --> 00:32:57.130 is to make phosphoribosylamine. 00:32:57.130 --> 00:32:59.830 So the interesting thing about this pathway-- 00:32:59.830 --> 00:33:02.110 so is you start out-- 00:33:05.270 --> 00:33:09.880 and again, the nomenclature, I've written out. 00:33:09.880 --> 00:33:14.580 On the exam, you probably will have something about purines 00:33:14.580 --> 00:33:15.080 there. 00:33:15.080 --> 00:33:17.860 I will give you the pathway, and I will give you 00:33:17.860 --> 00:33:19.390 all the names and the enzymes. 00:33:19.390 --> 00:33:22.180 So you don't need to memorize that. 00:33:22.180 --> 00:33:25.030 I'm probably the only one that knows the names, 00:33:25.030 --> 00:33:26.500 because I've worked on it. 00:33:26.500 --> 00:33:27.400 Very confusing. 00:33:31.470 --> 00:33:35.220 So what's unique, again, and we've already mentioned this, 00:33:35.220 --> 00:33:39.812 is you start out with ribosyl phosphate. 00:33:39.812 --> 00:33:41.020 And what you're going to do-- 00:33:41.020 --> 00:33:43.470 and this is what we're going to walk through-- 00:33:43.470 --> 00:33:45.390 is that the first thing you do is 00:33:45.390 --> 00:33:49.530 you build up the imidazole moeity of your purine. 00:33:49.530 --> 00:33:53.520 So using sort of basic metabolites in ATP-- 00:33:53.520 --> 00:33:57.750 there are five steps out of the 10 that use ATP-- 00:33:57.750 --> 00:34:02.580 you make this amino imidazole ribonucelotide. 00:34:02.580 --> 00:34:05.940 And then what you do again, step by step, 00:34:05.940 --> 00:34:09.630 is convert this into the pyrimidine moiety. 00:34:09.630 --> 00:34:11.610 So you make your purine. 00:34:11.610 --> 00:34:14.460 So that's a step, one step at a time. 00:34:14.460 --> 00:34:20.219 And this was unraveled using metabolic labeling experiments. 00:34:20.219 --> 00:34:23.489 So the first enzyme I'll spend a little bit of time 00:34:23.489 --> 00:34:27.030 on, because I think it's a paradigm for many enzymes 00:34:27.030 --> 00:34:31.739 in metabolism in general, where do you get ammonia 00:34:31.739 --> 00:34:32.909 from most of the time? 00:34:32.909 --> 00:34:36.420 The major source of ammonia is glutamine. 00:34:36.420 --> 00:34:42.000 So that's something that you see in this pathway. 00:34:42.000 --> 00:34:48.530 So glutamine-- you all know glutamine has this part in this 00:34:48.530 --> 00:34:50.520 side chain-- 00:34:50.520 --> 00:34:53.190 is going to glutamate. 00:34:53.190 --> 00:34:56.639 And so you form glutamic acid. 00:34:56.639 --> 00:34:59.280 And the ammonia from the amid is going 00:34:59.280 --> 00:35:03.150 to interact with phosphoribosyl pyrophosphate, which 00:35:03.150 --> 00:35:11.010 is always bound to magnesium to form phosphoribosylamine. 00:35:11.010 --> 00:35:20.073 And so I'm now going to start being sloppier. 00:35:20.073 --> 00:35:21.490 Instead of writing phosphate here, 00:35:21.490 --> 00:35:23.782 I'm going to have a phosphorus with a circle around it. 00:35:23.782 --> 00:35:26.340 That means we always have the five prime phosphate. 00:35:26.340 --> 00:35:29.700 And furthermore, what I'm going to do 00:35:29.700 --> 00:35:32.220 is replace all of this with an R group, 00:35:32.220 --> 00:35:36.180 ribosylphosphate is present at every single step 00:35:36.180 --> 00:35:37.410 in the pathway. 