WEBVTT 00:00:00.000 --> 00:00:00.500 align:middle line:90% 00:00:00.500 --> 00:00:02.830 align:middle line:84% The following content is provided under a Creative 00:00:02.830 --> 00:00:04.370 align:middle line:90% Commons license. 00:00:04.370 --> 00:00:06.670 align:middle line:84% Your support will help MIT OpenCourseWare 00:00:06.670 --> 00:00:11.030 align:middle line:84% continue to offer high quality educational resources for free. 00:00:11.030 --> 00:00:13.660 align:middle line:84% To make a donation or view additional materials 00:00:13.660 --> 00:00:17.610 align:middle line:84% from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.610 --> 00:00:18.520 align:middle line:90% at ocw.mit.edu. 00:00:18.520 --> 00:00:25.710 align:middle line:90% 00:00:25.710 --> 00:00:28.740 align:middle line:84% JOANNE STUBBE: What I want to do is sort of introduce you 00:00:28.740 --> 00:00:31.890 align:middle line:84% to the second half of the course, where we're going, 00:00:31.890 --> 00:00:33.850 align:middle line:84% and what topics we're going to be covering. 00:00:33.850 --> 00:00:36.600 align:middle line:90% And then I'll start in module 5. 00:00:36.600 --> 00:00:42.180 align:middle line:84% So as with the first half of the course, we have four modules. 00:00:42.180 --> 00:00:45.790 align:middle line:84% The first half the courses was pretty well organized-- 00:00:45.790 --> 00:00:47.910 align:middle line:84% that is, you went from here to here to here. 00:00:47.910 --> 00:00:49.140 align:middle line:90% It all sort of made sense. 00:00:49.140 --> 00:00:51.750 align:middle line:84% This half of the course doesn't do that. 00:00:51.750 --> 00:00:53.520 align:middle line:84% We're talking about-- there are hundreds 00:00:53.520 --> 00:00:57.660 align:middle line:84% of topics in biochemistry, and any one of them 00:00:57.660 --> 00:00:59.880 align:middle line:90% is exciting and important. 00:00:59.880 --> 00:01:03.300 align:middle line:84% And these are the ones we're talking about this semester. 00:01:03.300 --> 00:01:04.890 align:middle line:84% And again, the focus will be sort 00:01:04.890 --> 00:01:07.380 align:middle line:84% of trying to get you to think about, 00:01:07.380 --> 00:01:10.180 align:middle line:84% what is the evidence that supports 00:01:10.180 --> 00:01:12.310 align:middle line:90% the model I'm going to present. 00:01:12.310 --> 00:01:18.120 align:middle line:84% So in module 5, we're going to be talking about the terpenome. 00:01:18.120 --> 00:01:20.460 align:middle line:84% And I'm going to be talking about, most of you 00:01:20.460 --> 00:01:25.320 align:middle line:84% have seen in the last module, with polyketide synthases, 00:01:25.320 --> 00:01:27.840 align:middle line:84% you have aldol reactions and claison reactions 00:01:27.840 --> 00:01:29.940 align:middle line:90% to form carbon carbon bonds. 00:01:29.940 --> 00:01:33.510 align:middle line:84% In this module, you're going to be introduced to another way 00:01:33.510 --> 00:01:35.410 align:middle line:90% to form carbon carbon bonds. 00:01:35.410 --> 00:01:38.610 align:middle line:90% And that's through C5 units. 00:01:38.610 --> 00:01:43.110 align:middle line:84% And C5 units are the basis for forming cholesterol, which is 00:01:43.110 --> 00:01:46.520 align:middle line:90% really the focus of module 5. 00:01:46.520 --> 00:01:48.540 align:middle line:84% And what we're going to be talking about 00:01:48.540 --> 00:01:51.300 align:middle line:84% is initially cholesterol biosynthesis, 00:01:51.300 --> 00:01:53.910 align:middle line:84% that will be this lecture and probably 00:01:53.910 --> 00:01:56.190 align:middle line:90% most of the next lecture. 00:01:56.190 --> 00:01:59.370 align:middle line:84% And then you all know from eating McDonald's hamburgers, 00:01:59.370 --> 00:02:01.830 align:middle line:84% you get a lot of cholesterol in your diet. 00:02:01.830 --> 00:02:05.760 align:middle line:84% And the question is, how do you control cholesterol levels? 00:02:05.760 --> 00:02:08.550 align:middle line:84% And so this semester, the second part of the semester 00:02:08.550 --> 00:02:11.370 align:middle line:84% is really focus on the question of homeostasis. 00:02:11.370 --> 00:02:12.660 align:middle line:90% Cholesterol is essential. 00:02:12.660 --> 00:02:14.460 align:middle line:84% If you have too much you've got problems. 00:02:14.460 --> 00:02:17.130 align:middle line:84% Doesn't matter what pathway you're talking about, 00:02:17.130 --> 00:02:20.430 align:middle line:84% if you have too much or you have too little, you have problems. 00:02:20.430 --> 00:02:22.668 align:middle line:84% So how do you control everything? 00:02:22.668 --> 00:02:24.960 align:middle line:84% And we're going to be talking about cholesterol sensing 00:02:24.960 --> 00:02:26.760 align:middle line:90% and regulation. 00:02:26.760 --> 00:02:29.700 align:middle line:84% And we're going to come back to a topic you covered 00:02:29.700 --> 00:02:34.920 align:middle line:84% in the first half of the course with ClpX and ClpP protein 00:02:34.920 --> 00:02:36.660 align:middle line:84% mediated degradation, because it plays 00:02:36.660 --> 00:02:41.460 align:middle line:84% a central role in controlling cholesterol homeostasis. 00:02:41.460 --> 00:02:46.080 align:middle line:84% So that will be the first module, module 5. 00:02:46.080 --> 00:02:51.180 align:middle line:84% Module 6 is also going to be a module on homeostasis. 00:02:51.180 --> 00:02:53.580 align:middle line:84% It's my feeling that in introductory courses, 00:02:53.580 --> 00:02:57.300 align:middle line:84% people don't get introduced enough to metals. 00:02:57.300 --> 00:03:01.410 align:middle line:84% And 50% of all the proteins inside of us 00:03:01.410 --> 00:03:04.090 align:middle line:84% bind metals and do something with them. 00:03:04.090 --> 00:03:05.790 align:middle line:84% And so in this module, I'm going to talk 00:03:05.790 --> 00:03:07.680 align:middle line:90% about metal homeostasis. 00:03:07.680 --> 00:03:10.950 align:middle line:84% And I'm really going to focus on iron. 00:03:10.950 --> 00:03:12.750 align:middle line:84% And you'll see why I'm going to focus 00:03:12.750 --> 00:03:14.110 align:middle line:90% on iron when we get there. 00:03:14.110 --> 00:03:17.880 align:middle line:84% But we're going to talk about iron sensing and regulation, 00:03:17.880 --> 00:03:20.730 align:middle line:84% initially in humans, and then we're 00:03:20.730 --> 00:03:24.435 align:middle line:84% going to focus on the war between pathogenic organisms 00:03:24.435 --> 00:03:27.570 align:middle line:84% for iron and humans for iron, since iron 00:03:27.570 --> 00:03:33.000 align:middle line:84% is essential for almost all organisms to survive. 00:03:33.000 --> 00:03:35.850 align:middle line:90% The third module is sort of-- 00:03:35.850 --> 00:03:40.320 align:middle line:84% module 7-- sort of follows from module 6. 00:03:40.320 --> 00:03:45.780 align:middle line:84% And it's a topic that I've been following for 30 years, 00:03:45.780 --> 00:03:48.750 align:middle line:84% and it irritates the hell out of me. 00:03:48.750 --> 00:03:52.650 align:middle line:84% So everybody talks about reactive oxygen species, 00:03:52.650 --> 00:03:54.150 align:middle line:90% and how bad they are. 00:03:54.150 --> 00:03:56.020 align:middle line:84% You can't-- you can listen to NPR, 00:03:56.020 --> 00:03:58.320 align:middle line:84% you can read it in The New York Times. 00:03:58.320 --> 00:04:00.600 align:middle line:84% What are reactive oxygen species? 00:04:00.600 --> 00:04:02.700 align:middle line:90% As a chemist, what are they? 00:04:02.700 --> 00:04:06.930 align:middle line:84% So we'll define what reactive oxygen species are. 00:04:06.930 --> 00:04:09.010 align:middle line:84% Many of you know that they're bad. 00:04:09.010 --> 00:04:13.200 align:middle line:84% That's why you eat vitamin C and vitamin E. 00:04:13.200 --> 00:04:17.190 align:middle line:84% And they're involved, in fact, in defense 00:04:17.190 --> 00:04:19.410 align:middle line:90% against some microorganisms. 00:04:19.410 --> 00:04:21.279 align:middle line:90% But also they're good. 00:04:21.279 --> 00:04:23.850 align:middle line:84% They're now known to be involved in signaling. 00:04:23.850 --> 00:04:26.820 align:middle line:84% So again, it's a question of homeostasis. 00:04:26.820 --> 00:04:29.130 align:middle line:84% And so you need to understand the chemistry 00:04:29.130 --> 00:04:33.480 align:middle line:84% of what these species do, and where they can go astray, 00:04:33.480 --> 00:04:35.430 align:middle line:84% or where you can harness the reactivity 00:04:35.430 --> 00:04:37.690 align:middle line:90% to do something important. 00:04:37.690 --> 00:04:40.380 align:middle line:84% And the last section-- hopefully we'll get there this year, 00:04:40.380 --> 00:04:41.520 align:middle line:90% I'm trying my best-- 00:04:41.520 --> 00:04:44.080 align:middle line:84% this is the area I know most about, 00:04:44.080 --> 00:04:47.100 align:middle line:84% we're going to talk about nucleotide metabolism. 