00:35:37.410 --> 00:35:39.870 And in fact, one of the reasons I thought this pathway was 00:35:39.870 --> 00:35:42.690 interesting, every enzyme in the pathway 00:35:42.690 --> 00:35:45.323 has to have a binding site for ribosylphosphate. 00:35:45.323 --> 00:35:46.740 Well, have any of you ever thought 00:35:46.740 --> 00:35:51.340 about how metabolic pathways evolved? 00:35:51.340 --> 00:35:52.430 Where does it come from? 00:35:52.430 --> 00:35:54.610 You have these really complicated pathways. 00:35:54.610 --> 00:35:55.820 Where do you start? 00:35:55.820 --> 00:35:57.070 How do you think about that? 00:35:57.070 --> 00:35:59.830 Well, this might be a fantastic place to look at that. 00:35:59.830 --> 00:36:00.490 Why? 00:36:00.490 --> 00:36:03.610 Because you might have a ribosyl binding site for everything. 00:36:03.610 --> 00:36:05.020 So maybe it starts with something 00:36:05.020 --> 00:36:07.030 that binds ribosylphosphate. 00:36:07.030 --> 00:36:08.945 Anyhow, this is an unusual pathway 00:36:08.945 --> 00:36:10.070 in that you have something. 00:36:10.070 --> 00:36:13.482 You have a really good handle on to hang on to. 00:36:13.482 --> 00:36:15.190 And as we already talked about-- so this, 00:36:15.190 --> 00:36:17.410 we're going to call R-- 00:36:17.410 --> 00:36:19.188 what's unusual, there are a couple things 00:36:19.188 --> 00:36:20.230 I want to say about this. 00:36:20.230 --> 00:36:24.250 But we already talked about this a little in terms of channeling 00:36:24.250 --> 00:36:26.200 and this question of why you would ever want 00:36:26.200 --> 00:36:29.680 to have clustering enzymes. 00:36:29.680 --> 00:36:33.370 And that's because the half-life of this 00:36:33.370 --> 00:36:37.320 is about 10 seconds at 37 degrees. 00:36:37.320 --> 00:36:39.943 So it took a lot of effort to see this thing. 00:36:39.943 --> 00:36:41.860 I mean, you couldn't see it by normal methods. 00:36:41.860 --> 00:36:45.190 People inferred its presence because Buchanan actually 00:36:45.190 --> 00:36:48.730 was able to see the next intermediate in the pathway 00:36:48.730 --> 00:36:51.100 and inferred the existence of this. 00:36:51.100 --> 00:36:53.260 And many of these intermediates in the pathway, 00:36:53.260 --> 00:36:58.060 which is why Benkovic focused on this, are chemically unstable. 00:36:58.060 --> 00:36:59.727 Let's see if I have one of these. 00:36:59.727 --> 00:37:01.060 I don't have it in this pathway. 00:37:01.060 --> 00:37:02.410 Anyhow, I'll show you another one, which 00:37:02.410 --> 00:37:05.170 has a half-life of five seconds or something like that, that 00:37:05.170 --> 00:37:07.010 took forever for people to identify it, 00:37:07.010 --> 00:37:09.010 because when you try to work it up as a chemist, 00:37:09.010 --> 00:37:10.720 it falls apart. 00:37:10.720 --> 00:37:13.510 And I would say this is something any of you 00:37:13.510 --> 00:37:17.840 get into metabolomics, people are looking for metabolites 00:37:17.840 --> 00:37:18.710 now. 00:37:18.710 --> 00:37:20.530 There's one metabolite that people 00:37:20.530 --> 00:37:22.330 have found quite frequently, and it 00:37:22.330 --> 00:37:26.290 seems to be involved in regulation of glycolysis. 00:37:26.290 --> 00:37:29.390 It's this one. 00:37:29.390 --> 00:37:30.650 See, where am I? 00:37:33.390 --> 00:37:37.980 Aminoimidazole ribo-- this one. 00:37:37.980 --> 00:37:40.260 And that's because it's stable. 00:37:40.260 --> 00:37:42.550 And the lot of the other ones are not very stable. 00:37:42.550 --> 00:37:45.060 So I wouldn't be surprised if you ended up 00:37:45.060 --> 00:37:46.800 finding a lot more metabolites that 00:37:46.800 --> 00:37:50.430 are playing a central role in regulating enzymes 00:37:50.430 --> 00:37:52.920 in primary metabolism, because where 00:37:52.920 --> 00:37:54.660 does the serine come from? 00:37:54.660 --> 00:37:56.160 Does anybody know where the serine 00:37:56.160 --> 00:38:01.440 comes from that plays a key role in making this folate analog? 00:38:01.440 --> 00:38:02.790 Anybody have any idea? 00:38:06.980 --> 00:38:11.170 So serine is three phosphoglyceric acid 00:38:11.170 --> 00:38:12.740 in the glycolysis pathway. 00:38:12.740 --> 00:38:14.360 It's actually very straightforward 00:38:14.360 --> 00:38:17.180 to write a mechanism of how you get there. 00:38:17.180 --> 00:38:22.130 Intimately links the glycolysis pathway to purine metabolism. 00:38:22.130 --> 00:38:25.310 And we'll also see here of course, this is folate, 00:38:25.310 --> 00:38:26.690 but we also need glycine. 00:38:26.690 --> 00:38:30.242 That's the next step in small molecule in this pathway. 00:38:30.242 --> 00:38:30.950 It needs glycine. 00:38:30.950 --> 00:38:32.290 So everything is integrated. 00:38:32.290 --> 00:38:34.490 Once you see-- you sort of see the big picture 00:38:34.490 --> 00:38:37.280 and have central pictures of primary metabolism, 00:38:37.280 --> 00:38:41.420 everything becomes much more integrated. 00:38:41.420 --> 00:38:43.520 So how does this happen? 00:38:43.520 --> 00:38:46.130 So what I want to do is I want to talk 00:38:46.130 --> 00:38:47.510 a little bit about this enzyme. 00:38:50.890 --> 00:38:53.810 So here, let me just talk about this this. 00:38:53.810 --> 00:38:59.030 So if we call this Pur F, just so we have a name, 00:38:59.030 --> 00:39:03.875 Pur F is called an amidotransferase. 00:39:09.550 --> 00:39:15.535 And what it's going to do is it's going to take glutamine-- 00:39:24.420 --> 00:39:29.100 and it turns out these enzymes have a domain. 00:39:31.650 --> 00:39:33.960 They always have multiple domains. 00:39:33.960 --> 00:39:37.830 And the domain that uses the glutamine can be the same. 00:39:37.830 --> 00:39:40.860 There are actually two different convergent evolutions 00:39:40.860 --> 00:39:45.480 of glutamine binding domains that do the same chemistry. 00:39:45.480 --> 00:39:49.905 So what you do is-- we've seen this again many, many times-- 00:39:53.490 --> 00:40:00.570 so you form a covalent intermediate, 00:40:00.570 --> 00:40:04.370 which then hydrolyzes to glutamate regenerating ESH. 00:40:07.620 --> 00:40:14.560 And what happened during this reaction, you generate ammonia. 00:40:14.560 --> 00:40:18.130 So the goal of these amidotransferases in general, 00:40:18.130 --> 00:40:21.910 in many, many metabolic pathways, 00:40:21.910 --> 00:40:23.030 is to generate ammonia. 00:40:25.990 --> 00:40:27.970 And so to me, what's striking about 00:40:27.970 --> 00:40:33.310 this is the way nature evolved these metabolic enzymes 00:40:33.310 --> 00:40:34.780 that generate ammonia. 00:40:34.780 --> 00:40:36.970 And so what you see in a cartoon view-- 00:40:36.970 --> 00:40:41.950 so we are always going to have all of these enzymes. 00:40:41.950 --> 00:40:43.720 They may be a single polypeptide. 00:40:43.720 --> 00:40:46.780 They may be two polypeptides, but they all 00:40:46.780 --> 00:40:49.465 have a glutaminase domain. 