00:04:47.100 --> 00:04:50.640 align:middle line:84% In 5.07, they don't talk about nucleotide metabolism at all. 00:04:50.640 --> 00:04:52.710 align:middle line:84% And I would say in the next decade, 00:04:52.710 --> 00:04:56.220 align:middle line:84% you're going to see a lot about nucleotide metabolism. 00:04:56.220 --> 00:04:59.670 align:middle line:84% Where have you seen ATP and GTP in the first part 00:04:59.670 --> 00:05:00.560 align:middle line:90% of this course? 00:05:00.560 --> 00:05:01.990 align:middle line:90% Everywhere. 00:05:01.990 --> 00:05:03.720 align:middle line:90% How do we control all of that? 00:05:03.720 --> 00:05:05.070 align:middle line:90% Pretty important. 00:05:05.070 --> 00:05:07.170 align:middle line:84% What are the questions we're going to be asking? 00:05:07.170 --> 00:05:08.190 align:middle line:90% Where does it come from? 00:05:08.190 --> 00:05:10.080 align:middle line:84% And how do we control the levels, 00:05:10.080 --> 00:05:13.050 align:middle line:84% which is central to all of metabolism? 00:05:13.050 --> 00:05:15.570 align:middle line:84% Hopefully we'll get there and discuss that. 00:05:15.570 --> 00:05:18.170 align:middle line:90% 00:05:18.170 --> 00:05:24.110 align:middle line:84% So the required reading has now been posted on Stellar. 00:05:24.110 --> 00:05:28.330 align:middle line:84% And there are-- there is really sort of three 00:05:28.330 --> 00:05:29.330 align:middle line:90% things you need to read. 00:05:29.330 --> 00:05:32.872 align:middle line:84% One is about a short review on the terpenome, which 00:05:32.872 --> 00:05:34.580 align:middle line:84% is what I'm going to start talking about. 00:05:34.580 --> 00:05:38.270 align:middle line:84% The second is lipid metabolism that's 00:05:38.270 --> 00:05:40.400 align:middle line:84% been taken from Voet & Voet, you can 00:05:40.400 --> 00:05:44.795 align:middle line:84% go back in any basic textbook and read the section, 00:05:44.795 --> 00:05:46.670 align:middle line:84% because it's central to thinking about what's 00:05:46.670 --> 00:05:49.730 align:middle line:90% going on with cholesterol. 00:05:49.730 --> 00:05:52.520 align:middle line:84% And then you'll see that these are 00:05:52.520 --> 00:05:55.190 align:middle line:84% two of my favorite guys, Brown and Goldstein. 00:05:55.190 --> 00:05:57.050 align:middle line:84% They won the Nobel Prize for their work, 00:05:57.050 --> 00:06:01.310 align:middle line:84% but in reality they should have won at least two Nobel prizes 00:06:01.310 --> 00:06:02.120 align:middle line:90% for their work. 00:06:02.120 --> 00:06:05.210 align:middle line:84% I mean, you can never not listen to them talk 00:06:05.210 --> 00:06:06.650 align:middle line:90% and not get excited. 00:06:06.650 --> 00:06:08.540 align:middle line:84% I mean, they always have something 00:06:08.540 --> 00:06:11.330 align:middle line:90% important and exciting to say. 00:06:11.330 --> 00:06:13.890 align:middle line:84% So they-- last year we used this review. 00:06:13.890 --> 00:06:15.440 align:middle line:90% There's a new review. 00:06:15.440 --> 00:06:18.080 align:middle line:84% They're different, they're both pretty short. 00:06:18.080 --> 00:06:21.530 align:middle line:84% Pick which one you want, they cover the material. 00:06:21.530 --> 00:06:25.610 align:middle line:84% And then this one doesn't cover the most recent material 00:06:25.610 --> 00:06:26.200 align:middle line:90% as well. 00:06:26.200 --> 00:06:30.170 align:middle line:84% So here's another short review that covers that material. 00:06:30.170 --> 00:06:33.710 align:middle line:84% So these guys here will give you an overview of what 00:06:33.710 --> 00:06:35.932 align:middle line:90% I'm going to be talking about. 00:06:35.932 --> 00:06:40.310 align:middle line:84% And they've all been posted on the website already. 00:06:40.310 --> 00:06:43.730 align:middle line:90% So cholesterol homeostasis-- 00:06:43.730 --> 00:06:46.070 align:middle line:90% I never end a lecture on time. 00:06:46.070 --> 00:06:48.920 align:middle line:84% You'll find out that I'm trying my best, anyhow. 00:06:48.920 --> 00:06:52.050 align:middle line:84% We have about five lectures we're going to be covering. 00:06:52.050 --> 00:06:55.040 align:middle line:90% And this is where we're going. 00:06:55.040 --> 00:06:56.900 align:middle line:84% And the first one, today's lecture, we're 00:06:56.900 --> 00:06:59.660 align:middle line:84% going to be talking about a new way 00:06:59.660 --> 00:07:04.070 align:middle line:84% to form carbon carbon bonds through C5 units. 00:07:04.070 --> 00:07:09.920 align:middle line:84% And we'll be talking about the terpenome, where 00:07:09.920 --> 00:07:13.400 align:middle line:84% there are a huge number of natural products, 00:07:13.400 --> 00:07:15.470 align:middle line:84% distinct from polyketide synthases 00:07:15.470 --> 00:07:19.100 align:middle line:84% and non-ribosomal polypeptide synthases 00:07:19.100 --> 00:07:21.770 align:middle line:84% that you just finished talking about. 00:07:21.770 --> 00:07:26.570 align:middle line:84% We want to get to-- we'll see in the biosynthetic pathway 00:07:26.570 --> 00:07:30.590 align:middle line:84% to get to these C5 building blocks, 00:07:30.590 --> 00:07:34.880 align:middle line:84% we need to get to a metabolite called mevalonic acid, that's 00:07:34.880 --> 00:07:40.040 align:middle line:84% front and central in cholesterol biosynthesis. 00:07:40.040 --> 00:07:41.690 align:middle line:90% So we need to get that far. 00:07:41.690 --> 00:07:44.960 align:middle line:84% And then we need to get from mevalonic acid 00:07:44.960 --> 00:07:47.480 align:middle line:90% into cholesterol. 00:07:47.480 --> 00:07:50.420 align:middle line:84% And so we'll be talking about this the first couple 00:07:50.420 --> 00:07:52.620 align:middle line:90% of lectures. 00:07:52.620 --> 00:07:54.800 align:middle line:84% In addition to making cholesterol, 00:07:54.800 --> 00:07:57.410 align:middle line:84% you get a lot of cholesterol from your diet. 00:07:57.410 --> 00:08:00.440 align:middle line:84% And so the question is, how does it 00:08:00.440 --> 00:08:04.700 align:middle line:84% get from your diet, transferred through the plasma, 00:08:04.700 --> 00:08:06.500 align:middle line:90% and taken up into cells? 00:08:06.500 --> 00:08:10.430 align:middle line:84% And this section, we'll describe the discovery 00:08:10.430 --> 00:08:13.880 align:middle line:84% of receptor mediated endocytosis, 00:08:13.880 --> 00:08:16.490 align:middle line:84% by Brown and Goldstein, that's now known 00:08:16.490 --> 00:08:17.840 align:middle line:90% to be prevalent everywhere. 00:08:17.840 --> 00:08:22.130 align:middle line:84% So the module-- module 6, you take up 00:08:22.130 --> 00:08:26.900 align:middle line:84% iron by receptor mediated endocytosis. 00:08:26.900 --> 00:08:32.795 align:middle line:84% In module 7, growth factors are involved in receptor mediated 00:08:32.795 --> 00:08:34.059 align:middle line:90% endocytosis. 00:08:34.059 --> 00:08:37.580 align:middle line:84% So it represents a general paradigm 00:08:37.580 --> 00:08:41.330 align:middle line:84% that happens all the time over and over again in biology. 00:08:41.330 --> 00:08:43.789 align:middle line:84% And then we're going to ask the question, 00:08:43.789 --> 00:08:45.590 align:middle line:84% how do you sense the cholesterol? 00:08:45.590 --> 00:08:49.220 align:middle line:84% And we're going to be doing two recitations on this. 00:08:49.220 --> 00:08:53.600 align:middle line:84% And then we'll have a few lectures on the machinery 00:08:53.600 --> 00:08:58.460 align:middle line:84% that sense cholesterol sterile responsive binding proteins, 00:08:58.460 --> 00:09:02.210 align:middle line:84% in a molecule called SCAP, and another molecule-- 00:09:02.210 --> 00:09:05.750 align:middle line:84% both of these are proteins-- called INSIGs. 00:09:05.750 --> 00:09:08.120 align:middle line:84% Everything is located in a membrane-- that's something 00:09:08.120 --> 00:09:10.190 align:middle line:90% you haven't been exposed to. 00:09:10.190 --> 00:09:12.110 align:middle line:84% How do you control everything when you 00:09:12.110 --> 00:09:14.792 align:middle line:90% have stuff stuck in a membrane. 00:09:14.792 --> 00:09:16.250 align:middle line:84% And then we'll come back at the end 00:09:16.250 --> 00:09:20.810 align:middle line:84% to look at the rate limiting step 00:09:20.810 --> 00:09:26.480 align:middle line:84% in formation of mevalonic acid, HMGCoA reductase, 00:09:26.480 --> 00:09:29.900 align:middle line:84% and how that plays a key role, since it's 00:09:29.900 --> 00:09:32.180 align:middle line:90% involved in making cholesterol. 00:09:32.180 --> 00:09:34.970 align:middle line:84% How do we control and regulate that protein, also 00:09:34.970 --> 00:09:37.160 align:middle line:90% in the ER membrane. 00:09:37.160 --> 00:09:41.390 align:middle line:84% And we'll see it requires ubiquitin mediated protein 00:09:41.390 --> 00:09:42.150 align:middle line:90% degradation. 00:09:42.150 --> 00:09:44.330 align:middle line:84% And so that's why I'm going to come back, 00:09:44.330 --> 00:09:46.520 align:middle line:84% and we're going to spend a little bit of time 00:09:46.520 --> 00:09:49.880 align:middle line:84% talking about this process in eukaryotic systems. 00:09:49.880 --> 00:09:52.440 align:middle line:84% And actually, in all the other modules, 00:09:52.440 --> 00:09:55.880 align:middle line:84% you're going to see ubiquitin mediated protein degradation. 00:09:55.880 --> 00:10:01.520 align:middle line:84% And finally, this week in recitation, there's a new-- 00:10:01.520 --> 00:10:03.710 align:middle line:84% not new, it was discovered in 2003-- 00:10:03.710 --> 00:10:09.860 align:middle line:84% a new target for drug therapy in controlling cholesterol levels. 00:10:09.860 --> 00:10:13.340 align:middle line:84% And there was a paper published this year 00:10:13.340 --> 00:10:16.260 align:middle line:84% to show that this is, in fact, a good target. 00:10:16.260 --> 00:10:21.670 align:middle line:84% And this paper used CRISPR-Cas9 technology. 00:10:21.670 --> 00:10:22.780 align:middle line:90% So I'm going to-- 00:10:22.780 --> 00:10:25.400 align:middle line:84% even though I'm not an expert in that, my lab hasn't used it-- 00:10:25.400 --> 00:10:27.400 align:middle line:84% I'm going to introduce you to this technology, 00:10:27.400 --> 00:10:31.210 align:middle line:84% and then focus on why we think this is a good new target, 00:10:31.210 --> 00:10:33.010 align:middle line:90% and what the targeting is. 00:10:33.010 --> 00:10:35.890 align:middle line:90% So that's where we're going. 00:10:35.890 --> 00:10:39.250 align:middle line:84% So the terpenome-- let's see if I can 00:10:39.250 --> 00:10:40.750 align:middle line:90% remember what I'm going to say. 00:10:40.750 --> 00:10:44.750 align:middle line:84% The first thing I want to say is-- let me just get all this-- 00:10:44.750 --> 00:10:47.000 align:middle line:84% OK, the first thing I want to tell you something about 00:10:47.000 --> 00:10:48.110 align:middle line:90% is the nomenclature-- 00:10:48.110 --> 00:10:52.250 align:middle line:90% 00:10:52.250 --> 00:10:55.760 align:middle line:84% and all terpenes are either called 00:10:55.760 --> 00:11:07.510 align:middle line:84% isoprenoids or terpenoids, and they're all made from C5-- 00:11:07.510 --> 00:11:10.535 align:middle line:90% a C5 hydrocarbon skeleton. 00:11:10.535 --> 00:11:14.040 align:middle line:90% 00:11:14.040 --> 00:11:19.910 align:middle line:84% And this C5 hydrocarbon skeleton is an isoprene. 00:11:19.910 --> 00:11:22.490 align:middle line:84% So this is the key building block 00:11:22.490 --> 00:11:25.070 align:middle line:84% that you're going to see over and over again 00:11:25.070 --> 00:11:29.690 align:middle line:84% over the course of the first couple of lectures. 00:11:29.690 --> 00:11:34.355 align:middle line:84% So an isoprenoid is, in general, linear. 00:11:34.355 --> 00:11:37.610 align:middle line:90% 00:11:37.610 --> 00:11:40.640 align:middle line:84% And it's made of n of these C5 units. 00:11:40.640 --> 00:11:49.010 align:middle line:90% So n can be 2 to thousands. 00:11:49.010 --> 00:11:51.130 align:middle line:84% And I'll give you examples of these. 00:11:51.130 --> 00:11:56.040 align:middle line:84% And again-- and then the terpenoids-- 00:11:56.040 --> 00:12:00.110 align:middle line:84% so let's just use terpenoids over here-- 00:12:00.110 --> 00:12:05.360 align:middle line:90% in general are also made of C5-- 00:12:05.360 --> 00:12:08.420 align:middle line:90% C5 units. 00:12:08.420 --> 00:12:18.500 align:middle line:84% But often, they're oxidized, cyclized, 00:12:18.500 --> 00:12:20.100 align:middle line:90% and sometimes rearranged. 00:12:20.100 --> 00:12:23.820 align:middle line:90% 00:12:23.820 --> 00:12:26.850 align:middle line:84% So my goal today really sort of is introduce you 00:12:26.850 --> 00:12:30.990 align:middle line:84% to this huge class of natural products. 00:12:30.990 --> 00:12:32.550 align:middle line:84% And give you some examples of this, 00:12:32.550 --> 00:12:36.780 align:middle line:84% and then start focusing on how we get the building blocks. 00:12:36.780 --> 00:12:38.550 align:middle line:90% Do you think iso-- 00:12:38.550 --> 00:12:40.335 align:middle line:84% this isoprene can be a building block? 00:12:40.335 --> 00:12:42.060 align:middle line:84% It's chemically not very reactive. 00:12:42.060 --> 00:12:43.740 align:middle line:90% So no, we have to convert-- 00:12:43.740 --> 00:12:47.460 align:middle line:84% we have to convert this into the chemically reactive building 00:12:47.460 --> 00:12:48.390 align:middle line:90% block. 00:12:48.390 --> 00:12:54.500 align:middle line:84% So while isoprene gives you the C5 unit, 00:12:54.500 --> 00:12:59.235 align:middle line:84% our focus today is going to be on creating the building 00:12:59.235 --> 00:12:59.735 align:middle line:90% blocks. 00:12:59.735 --> 00:13:09.550 align:middle line:90% 00:13:09.550 --> 00:13:13.120 align:middle line:84% And again, the building blocks are going to be-- 00:13:13.120 --> 00:13:15.610 align:middle line:84% these are the guys you're going to see over and over again. 00:13:15.610 --> 00:13:20.310 align:middle line:90% 1, 2, 3, 4, 5-- 00:13:20.310 --> 00:13:22.785 align:middle line:84% so does everybody know what PPI means, so I 00:13:22.785 --> 00:13:23.910 align:middle line:90% don't have to write it out? 00:13:23.910 --> 00:13:26.710 align:middle line:84% pyrophosphate-- we're going to see this over and over again. 00:13:26.710 --> 00:13:29.730 align:middle line:84% You've seen this in the first part of the semester. 00:13:29.730 --> 00:13:35.490 align:middle line:84% And this isopentenyl pyrophosphate, or IPP. 00:13:35.490 --> 00:13:38.220 align:middle line:90% 00:13:38.220 --> 00:13:39.750 align:middle line:90% And we're going to see this-- 00:13:39.750 --> 00:13:42.660 align:middle line:84% if you look at this this hydrogen, what's 00:13:42.660 --> 00:13:47.210 align:middle line:84% interesting about this hydrogen, chemically? 00:13:47.210 --> 00:13:50.600 align:middle line:84% What's the pKa of that hydrogen, compared 00:13:50.600 --> 00:13:52.895 align:middle line:90% to if it was this hydrogen? 00:13:52.895 --> 00:13:55.630 align:middle line:90% 00:13:55.630 --> 00:13:56.370 align:middle line:90% It's much lower. 00:13:56.370 --> 00:14:01.390 align:middle line:84% Yeah, so it can form allylic cations or anions. 00:14:01.390 --> 00:14:03.670 align:middle line:84% And so this can readily isomerize 00:14:03.670 --> 00:14:10.340 align:middle line:84% to form this species, which also plays, 00:14:10.340 --> 00:14:17.720 align:middle line:84% which is dimethylalyl pyrophosphate, which 00:14:17.720 --> 00:14:21.170 align:middle line:84% is the other key building block that we're 00:14:21.170 --> 00:14:22.140 align:middle line:90% going to be looking at. 00:14:22.140 --> 00:14:26.420 align:middle line:84% So currently, it's estimated from the latest paper 00:14:26.420 --> 00:14:30.170 align:middle line:84% that I've read that these are the building blocks for what 00:14:30.170 --> 00:14:31.550 align:middle line:90% we call the terpenome. 00:14:31.550 --> 00:14:34.610 align:middle line:90% 00:14:34.610 --> 00:14:39.290 align:middle line:84% And it's estimated that there are greater than 70,000 00:14:39.290 --> 00:14:40.055 align:middle line:90% natural products. 00:14:40.055 --> 00:14:45.480 align:middle line:90% 00:14:45.480 --> 00:14:47.340 align:middle line:84% Now in contrast to what you've learned 00:14:47.340 --> 00:14:50.620 align:middle line:84% about with non-ribosomal polypeptides 00:14:50.620 --> 00:14:55.710 align:middle line:84% synthases and polyketide synthases, 00:14:55.710 --> 00:14:58.500 align:middle line:84% where you sort of can find everything in an operon, 00:14:58.500 --> 00:15:01.830 align:middle line:84% and you can sort of understand how your molecules could 00:15:01.830 --> 00:15:03.030 align:middle line:90% be put together. 00:15:03.030 --> 00:15:04.830 align:middle line:84% It's not so trivial with terpenes. 00:15:04.830 --> 00:15:09.480 align:middle line:84% There is no such logic in these systems. 00:15:09.480 --> 00:15:14.280 align:middle line:84% So let's just look at what some of these molecules 00:15:14.280 --> 00:15:18.860 align:middle line:84% actually are so you know why they're important. 00:15:18.860 --> 00:15:26.980 align:middle line:84% And, OK, so so in the center of everything 00:15:26.980 --> 00:15:30.600 align:middle line:84% are these two guys, our two building blocks. 00:15:30.600 --> 00:15:39.810 align:middle line:84% And these building blocks can go to the fat soluble vitamins-- 00:15:39.810 --> 00:15:44.110 align:middle line:84% so for example A and K. So if you look over here, 00:15:44.110 --> 00:15:45.545 align:middle line:90% you have vitamin A-- 00:15:45.545 --> 00:15:47.020 align:middle line:90% I knew this was going to happen. 