00:40:55.470 --> 00:40:59.730 So the glutaminase is just generating the ammonia. 00:40:59.730 --> 00:41:01.360 But what do we have? 00:41:01.360 --> 00:41:03.600 We start out with phosphoribosyl pyrophosphate. 00:41:03.600 --> 00:41:06.450 So once we generate the ammonia, what can happen? 00:41:06.450 --> 00:41:10.990 You can now by-- it turns out by dissociated mechanism-- 00:41:10.990 --> 00:41:14.790 displace a pyrophosphate to form phosphoribosylamine. 00:41:14.790 --> 00:41:17.910 So all of these kinds of reactions 00:41:17.910 --> 00:41:21.850 involve dissociative rather than associative transition states. 00:41:21.850 --> 00:41:24.110 That's not important. 00:41:24.110 --> 00:41:34.545 But what it what's amazing about this is that PRPP, 00:41:34.545 --> 00:41:37.950 in this case, binds to one domain, 00:41:37.950 --> 00:41:42.360 and the glutamine binds to this second domain. 00:41:42.360 --> 00:41:44.400 So ammonia, what would happen to ammonia 00:41:44.400 --> 00:41:46.698 if it went out into solution? 00:41:46.698 --> 00:41:47.607 STUDENT: Protonated. 00:41:47.607 --> 00:41:48.440 JOANNE STUBBE: Yeah. 00:41:48.440 --> 00:41:51.240 Gets protonated really rapidly, becomes unreactive. 00:41:51.240 --> 00:41:53.130 I don't know why nature designed this. 00:41:53.130 --> 00:41:57.540 But what you see with all these enzymes is she 00:41:57.540 --> 00:42:01.860 makes a tunnel across the domain interface that's 00:42:01.860 --> 00:42:04.830 about 25 to 40 angstroms long. 00:42:04.830 --> 00:42:07.790 So the ammonia this released never gets out into solutions. 00:42:07.790 --> 00:42:09.540 This is another example of channeling 00:42:09.540 --> 00:42:13.500 a reactive intermediate, which we talked about as potentially 00:42:13.500 --> 00:42:16.200 a reason for channeling in the purine pathway. 00:42:16.200 --> 00:42:20.250 So there's a tunnel. 00:42:20.250 --> 00:42:23.790 And the tunnel can be 25-- 00:42:23.790 --> 00:42:31.125 we have a number of structures in the ammonia channels. 00:42:34.770 --> 00:42:36.280 And I have no idea-- 00:42:36.280 --> 00:42:38.620 I mean, this surprised the heck out of me. 00:42:38.620 --> 00:42:40.750 I thought the way nature would hold 00:42:40.750 --> 00:42:45.380 on to this is by hanging on to not the covalent intermediate, 00:42:45.380 --> 00:42:47.500 but the preceding tetrahedral intermediate. 00:42:47.500 --> 00:42:49.840 And then when the white substrate was there, 00:42:49.840 --> 00:42:53.320 release it and then bind it, sitting right next to it. 00:42:53.320 --> 00:42:56.620 But nature, in all designs, has done this thing 00:42:56.620 --> 00:42:58.390 where you have this channel. 00:42:58.390 --> 00:43:03.010 And here is an example of Pur F. 00:43:03.010 --> 00:43:05.630 This is the glutaminase domain up here. 00:43:05.630 --> 00:43:08.680 And here is where the phosphoribosyl pyrophosphate 00:43:08.680 --> 00:43:09.880 binds down here. 00:43:09.880 --> 00:43:12.100 You can't see the channel, but this 00:43:12.100 --> 00:43:14.170 is work of Jan Smith a number of years ago, 00:43:14.170 --> 00:43:18.850 was the first one that showed the channel in this pathway. 00:43:18.850 --> 00:43:20.350 So that's common. 00:43:20.350 --> 00:43:23.200 And we're going to look at another glutamine requiring 00:43:23.200 --> 00:43:24.370 enzyme in this pathway. 00:43:24.370 --> 00:43:26.200 It's the fourth enzyme in the pathway. 