00:15:47.020 --> 00:15:50.820 align:middle line:84% My late-- my pointers are not working very well. 00:15:50.820 --> 00:15:54.390 align:middle line:84% I need-- OK, so anyhow you have vitamin A and you have vitamin 00:15:54.390 --> 00:15:55.860 align:middle line:84% K. And where do you see-- you can 00:15:55.860 --> 00:15:59.430 align:middle line:84% see here readily that you have these C5 units somehow 00:15:59.430 --> 00:16:00.110 align:middle line:90% stuck together. 00:16:00.110 --> 00:16:01.720 align:middle line:84% And you have to ask the question, 00:16:01.720 --> 00:16:04.120 align:middle line:84% where does the rest of this come from? 00:16:04.120 --> 00:16:06.840 align:middle line:84% So fat soluble vitamins, which we're not 00:16:06.840 --> 00:16:12.180 align:middle line:84% going to talk about, what you also have 00:16:12.180 --> 00:16:13.725 align:middle line:90% is prenylated proteins. 00:16:13.725 --> 00:16:16.670 align:middle line:90% 00:16:16.670 --> 00:16:21.270 align:middle line:84% And prenylated proteins are shown here. 00:16:21.270 --> 00:16:25.230 align:middle line:84% So quite frequently, you have small little g 00:16:25.230 --> 00:16:28.140 align:middle line:84% proteins, GTPases, you've seen these before. 00:16:28.140 --> 00:16:31.230 align:middle line:84% EFTU, EFGG, they're all over the place. 00:16:31.230 --> 00:16:32.760 align:middle line:84% There are hundreds of g proteins-- 00:16:32.760 --> 00:16:38.220 align:middle line:84% we talked about them in the recitation section on Rodnina 00:16:38.220 --> 00:16:39.390 align:middle line:90% that I gave you. 00:16:39.390 --> 00:16:43.080 align:middle line:84% Anyhow, a lot of those little g proteins go to the membrane 00:16:43.080 --> 00:16:44.760 align:middle line:90% and come away from the membrane. 00:16:44.760 --> 00:16:47.160 align:middle line:84% They do that by sticking on a tag. 00:16:47.160 --> 00:16:51.820 align:middle line:84% This tag can be geranylated or gerynalgeranylated-- 00:16:51.820 --> 00:16:53.850 align:middle line:84% it just the hydrophobic tag that allows 00:16:53.850 --> 00:16:57.720 align:middle line:84% things to interact with the membrane, 00:16:57.720 --> 00:17:00.960 align:middle line:84% increasing the effective molarity. 00:17:00.960 --> 00:17:04.890 align:middle line:84% Nature uses this trick over and over and over again. 00:17:04.890 --> 00:17:12.170 align:middle line:84% Another thing you can generate is natural products 00:17:12.170 --> 00:17:13.620 align:middle line:90% of medicinal interest. 00:17:13.620 --> 00:17:18.750 align:middle line:90% 00:17:18.750 --> 00:17:25.204 align:middle line:84% And I just show here taxol and artemisinin. 00:17:25.204 --> 00:17:29.150 align:middle line:90% 00:17:29.150 --> 00:17:35.900 align:middle line:84% So taxol is used in the treatment of breast cancer. 00:17:35.900 --> 00:17:39.300 align:middle line:84% Artemisinin, anybody heard of that? 00:17:39.300 --> 00:17:41.760 align:middle line:84% Yeah, so this has been the major target-- 00:17:41.760 --> 00:17:43.830 align:middle line:84% in fact, this pathway we're talking about today 00:17:43.830 --> 00:17:47.520 align:middle line:84% has been a major focus of many synthetic biologies, 00:17:47.520 --> 00:17:50.490 align:middle line:84% trying to make this mevalonic acid pathway 00:17:50.490 --> 00:17:56.100 align:middle line:84% so they can make potential drugs, but also jet fuels-- 00:17:56.100 --> 00:17:58.490 align:middle line:84% which, again, you want some kind of hydrocarbon. 00:17:58.490 --> 00:18:01.800 align:middle line:84% So this pathway has been studied a lot 00:18:01.800 --> 00:18:06.180 align:middle line:84% from a point of view of metabolic engineering. 00:18:06.180 --> 00:18:10.290 align:middle line:90% It's also involved in-- 00:18:10.290 --> 00:18:12.600 align:middle line:84% this is one of my favorite-- the perfume. 00:18:12.600 --> 00:18:18.120 align:middle line:84% You can tell from the way I smell the perfume industry. 00:18:18.120 --> 00:18:21.110 align:middle line:84% Any of you ever break a pine cone? 00:18:21.110 --> 00:18:22.500 align:middle line:90% A pine needle? 00:18:22.500 --> 00:18:24.670 align:middle line:90% Yeah, doesn't it smell great? 00:18:24.670 --> 00:18:25.772 align:middle line:90% No, you don't think so? 00:18:25.772 --> 00:18:26.730 align:middle line:90% I think it's wonderful. 00:18:26.730 --> 00:18:27.630 align:middle line:90% It's called pinene. 00:18:27.630 --> 00:18:28.560 align:middle line:90% Anyhow, it has-- 00:18:28.560 --> 00:18:30.580 align:middle line:84% I think terpenes wonderful smells, 00:18:30.580 --> 00:18:34.800 align:middle line:84% and it's the hallmark of the fragrance industry. 00:18:34.800 --> 00:18:35.510 align:middle line:90% Is that on here? 00:18:35.510 --> 00:18:36.720 align:middle line:90% Yeah, so menthol. 00:18:36.720 --> 00:18:41.460 align:middle line:84% Limonene is orange-- in fact, if you were here when Barry 00:18:41.460 --> 00:18:42.630 align:middle line:90% Sharpless used to teach-- 00:18:42.630 --> 00:18:44.572 align:middle line:84% I can't digress, because that's why I never 00:18:44.572 --> 00:18:45.780 align:middle line:90% get to the end of the course. 00:18:45.780 --> 00:18:49.470 align:middle line:84% But anyhow, Barry used to bring to organic class-- 00:18:49.470 --> 00:18:51.870 align:middle line:90% he had boxes of smells. 00:18:51.870 --> 00:18:53.820 align:middle line:84% And he used to pass around the smells, 00:18:53.820 --> 00:18:55.230 align:middle line:90% and they were all wonderful. 00:18:55.230 --> 00:18:59.280 align:middle line:84% And they were almost all terpenes. 00:18:59.280 --> 00:19:05.130 align:middle line:84% And we're going to be looking at things 00:19:05.130 --> 00:19:07.620 align:middle line:84% like dolichol-- we aren't going to be looking at it. 00:19:07.620 --> 00:19:10.200 align:middle line:90% We will see it a little bit. 00:19:10.200 --> 00:19:13.500 align:middle line:84% But what you can see here, Suzanne Walker 00:19:13.500 --> 00:19:15.900 align:middle line:90% is giving a talk here April 4th. 00:19:15.900 --> 00:19:17.160 align:middle line:90% And she works on-- 00:19:17.160 --> 00:19:20.340 align:middle line:84% one of the things she works on is peptidoglycan biosynthesis. 00:19:20.340 --> 00:19:24.990 align:middle line:84% And so sugars are carried around on these lipids. 00:19:24.990 --> 00:19:28.370 align:middle line:90% Some of them are C19 to C55. 00:19:28.370 --> 00:19:31.320 align:middle line:84% If you look at these, you can see these little units 00:19:31.320 --> 00:19:32.190 align:middle line:90% stuck together. 00:19:32.190 --> 00:19:35.940 align:middle line:84% Barbara Imperiali, in our department, the biology 00:19:35.940 --> 00:19:38.940 align:middle line:84% department, works on a asparagine-linked 00:19:38.940 --> 00:19:40.650 align:middle line:90% glycosylation. 00:19:40.650 --> 00:19:43.420 align:middle line:84% Again, the sugars are carried around 00:19:43.420 --> 00:19:45.230 align:middle line:90% by these kinds of terpenes. 00:19:45.230 --> 00:19:51.820 align:middle line:84% So plays a central role in putting sugars onto systems. 00:19:51.820 --> 00:19:56.360 align:middle line:84% And then what we're going to be focused on today-- 00:19:56.360 --> 00:19:59.380 align:middle line:84% and this is the focus in general-- 00:19:59.380 --> 00:20:02.605 align:middle line:90% is on cholesterol. 00:20:02.605 --> 00:20:03.910 align:middle line:90% Do I have that up there? 00:20:03.910 --> 00:20:05.350 align:middle line:90% I think so. 00:20:05.350 --> 00:20:07.730 align:middle line:84% So what we're going to-- do I have cholesterol up there? 00:20:07.730 --> 00:20:08.285 align:middle line:90% Yeah. 00:20:08.285 --> 00:20:08.910 align:middle line:90% OK, here it is. 00:20:08.910 --> 00:20:09.970 align:middle line:90% Cholesterol. 00:20:09.970 --> 00:20:12.110 align:middle line:84% That's what we're going to be focusing on. 00:20:12.110 --> 00:20:15.350 align:middle line:90% And that's not a C5, But a C30. 00:20:15.350 --> 00:20:21.290 align:middle line:84% So how do we get from these C5 units into the C30 units? 00:20:21.290 --> 00:20:26.240 align:middle line:84% So that's an introduction to the terpenome. 00:20:26.240 --> 00:20:27.720 align:middle line:90% They're everywhere. 00:20:27.720 --> 00:20:30.420 align:middle line:84% And so you can't become a biochemist 00:20:30.420 --> 00:20:34.710 align:middle line:84% without seeing carbon carbon bond formation by these C5 00:20:34.710 --> 00:20:37.740 align:middle line:84% units, in many, many kinds of reactions-- 00:20:37.740 --> 00:20:40.470 align:middle line:84% in both primary and secondary metabolism. 00:20:40.470 --> 00:20:42.860 align:middle line:90% They're very important. 00:20:42.860 --> 00:20:45.090 align:middle line:84% So what I want to do before we get 00:20:45.090 --> 00:20:48.600 align:middle line:84% into looking at one of the pathways 00:20:48.600 --> 00:20:54.390 align:middle line:84% that you can make the building blocks, IPP and DMAPP-- 00:20:54.390 --> 00:20:57.350 align:middle line:90% 00:20:57.350 --> 00:21:00.330 align:middle line:90% this is abbreviated DMAPP. 00:21:00.330 --> 00:21:05.210 align:middle line:90% 00:21:05.210 --> 00:21:08.210 align:middle line:84% One of the ways is through the mevalonic acid pathway, 00:21:08.210 --> 00:21:11.780 align:middle line:84% and here's a picture of a cell that I took from something 00:21:11.780 --> 00:21:12.860 align:middle line:90% off the web. 00:21:12.860 --> 00:21:16.250 align:middle line:84% But I want to introduce you to where we're going to be going, 00:21:16.250 --> 00:21:18.680 align:middle line:90% cholesterol biosynthesis. 00:21:18.680 --> 00:21:22.100 align:middle line:84% So where do we break down fatty acids? 00:21:22.100 --> 00:21:25.100 align:middle line:90% 00:21:25.100 --> 00:21:25.940 align:middle line:90% Does anybody know? 00:21:25.940 --> 00:21:28.550 align:middle line:84% You remember from your introductory course? 00:21:28.550 --> 00:21:31.760 align:middle line:84% I want to try to put this into the big picture on metabolism, 00:21:31.760 --> 00:21:35.270 align:middle line:84% so you're not-- we're just not pulling it out of the air. 00:21:35.270 --> 00:21:38.570 align:middle line:84% Anybody know where you break down fatty acids from the diet? 00:21:38.570 --> 00:21:41.986 align:middle line:90% 00:21:41.986 --> 00:21:44.069 align:middle line:84% AUDIENCE: You're asking in the cell, specifically? 00:21:44.069 --> 00:21:45.444 align:middle line:84% JOANNE STUBBE: There in the cell. 00:21:45.444 --> 00:21:46.490 align:middle line:90% Yeah, where in the cell? 00:21:46.490 --> 00:21:49.420 align:middle line:84% These are-- we're talking about eukaryotes now. 00:21:49.420 --> 00:21:50.760 align:middle line:90% Bacteria don't make cholesterol. 00:21:50.760 --> 00:21:51.260 align:middle line:90% Yeah. 00:21:51.260 --> 00:21:53.330 align:middle line:90% What? 00:21:53.330 --> 00:21:54.540 align:middle line:90% OK, you don't even know that. 00:21:54.540 --> 00:21:57.610 align:middle line:84% OK, so you should go back and read the chapter 00:21:57.610 --> 00:22:00.760 align:middle line:84% on fatty acid biosynthesis and degradation. 00:22:00.760 --> 00:22:02.920 align:middle line:84% That would be a good thing for you to do. 00:22:02.920 --> 00:22:06.520 align:middle line:84% Anyhow, fatty acids are broken down in the mitochondria. 00:22:06.520 --> 00:22:08.150 align:middle line:90% We'll see this in a second. 00:22:08.150 --> 00:22:10.510 align:middle line:90% So the mitochondria play a role. 00:22:10.510 --> 00:22:12.670 align:middle line:90% We're going to see today-- 00:22:12.670 --> 00:22:17.110 align:middle line:84% so here's the nucleus, here's the endoplasmic reticulum. 00:22:17.110 --> 00:22:20.830 align:middle line:84% The endoplasmic reticulum is the key sensor 00:22:20.830 --> 00:22:23.830 align:middle line:90% in cholesterol homeostasis. 00:22:23.830 --> 00:22:25.930 align:middle line:84% And so we're going to-- and we're 00:22:25.930 --> 00:22:29.410 align:middle line:84% going to see that it controls transcription factors. 00:22:29.410 --> 00:22:33.160 align:middle line:84% Transcription factors are stuck in a membrane in the ER. 00:22:33.160 --> 00:22:34.590 align:middle line:90% That's-- how weird is that? 00:22:34.590 --> 00:22:38.600 align:middle line:84% Because where do transcription factors need to go? 00:22:38.600 --> 00:22:40.582 align:middle line:90% They need to go to the nucleus. 00:22:40.582 --> 00:22:41.540 align:middle line:90% So how can you do that? 00:22:41.540 --> 00:22:43.457 align:middle line:84% How can you take something stuck in a membrane 00:22:43.457 --> 00:22:44.870 align:middle line:90% and get it to the nucleus? 00:22:44.870 --> 00:22:48.740 align:middle line:84% So they need to go through a golgi stack. 00:22:48.740 --> 00:22:51.650 align:middle line:84% They do some stuff we're going to learn about 00:22:51.650 --> 00:22:54.830 align:middle line:84% to eventually get into the nucleus, where they control 00:22:54.830 --> 00:22:59.030 align:middle line:84% not only the levels of cholesterol proteins, 00:22:59.030 --> 00:23:03.560 align:middle line:84% but also of metabolism of phospholipids, 00:23:03.560 --> 00:23:05.120 align:middle line:90% triacylglycerols. 00:23:05.120 --> 00:23:09.182 align:middle line:84% so this takes us into the big realm of all lipid metabolism, 00:23:09.182 --> 00:23:10.640 align:middle line:84% which most of the time people don't 00:23:10.640 --> 00:23:13.280 align:middle line:84% spend a lot of time talking about 00:23:13.280 --> 00:23:15.320 align:middle line:90% in an introductory course. 00:23:15.320 --> 00:23:17.840 align:middle line:84% And I mean, one of the interesting questions-- 00:23:17.840 --> 00:23:21.920 align:middle line:84% we're going to see the key rate limiting step 00:23:21.920 --> 00:23:24.910 align:middle line:90% in cholesterol homeostasis. 00:23:24.910 --> 00:23:29.120 align:middle line:84% The protein is bound to the ER membrane, 00:23:29.120 --> 00:23:30.800 align:middle line:84% and a lot of the proteins involved 00:23:30.800 --> 00:23:35.150 align:middle line:84% in cholesterol biosynthesis are in the ER membrane. 00:23:35.150 --> 00:23:40.230 align:middle line:84% 50% of all the cholesterol ends up in the plasma membrane. 00:23:40.230 --> 00:23:41.600 align:middle line:90% how does it get there? 00:23:41.600 --> 00:23:43.250 align:middle line:84% Does it just go through solution? 00:23:43.250 --> 00:23:46.590 align:middle line:84% You need to think about the properties of cholesterol. 00:23:46.590 --> 00:23:48.770 align:middle line:84% So when you get confused about where we're going, 00:23:48.770 --> 00:23:50.270 align:middle line:90% come back to the picture. 00:23:50.270 --> 00:23:54.260 align:middle line:84% And I'm going to show you one other big picture, which 00:23:54.260 --> 00:23:55.920 align:middle line:90% we use when I teach-- 00:23:55.920 --> 00:23:58.760 align:middle line:84% when I've taught 5.07 with Essigmann-- again, 00:23:58.760 --> 00:24:00.470 align:middle line:84% this is the picture we can back to over 00:24:00.470 --> 00:24:02.120 align:middle line:90% and over and over again. 00:24:02.120 --> 00:24:04.700 align:middle line:84% Because everything is interconnected. 00:24:04.700 --> 00:24:08.540 align:middle line:84% So we're going to be talking about cholesterol biosynthesis. 00:24:08.540 --> 00:24:12.300 align:middle line:84% We're going to see a key player is acetyl-CoA. 00:24:12.300 --> 00:24:13.430 align:middle line:90% Where have you seen that? 00:24:13.430 --> 00:24:17.130 align:middle line:84% You've learned a lot about acetyl-CoA in biosynthesis, 00:24:17.130 --> 00:24:20.460 align:middle line:84% and use in biosynthesis and polyketide. 00:24:20.460 --> 00:24:26.690 align:middle line:84% Synthases, you talked about biosynthesis of fatty acids. 00:24:26.690 --> 00:24:30.470 align:middle line:84% So fatty acids are biosynthesized in the cytosol. 00:24:30.470 --> 00:24:33.500 align:middle line:90% 00:24:33.500 --> 00:24:37.040 align:middle line:84% But I just told you fatty acids are 00:24:37.040 --> 00:24:40.970 align:middle line:90% degraded in the mitochondria. 00:24:40.970 --> 00:24:42.350 align:middle line:90% What are they degraded to? 00:24:42.350 --> 00:24:45.170 align:middle line:90% Degraded to acetyl-CoA. 00:24:45.170 --> 00:24:50.450 align:middle line:84% Can acetyl-CoA get from the mitochondria to the cytosol? 00:24:50.450 --> 00:24:58.820 align:middle line:90% 00:24:58.820 --> 00:25:02.030 align:middle line:90% Nobody knows? 00:25:02.030 --> 00:25:04.910 align:middle line:90% Let's get some energy, you guys. 00:25:04.910 --> 00:25:07.514 align:middle line:84% What do we know about acetyl-CoA? 00:25:07.514 --> 00:25:09.958 align:middle line:90% AUDIENCE: [INAUDIBLE] 00:25:09.958 --> 00:25:11.000 align:middle line:90% JOANNE STUBBE: It's what? 00:25:11.000 --> 00:25:13.295 align:middle line:84% AUDIENCE: A transport system [INAUDIBLE]---- 00:25:13.295 --> 00:25:15.128 align:middle line:84% JOANNE STUBBE: So you think is the transport 00:25:15.128 --> 00:25:17.850 align:middle line:84% system that takes it from the mitochondria to the cytosol. 00:25:17.850 --> 00:25:19.910 align:middle line:90% So that's wrong. 00:25:19.910 --> 00:25:21.890 align:middle line:84% And in fact, this is again another thing 00:25:21.890 --> 00:25:24.380 align:middle line:84% that I think maybe isn't emphasized enough 00:25:24.380 --> 00:25:25.850 align:middle line:90% in an introductory course. 00:25:25.850 --> 00:25:28.460 align:middle line:84% A lot of these things cannot transfer across these 00:25:28.460 --> 00:25:29.640 align:middle line:90% membranes. 00:25:29.640 --> 00:25:31.880 align:middle line:84% So-- and this may or may not be logical to you, 00:25:31.880 --> 00:25:33.020 align:middle line:84% when you take this and you're saying, 00:25:33.020 --> 00:25:34.437 align:middle line:84% oh my god, this is so complicated. 