00:43:26.200 --> 00:43:27.430 Also is a channel. 00:43:27.430 --> 00:43:29.800 Again, it's distinct. 00:43:29.800 --> 00:43:34.000 It all does this glutaminase covalent intermediate, 00:43:34.000 --> 00:43:39.520 but the structure of the glutaminase domain is distinct. 00:43:39.520 --> 00:43:41.360 So what's the next enzyme in the pathway? 00:43:41.360 --> 00:43:43.900 So the next enzyme in the pathway again, 00:43:43.900 --> 00:43:47.050 is a paradigm for many, many unsungs 00:43:47.050 --> 00:43:49.630 and primary metabolic pathways. 00:43:49.630 --> 00:43:51.040 And if you look at the structure, 00:43:51.040 --> 00:43:57.370 let's just go back to the pathway. 00:43:57.370 --> 00:44:01.240 If you look at this pathway, what you now want to do-- 00:44:01.240 --> 00:44:03.940 so we keep the ribose 5-phosphate all the way 00:44:03.940 --> 00:44:05.920 through the whole thing. 00:44:05.920 --> 00:44:07.540 That's the scaffold. 00:44:07.540 --> 00:44:08.830 Now what are you going to add? 00:44:08.830 --> 00:44:11.230 You're going to add glycine. 00:44:11.230 --> 00:44:13.140 So here is your phosphoribosylamine. 00:44:13.140 --> 00:44:15.340 And you're going to add glycine. 00:44:15.340 --> 00:44:19.060 How do you inactivate an amino acid? 00:44:19.060 --> 00:44:21.790 You've seen activation of amino acids now many times. 00:44:21.790 --> 00:44:24.858 What are the two ways you can activate amino acids? 00:44:24.858 --> 00:44:26.142 STUDENT: [INAUDIBLE]. 00:44:26.142 --> 00:44:29.100 JOANNE STUBBE: So either adenylate or phosphorylate. 00:44:29.100 --> 00:44:31.750 So that's a paradigm that you see over and over again 00:44:31.750 --> 00:44:33.910 in nature. 00:44:33.910 --> 00:44:37.290 This enzyme uses ATP. 00:44:37.290 --> 00:44:40.300 This is one of the five enzymes. 00:44:40.300 --> 00:44:42.670 And it forms inorganic phosphate. 00:44:42.670 --> 00:44:46.510 So you're phosphorylating, not adenylating. 00:44:46.510 --> 00:44:51.202 And so I'll show you what the mechanism is up there. 00:44:51.202 --> 00:44:52.660 You've already seen this mechanism, 00:44:52.660 --> 00:44:54.490 but the idea is you phosphorylate this. 00:44:54.490 --> 00:44:57.400 You're going to form the phosphoanhydride. 00:44:57.400 --> 00:45:03.340 And then the phosphoanhydride can react with the amino group. 00:45:03.340 --> 00:45:06.198 And kinetically-- this is something that's one 00:45:06.198 --> 00:45:07.990 of my students working on a long time ago-- 00:45:07.990 --> 00:45:10.150 there was evidence that this intermediate, which 00:45:10.150 --> 00:45:12.910 is chemically unstable, could channel between the two 00:45:12.910 --> 00:45:13.430 proteins. 00:45:13.430 --> 00:45:15.100 So you don't generate this own solution 00:45:15.100 --> 00:45:17.230 where it can fall apart and it can anomerize. 00:45:17.230 --> 00:45:18.580 It gets transferred directly. 00:45:18.580 --> 00:45:22.310 In fact, in the early days when we 00:45:22.310 --> 00:45:25.290 invented the first biochemistry labs at MIT, 00:45:25.290 --> 00:45:27.363 they used this system. 00:45:27.363 --> 00:45:28.780 I really pushed them to the limit, 00:45:28.780 --> 00:45:30.760 because they were dealing with the substrate. 00:45:30.760 --> 00:45:32.770 They had a very short half-life Anyhow, 00:45:32.770 --> 00:45:35.440 they learned a lot from the exercise. 