00:25:34.437 --> 00:25:36.110 align:middle line:84% It's really not that complicated when 00:25:36.110 --> 00:25:40.600 align:middle line:84% you put all primary metabolism into the big picture. 00:25:40.600 --> 00:25:44.180 align:middle line:84% Acetyl-CoA goes into the TCA cycle, 00:25:44.180 --> 00:25:46.790 align:middle line:84% and it condenses with oxaloacetic acid 00:25:46.790 --> 00:25:47.960 align:middle line:90% to form citric acid. 00:25:47.960 --> 00:25:51.440 align:middle line:84% We're going to see citric acid again with iron homeostasis. 00:25:51.440 --> 00:25:55.190 align:middle line:84% Anyhow, it's citrate that is able to go 00:25:55.190 --> 00:25:57.050 align:middle line:84% across the mitochondrial membrane, 00:25:57.050 --> 00:26:00.310 align:middle line:90% as is malate, as is pyruvate. 00:26:00.310 --> 00:26:03.260 align:middle line:84% Acetyl-CoA is not able to do that. 00:26:03.260 --> 00:26:05.420 align:middle line:84% And so to get acetyl-CoA, then you 00:26:05.420 --> 00:26:08.228 align:middle line:84% have to enzymatically break down citrate. 00:26:08.228 --> 00:26:09.770 align:middle line:84% So if you don't know what citrate is, 00:26:09.770 --> 00:26:11.030 align:middle line:90% it's a central metabolite. 00:26:11.030 --> 00:26:12.770 align:middle line:84% You're going to see it again and again. 00:26:12.770 --> 00:26:14.150 align:middle line:90% Pull it up, Google it. 00:26:14.150 --> 00:26:16.280 align:middle line:90% Put it in your brain. 00:26:16.280 --> 00:26:18.890 align:middle line:90% You form acetyl-CoA. 00:26:18.890 --> 00:26:22.260 align:middle line:84% An acetyl-CoA, as you learned in the first part of course, 00:26:22.260 --> 00:26:24.260 align:middle line:90% can form fatty acids. 00:26:24.260 --> 00:26:26.180 align:middle line:90% Where do fatty acids go? 00:26:26.180 --> 00:26:29.450 align:middle line:90% They can attach to glycerol. 00:26:29.450 --> 00:26:31.230 align:middle line:84% And where does glycerol come from? 00:26:31.230 --> 00:26:32.660 align:middle line:84% It comes from breakdown of sugars 00:26:32.660 --> 00:26:35.240 align:middle line:90% through the glycolosis pathway. 00:26:35.240 --> 00:26:39.017 align:middle line:84% And they come together to form phospholipids, which 00:26:39.017 --> 00:26:40.100 align:middle line:90% make up all our membranes. 00:26:40.100 --> 00:26:42.020 align:middle line:90% Pretty important. 00:26:42.020 --> 00:26:44.030 align:middle line:90% What else can fatty acids do? 00:26:44.030 --> 00:26:47.970 align:middle line:84% They can interact with glycerol without a phosphate, 00:26:47.970 --> 00:26:50.840 align:middle line:90% to triacylglycerols. 00:26:50.840 --> 00:26:54.020 align:middle line:84% Triacyl-- esterified triacylgycerols. 00:26:54.020 --> 00:26:57.400 align:middle line:84% Does everybody know what glycerol is? 00:26:57.400 --> 00:26:59.210 align:middle line:90% Everybody know? 00:26:59.210 --> 00:27:02.490 align:middle line:84% OK, so-- and this is another thing we're going to find. 00:27:02.490 --> 00:27:04.820 align:middle line:84% We have huge amounts of phospholipids 00:27:04.820 --> 00:27:07.970 align:middle line:84% and triacylglycerols in our diet. 00:27:07.970 --> 00:27:10.490 align:middle line:84% So we have to deal with those things. 00:27:10.490 --> 00:27:14.420 align:middle line:84% But acetyl-CoA, we'll also see, is the building block 00:27:14.420 --> 00:27:17.210 align:middle line:84% to form mevalonic acid through a pathway we're 00:27:17.210 --> 00:27:18.890 align:middle line:90% going to describe now. 00:27:18.890 --> 00:27:23.040 align:middle line:84% So mevalonic acid is a key player, 00:27:23.040 --> 00:27:25.100 align:middle line:84% and its formation is rate limiting 00:27:25.100 --> 00:27:27.620 align:middle line:90% in cholesterol biosynthesis. 00:27:27.620 --> 00:27:29.990 align:middle line:84% The enzyme that makes mevaloinc acid 00:27:29.990 --> 00:27:33.920 align:middle line:84% is located in this little messy thing here, and that's the ER. 00:27:33.920 --> 00:27:36.020 align:middle line:90% So it's bound to the ER. 00:27:36.020 --> 00:27:38.070 align:middle line:90% It makes cholesterol. 00:27:38.070 --> 00:27:42.530 align:middle line:84% And then ultimately, how does cholesterol move-- 00:27:42.530 --> 00:27:45.200 align:middle line:84% and a lot of the precursors to cholesterol stay 00:27:45.200 --> 00:27:47.720 align:middle line:84% solubilized, and then get distributed 00:27:47.720 --> 00:27:49.520 align:middle line:90% to all the membranes. 00:27:49.520 --> 00:27:51.830 align:middle line:84% 50% of the cholesterol, for example, 00:27:51.830 --> 00:27:54.800 align:middle line:90% is found in the plasma membrane. 00:27:54.800 --> 00:27:56.200 align:middle line:90% So that's the big picture. 00:27:56.200 --> 00:27:59.690 align:middle line:84% And so when you get confused about where we're going, 00:27:59.690 --> 00:28:02.570 align:middle line:84% you need to go back and see how central a player 00:28:02.570 --> 00:28:06.260 align:middle line:90% acetyl-CoA is to everything. 00:28:06.260 --> 00:28:09.350 align:middle line:84% And so its regulation, you're going to see, 00:28:09.350 --> 00:28:11.930 align:middle line:84% is governed by the same transcription 00:28:11.930 --> 00:28:15.562 align:middle line:84% factors that regulate cholesterol homeostasis. 00:28:15.562 --> 00:28:16.770 align:middle line:90% Because they were all linked. 00:28:16.770 --> 00:28:19.310 align:middle line:90% 00:28:19.310 --> 00:28:20.513 align:middle line:90% So where are we going? 00:28:20.513 --> 00:28:22.430 align:middle line:84% Let's see if I can remember where we're going. 00:28:22.430 --> 00:28:29.570 align:middle line:90% 00:28:29.570 --> 00:28:30.650 align:middle line:90% So where are we going? 00:28:30.650 --> 00:28:35.330 align:middle line:90% 00:28:35.330 --> 00:28:40.580 align:middle line:84% So I'm giving you an overview of where we're going. 00:28:40.580 --> 00:28:46.010 align:middle line:84% And I'm only-- this is a long pathway to get to lanosterol. 00:28:46.010 --> 00:28:48.380 align:middle line:84% I'm not going to look at all the steps in the pathway. 00:28:48.380 --> 00:28:52.280 align:middle line:84% I'm just going to tell you how we use these C5 00:28:52.280 --> 00:28:54.440 align:middle line:90% units to generate terpenes. 00:28:54.440 --> 00:28:56.840 align:middle line:84% So we've got to get to the C5 units over here. 00:28:56.840 --> 00:29:00.350 align:middle line:84% We've got to get to these two intermediates. 00:29:00.350 --> 00:29:05.060 align:middle line:84% And then we're going to use them to get eventually to a C30. 00:29:05.060 --> 00:29:06.710 align:middle line:84% And we'll see the same chemistry, 00:29:06.710 --> 00:29:10.070 align:middle line:84% once we know a few rules, just like with aldol reactions 00:29:10.070 --> 00:29:14.540 align:middle line:84% and claison reactions, are used over and over and over again. 00:29:14.540 --> 00:29:15.800 align:middle line:90% There are a few basic rules. 00:29:15.800 --> 00:29:17.840 align:middle line:84% Every protein is different, but I'm 00:29:17.840 --> 00:29:20.240 align:middle line:84% going to make sweeping generalizations, which 00:29:20.240 --> 00:29:22.390 align:middle line:90% is a good place to start. 00:29:22.390 --> 00:29:29.510 align:middle line:84% So we have to use three molecules of acetyl-CoA. 00:29:29.510 --> 00:29:31.250 align:middle line:84% So you're all-- you all should be 00:29:31.250 --> 00:29:34.520 align:middle line:90% very familiar with acetyl-CoA. 00:29:34.520 --> 00:29:41.160 align:middle line:84% And we're after trying to form C5. 00:29:41.160 --> 00:29:44.700 align:middle line:84% So three of these gives us C6, so we 00:29:44.700 --> 00:29:46.730 align:middle line:90% have to get rid of a carbon. 00:29:46.730 --> 00:29:51.600 align:middle line:84% So from this we need to lose whatever the pathway is. 00:29:51.600 --> 00:29:55.410 align:middle line:84% It turns out we lose one carbon as CO2. 00:29:55.410 --> 00:29:56.650 align:middle line:90% And this whole process-- 00:29:56.650 --> 00:29:59.510 align:middle line:90% so this can be multiple steps-- 00:29:59.510 --> 00:30:02.610 align:middle line:90% is called initiation. 00:30:02.610 --> 00:30:09.120 align:middle line:84% And it forms IPP, which can isomerize to form DMAPP. 00:30:09.120 --> 00:30:11.940 align:middle line:90% 00:30:11.940 --> 00:30:15.390 align:middle line:84% And then, again, this is our C5 unit that we're after. 00:30:15.390 --> 00:30:19.020 align:middle line:90% So this is C5. 00:30:19.020 --> 00:30:24.840 align:middle line:84% And we've lost one carbon as CO2. 00:30:24.840 --> 00:30:29.040 align:middle line:84% So then we're going to have what I'm going to call elongation. 00:30:29.040 --> 00:30:32.940 align:middle line:90% 00:30:32.940 --> 00:30:38.050 align:middle line:84% And what we're going to see is that to get to lanosterol, 00:30:38.050 --> 00:30:41.200 align:middle line:90% all we need to have a C30. 00:30:41.200 --> 00:30:42.740 align:middle line:90% So we need six of these guys. 00:30:42.740 --> 00:30:48.680 align:middle line:90% So we have six C5 to form a C30. 00:30:48.680 --> 00:30:53.540 align:middle line:90% 00:30:53.540 --> 00:30:54.630 align:middle line:90% Which is lanosterol. 00:30:54.630 --> 00:30:57.770 align:middle line:90% This is a precursor to steroids. 00:30:57.770 --> 00:31:03.980 align:middle line:84% [INAUDIBLE] talk about cholesterol-- uh oh. 00:31:03.980 --> 00:31:07.520 align:middle line:84% I knew that that was going to happen. 00:31:07.520 --> 00:31:08.180 align:middle line:90% I jump around. 00:31:08.180 --> 00:31:10.020 align:middle line:84% Usually this falls off, so you'll 00:31:10.020 --> 00:31:12.820 align:middle line:90% have to get used to this. 00:31:12.820 --> 00:31:13.660 align:middle line:90% It's a good thing-- 00:31:13.660 --> 00:31:18.374 align:middle line:84% I spent all morning trying to figure out where the-- 00:31:18.374 --> 00:31:20.650 align:middle line:84% where this cord came, because I knew 00:31:20.650 --> 00:31:23.310 align:middle line:84% my cord wasn't going to fit, and the cord was going to be shot, 00:31:23.310 --> 00:31:25.810 align:middle line:84% and then I was thinking, how am I going to run from one door 00:31:25.810 --> 00:31:27.070 align:middle line:90% to the next? 00:31:27.070 --> 00:31:31.210 align:middle line:90% I need to get this back on me. 00:31:31.210 --> 00:31:32.140 align:middle line:90% Can you hear me? 00:31:32.140 --> 00:31:32.640 align:middle line:90% OK. 00:31:32.640 --> 00:31:36.990 align:middle line:90% 00:31:36.990 --> 00:31:37.990 align:middle line:90% Let me get back on gear. 00:31:37.990 --> 00:31:40.540 align:middle line:84% So C30-- but then what's going to happen-- 00:31:40.540 --> 00:31:44.650 align:middle line:84% so we get to lanosterol, and then from lanosterol, 00:31:44.650 --> 00:31:49.930 align:middle line:84% and going to lanosterol here, we're going to have to do, 00:31:49.930 --> 00:31:52.690 align:middle line:84% like, I think, the most amazing chemistry in the whole world. 00:31:52.690 --> 00:31:59.650 align:middle line:84% We're going to have to do an oxidation and a cyclization. 00:31:59.650 --> 00:32:01.330 align:middle line:84% So this is going to be a terpene. 00:32:01.330 --> 00:32:03.435 align:middle line:84% So we're going to put these things together 00:32:03.435 --> 00:32:06.220 align:middle line:84% to form a C30, a linear C30, and then 00:32:06.220 --> 00:32:10.120 align:middle line:84% they have to come together to form this guy. 00:32:10.120 --> 00:32:12.640 align:middle line:84% So we're going to talk about this reaction, 00:32:12.640 --> 00:32:14.650 align:middle line:84% because it's such a cool reaction. 00:32:14.650 --> 00:32:17.800 align:middle line:84% Anyhow, we're going to talk about how this linear molecule 00:32:17.800 --> 00:32:18.790 align:middle line:90% gets to this. 00:32:18.790 --> 00:32:22.840 align:middle line:84% That's-- that is the coolest reaction, in my opinion, 00:32:22.840 --> 00:32:23.650 align:middle line:90% in biology. 00:32:23.650 --> 00:32:26.500 align:middle line:84% I remember when I first heard about this when I 00:32:26.500 --> 00:32:29.500 align:middle line:90% was in graduate school in 1968. 00:32:29.500 --> 00:32:30.640 align:middle line:90% A long time ago. 00:32:30.640 --> 00:32:32.200 align:middle line:84% That's what made me decide I didn't 00:32:32.200 --> 00:32:34.270 align:middle line:90% want to be an organic chemist. 00:32:34.270 --> 00:32:37.480 align:middle line:90% I said, how amazing is this? 00:32:37.480 --> 00:32:40.600 align:middle line:84% That you can do one step and you can 00:32:40.600 --> 00:32:45.580 align:middle line:84% put in all of these asymmetric centers and 100% yield. 00:32:45.580 --> 00:32:46.360 align:middle line:90% So that was it. 00:32:46.360 --> 00:32:48.520 align:middle line:84% That was a turning point in my life. 00:32:48.520 --> 00:32:51.490 align:middle line:84% Anyhow, hopefully it'll be a turning point in your life too. 00:32:51.490 --> 00:32:53.930 align:middle line:90% So we have C30. 00:32:53.930 --> 00:32:56.150 align:middle line:90% And then we're not there yet. 00:32:56.150 --> 00:32:58.420 align:middle line:84% So this is going to be, for us, the-- 00:32:58.420 --> 00:33:02.500 align:middle line:84% after the elongation, these cyclizations and oxidation 00:33:02.500 --> 00:33:07.970 align:middle line:84% is going to be the termination to get to this ring structure. 00:33:07.970 --> 00:33:12.490 align:middle line:84% But then to get to cholesterol, we have 19 more steps. 00:33:12.490 --> 00:33:15.610 align:middle line:90% 00:33:15.610 --> 00:33:18.910 align:middle line:84% So this is really complicated pathway. 00:33:18.910 --> 00:33:21.760 align:middle line:84% And I'll tell you what had the chemistry actually is not 00:33:21.760 --> 00:33:24.370 align:middle line:84% so hard to understand, but the details are really 00:33:24.370 --> 00:33:25.720 align:middle line:90% still not understood. 00:33:25.720 --> 00:33:30.710 align:middle line:84% Because all of the proteins are membrane bound. 00:33:30.710 --> 00:33:34.360 align:middle line:84% So what I want to do now is come back over here. 00:33:34.360 --> 00:33:39.080 align:middle line:84% And we're going to talk about initiation, elongation, 00:33:39.080 --> 00:33:40.450 align:middle line:90% and the termination steps. 00:33:40.450 --> 00:33:43.570 align:middle line:84% And I'm going to focus on a few of the reactions 00:33:43.570 --> 00:33:48.190 align:middle line:84% that I think are important, and a lot of the details 00:33:48.190 --> 00:33:50.120 align:middle line:90% are written down-- 00:33:50.120 --> 00:33:54.100 align:middle line:84% are written down on the PowerPoint. 00:33:54.100 --> 00:33:57.130 align:middle line:84% So let's look at the first few steps. 00:33:57.130 --> 00:33:59.455 align:middle line:90% And so let's start the pathway. 00:33:59.455 --> 00:34:02.230 align:middle line:90% 00:34:02.230 --> 00:34:08.484 align:middle line:84% And the first molecule we're dealing with is acetyl-CoA. 00:34:08.484 --> 00:34:11.040 align:middle line:90% 00:34:11.040 --> 00:34:13.860 align:middle line:84% And what is special about acetyl-CoA. 00:34:13.860 --> 00:34:17.370 align:middle line:84% Why is nature-- you've just had a whole bunch of units 00:34:17.370 --> 00:34:23.880 align:middle line:84% on acetyl-CoA-- why does nature use thioesters? 00:34:23.880 --> 00:34:25.560 align:middle line:84% What are the two key things you need 00:34:25.560 --> 00:34:28.230 align:middle line:84% to think about in terms of its reactivity? 00:34:28.230 --> 00:34:30.280 align:middle line:84% You guys should be experts on this now. 00:34:30.280 --> 00:34:30.780 align:middle line:90% Yeah? 00:34:30.780 --> 00:34:33.842 align:middle line:90% 00:34:33.842 --> 00:34:35.550 align:middle line:84% AUDIENCE: There's a low pKa [INAUDIBLE].. 00:34:35.550 --> 00:34:37.699 align:middle line:84% JOANNE STUBBE: Right, so you have a reduced pKa, 00:34:37.699 --> 00:34:40.850 align:middle line:90% is reduced from, say, 22 to 18. 00:34:40.850 --> 00:34:44.239 align:middle line:84% So this is the alpha hydrogen acidity. 00:34:44.239 --> 00:34:48.790 align:middle line:84% And what else does a thioester do? 00:34:48.790 --> 00:34:52.550 align:middle line:84% What is the other reactive part of CoA? 00:34:52.550 --> 00:34:54.929 align:middle line:84% This should be like the back of your hand. 00:34:54.929 --> 00:34:57.450 align:middle line:84% I mean, this is part-- this is central to everything 00:34:57.450 --> 00:34:58.300 align:middle line:90% in biochemistry. 00:34:58.300 --> 00:34:58.800 align:middle line:90% Yeah. 00:34:58.800 --> 00:35:00.230 align:middle line:90% AUDIENCE: [INAUDIBLE] 00:35:00.230 --> 00:35:00.670 align:middle line:90% JOANNE STUBBE: Pardon me? 00:35:00.670 --> 00:35:01.837 align:middle line:90% AUDIENCE: The leaving group. 00:35:01.837 --> 00:35:04.800 align:middle line:84% JOANNE STUBBE: The leaving group. 00:35:04.800 --> 00:35:06.480 align:middle line:84% You are going to have a leaving group. 00:35:06.480 --> 00:35:08.610 align:middle line:84% But that's-- and that is important, 00:35:08.610 --> 00:35:11.250 align:middle line:84% but that's not the key important thing. 00:35:11.250 --> 00:35:13.015 align:middle line:90% That is a part of the game. 00:35:13.015 --> 00:35:14.640 align:middle line:84% It can drive the reaction to the right, 00:35:14.640 --> 00:35:17.880 align:middle line:84% if you look at the free energy of hydrolysis. 00:35:17.880 --> 00:35:22.740 align:middle line:84% What else is activated when you have a sulfur ester as opposed 00:35:22.740 --> 00:35:24.220 align:middle line:90% to an oxygen ester? 00:35:24.220 --> 00:35:25.400 align:middle line:90% AUDIENCE: Carbonyl. 00:35:25.400 --> 00:35:27.930 align:middle line:84% JOANNE STUBBE: The carbonyl, because of the decreased 00:35:27.930 --> 00:35:29.910 align:middle line:90% resonance stabilization. 