00:45:35.440 --> 00:45:38.500 So what you're going to have then is ribos-- 00:45:38.500 --> 00:45:44.910 I'm just going to call it R. And so here's our glycine. 00:45:44.910 --> 00:45:45.856 Whoops. 00:45:45.856 --> 00:45:47.880 Guess I'd better get the structure right. 00:45:52.820 --> 00:45:56.970 So this is from lysine. 00:45:56.970 --> 00:46:03.008 So what we will see is that this is another-- 00:46:03.008 --> 00:46:04.550 we're not getting very far-- but this 00:46:04.550 --> 00:46:14.540 is a member of the ATP grasp superfamily of enzymes. 00:46:14.540 --> 00:46:15.920 They all do the same chemistry. 00:46:15.920 --> 00:46:19.400 So let me just move forward a little bit. 00:46:19.400 --> 00:46:20.930 I'm not going to draw this out. 00:46:20.930 --> 00:46:24.710 You guys have seen this chemistry many times. 00:46:24.710 --> 00:46:27.590 So what's happening in this chemistry 00:46:27.590 --> 00:46:30.140 is you have a carboxylate. 00:46:30.140 --> 00:46:36.360 ATP phosphorylates it, and then you attack by a nucleophile, 00:46:36.360 --> 00:46:40.730 in this case, the nucleophile is the amino group 00:46:40.730 --> 00:46:43.340 of phosphoribosylamine. 00:46:43.340 --> 00:46:46.400 So what I just want you to see here if you look at this, 00:46:46.400 --> 00:46:49.790 there are four enzymes that are involved in purine metabolism 00:46:49.790 --> 00:46:51.860 that all have the same structure. 00:46:51.860 --> 00:46:56.540 They all have ATP grasp structures. 00:46:56.540 --> 00:46:59.480 They all go through phosoanhydride intermediates. 00:46:59.480 --> 00:47:01.820 And you can, from bioinformatics, 00:47:01.820 --> 00:47:04.160 pick these structures out. 00:47:04.160 --> 00:47:05.720 So this is again, an example. 00:47:05.720 --> 00:47:09.560 Once people defined-- there's almost a no sequence homology 00:47:09.560 --> 00:47:11.060 between these proteins-- 00:47:11.060 --> 00:47:14.540 but by knowing this chemistry, you 00:47:14.540 --> 00:47:17.400 can actually pick out that these are going to be family members. 00:47:17.400 --> 00:47:19.010 And if you know if they are organized 00:47:19.010 --> 00:47:21.830 in bacteria and operons, you can even guess at the substrate. 00:47:21.830 --> 00:47:24.590 And then you can test this model that they 00:47:24.590 --> 00:47:28.520 go through phosoanhydride intermediates. 00:47:28.520 --> 00:47:31.025 And I'm over, but the next step in this pathway-- 00:47:36.570 --> 00:47:38.300 the next step in this pathway-- 00:47:38.300 --> 00:47:40.490 we're going to come back. 00:47:40.490 --> 00:47:42.300 And what are we going to use? 00:47:42.300 --> 00:47:44.660 We're going to use N10 formal tetrahydrofolate. 00:47:44.660 --> 00:47:46.280 That's why I went through this. 00:47:46.280 --> 00:47:49.430 We're going to put a formal group here. 00:47:49.430 --> 00:47:51.290 And again, the chemistry is just the same. 00:47:51.290 --> 00:47:54.020 Go home and think about the chemistry 00:47:54.020 --> 00:47:56.120 of how you generate all the different oxidation 00:47:56.120 --> 00:47:57.453 states of the carbon. 00:47:57.453 --> 00:47:59.870 And then I think you can see the chemistry in this pathway 00:47:59.870 --> 00:48:03.890 actually is pretty simple, once a few basic reactions. 00:48:03.890 --> 00:48:07.370 So the ATP grasp family is interesting. 00:48:07.370 --> 00:48:10.160 The amidotransferase and the channel 00:48:10.160 --> 00:48:14.320 is interesting as being general in metabolism.