00:35:29.910 --> 00:35:33.450 align:middle line:84% So what you've done then is you have activation 00:35:33.450 --> 00:35:35.880 align:middle line:90% for nucleophilic attack. 00:35:35.880 --> 00:35:38.010 align:middle line:84% And you see this-- nature uses this 00:35:38.010 --> 00:35:41.190 align:middle line:84% in cholesterol homeostasis as well, 00:35:41.190 --> 00:35:42.900 align:middle line:90% over and over and over again. 00:35:42.900 --> 00:35:45.360 align:middle line:84% So in the first step, and I'm not going to draw out 00:35:45.360 --> 00:35:50.550 align:middle line:84% the details, what you can see here is that you're taking two 00:35:50.550 --> 00:35:54.600 align:middle line:84% molecules of acetyl-CoA and you're forming 00:35:54.600 --> 00:35:56.010 align:middle line:90% acetoacetyl-CoA-- 00:35:56.010 --> 00:35:57.690 align:middle line:84% that should be good practice for you 00:35:57.690 --> 00:36:02.300 align:middle line:84% for thinking about the exam on Wednesday. 00:36:02.300 --> 00:36:05.520 align:middle line:90% 00:36:05.520 --> 00:36:08.480 align:middle line:84% And this is an example of a claison reaction, 00:36:08.480 --> 00:36:13.460 align:middle line:84% one of the three types of mechanisms, 00:36:13.460 --> 00:36:16.000 align:middle line:90% to form carbon carbon bonds. 00:36:16.000 --> 00:36:20.570 align:middle line:84% The next step, we need three acetyl-CoA's 00:36:20.570 --> 00:36:25.010 align:middle line:84% to get eventually to isopentenyl pyrophosphate and dimethylallyl 00:36:25.010 --> 00:36:25.940 align:middle line:90% pyrophosphate. 00:36:25.940 --> 00:36:29.770 align:middle line:84% So we're going to use another molecule on acetyl-CoA 00:36:29.770 --> 00:36:34.790 align:middle line:84% to form hydroxymethylglutaryl-CoA. 00:36:34.790 --> 00:36:37.430 align:middle line:84% So we need to add another one of these guys. 00:36:37.430 --> 00:36:40.010 align:middle line:90% 00:36:40.010 --> 00:36:42.030 align:middle line:90% And this is HMG-CoA synthase. 00:36:42.030 --> 00:36:45.850 align:middle line:90% 00:36:45.850 --> 00:36:48.870 align:middle line:84% So before I go there, let's go through what we know. 00:36:48.870 --> 00:36:53.740 align:middle line:84% So this is-- so we're starting here. 00:36:53.740 --> 00:36:57.640 align:middle line:84% So here-- acetyl-CoA plays a central role. 00:36:57.640 --> 00:36:58.810 align:middle line:90% Why thioesters? 00:36:58.810 --> 00:37:01.080 align:middle line:84% It's important in claison reactions. 00:37:01.080 --> 00:37:04.120 align:middle line:84% Here's the example of a claison reaction involving 00:37:04.120 --> 00:37:08.780 align:middle line:84% a carbanion intermediate that you guys should all be experts 00:37:08.780 --> 00:37:10.870 align:middle line:90% at at this stage. 00:37:10.870 --> 00:37:11.900 align:middle line:90% What about this step? 00:37:11.900 --> 00:37:12.700 align:middle line:90% The next step? 00:37:12.700 --> 00:37:16.690 align:middle line:84% Formation of hydroxymethylglutaryl-CoA? 00:37:16.690 --> 00:37:22.180 align:middle line:84% So here it turns out that this enzyme uses covalent catalysis. 00:37:22.180 --> 00:37:24.478 align:middle line:84% Frequently enzymes-- you've already seen this as well, 00:37:24.478 --> 00:37:26.770 align:middle line:84% we're going to see this again and again over the course 00:37:26.770 --> 00:37:28.270 align:middle line:90% of the rest of the semester. 00:37:28.270 --> 00:37:30.730 align:middle line:84% One of the major mechanisms of rate acceleration 00:37:30.730 --> 00:37:32.460 align:middle line:90% is covalent catalysis. 00:37:32.460 --> 00:37:36.340 align:middle line:84% Here the thioester-- the CoA ester has been removed 00:37:36.340 --> 00:37:40.090 align:middle line:84% and it's attached to a cysteine in the active site 00:37:40.090 --> 00:37:41.140 align:middle line:90% of the enzyme. 00:37:41.140 --> 00:37:44.170 align:middle line:84% And then this can react with, in this case, 00:37:44.170 --> 00:37:48.080 align:middle line:84% a ketone like molecule in an aldol reaction. 00:37:48.080 --> 00:37:50.830 align:middle line:90% So here's the second example. 00:37:50.830 --> 00:37:52.430 align:middle line:90% We take acetoacetyl-CoA. 00:37:52.430 --> 00:37:58.000 align:middle line:90% 00:37:58.000 --> 00:37:59.680 align:middle line:90% We add another CoA. 00:37:59.680 --> 00:38:04.160 align:middle line:84% And what we form, then, is hydroxymethylglutaryl-CoA. 00:38:04.160 --> 00:38:06.850 align:middle line:90% 00:38:06.850 --> 00:38:10.390 align:middle line:84% And what we're going to see is, during this reaction, 00:38:10.390 --> 00:38:14.620 align:middle line:84% we also have to do a hydrolosis reaction. 00:38:14.620 --> 00:38:18.160 align:middle line:84% Because we start out with acetyl-CoA and we only add-- 00:38:18.160 --> 00:38:21.550 align:middle line:90% end up with a single thioester. 00:38:21.550 --> 00:38:24.230 align:middle line:90% 00:38:24.230 --> 00:38:31.050 align:middle line:84% And this reaction forms hydroxymethylglutaryl-CoA. 00:38:31.050 --> 00:38:35.310 align:middle line:90% 00:38:35.310 --> 00:38:37.980 align:middle line:84% So we've lost-- in this reaction over here, 00:38:37.980 --> 00:38:39.360 align:middle line:90% you can see where this is lost. 00:38:39.360 --> 00:38:43.950 align:middle line:84% So you have an acetyl-CoA to form the thioester 00:38:43.950 --> 00:38:45.550 align:middle line:90% in the active site. 00:38:45.550 --> 00:38:47.550 align:middle line:90% You you've lost a CoA. 00:38:47.550 --> 00:38:49.330 align:middle line:90% Is everybody with me? 00:38:49.330 --> 00:38:52.560 align:middle line:84% So you're using the third molecule of acetyl-CoA. 00:38:52.560 --> 00:38:54.900 align:middle line:90% You've already lost the CoA. 00:38:54.900 --> 00:38:57.570 align:middle line:84% And then what you end up with in the end 00:38:57.570 --> 00:39:00.490 align:middle line:84% is you have to hydrolize this off. 00:39:00.490 --> 00:39:02.760 align:middle line:84% So if you didn't realize it went through a covalent 00:39:02.760 --> 00:39:04.470 align:middle line:84% intermediate, it would be like you just 00:39:04.470 --> 00:39:06.270 align:middle line:90% lost a CoA, which you did. 00:39:06.270 --> 00:39:09.420 align:middle line:84% But you lost it in this step, because you 00:39:09.420 --> 00:39:11.460 align:middle line:84% went through a covalent intermediate. 00:39:11.460 --> 00:39:13.890 align:middle line:84% And you're not responsible for the details. 00:39:13.890 --> 00:39:17.460 align:middle line:84% Many, many enzymes that use acetyl-CoA go through covalent 00:39:17.460 --> 00:39:19.600 align:middle line:90% intermediates just like this. 00:39:19.600 --> 00:39:23.170 align:middle line:84% But you have to study each one to figure out why they do that. 00:39:23.170 --> 00:39:24.150 align:middle line:90% Why do they do that? 00:39:24.150 --> 00:39:27.750 align:middle line:84% Because covalent catalysis gives us rate accelerations of 10 00:39:27.750 --> 00:39:29.710 align:middle line:90% to the 4th, 10 to the 5th. 00:39:29.710 --> 00:39:31.950 align:middle line:84% So nature has used that as a repertoire 00:39:31.950 --> 00:39:37.440 align:middle line:84% of defining how to catalyze reactions at amazing rates. 00:39:37.440 --> 00:39:39.930 align:middle line:90% So now we're at this stage. 00:39:39.930 --> 00:39:42.060 align:middle line:84% And the next step in this pathway-- 00:39:42.060 --> 00:39:46.020 align:middle line:84% so we're still trying to get to the C5 over here. 00:39:46.020 --> 00:39:50.900 align:middle line:90% 00:39:50.900 --> 00:39:53.510 align:middle line:84% And to get to this now, we're going 00:39:53.510 --> 00:39:56.370 align:middle line:90% to have to do a reduction. 00:39:56.370 --> 00:39:58.640 align:middle line:90% And we're trying to get to-- 00:39:58.640 --> 00:40:00.860 align:middle line:90% so we did this reaction here. 00:40:00.860 --> 00:40:03.900 align:middle line:90% 00:40:03.900 --> 00:40:05.410 align:middle line:90% I will fix my thing for next-- 00:40:05.410 --> 00:40:05.980 align:middle line:90% OK. 00:40:05.980 --> 00:40:08.710 align:middle line:84% So now what we're doing is we're going 00:40:08.710 --> 00:40:12.970 align:middle line:84% from hydroxymethylglutaryl-CoA to mevalonic acid. 00:40:12.970 --> 00:40:15.620 align:middle line:90% And this is wrong. 00:40:15.620 --> 00:40:17.990 align:middle line:90% They should all be NADPHs. 00:40:17.990 --> 00:40:20.720 align:middle line:84% When you're doing biosynthesis, what do you use? 00:40:20.720 --> 00:40:23.000 align:middle line:90% You don't use any NADH. 00:40:23.000 --> 00:40:27.980 align:middle line:84% You use NADPH in almost all biosynthetic pathways. 00:40:27.980 --> 00:40:29.240 align:middle line:90% So what happens? 00:40:29.240 --> 00:40:34.150 align:middle line:84% You're reducing, basically, the thioester down to-- 00:40:34.150 --> 00:40:37.910 align:middle line:90% a thioester down to an alcohol. 00:40:37.910 --> 00:40:41.030 align:middle line:84% Everybody should know at this stage how this kind of reaction 00:40:41.030 --> 00:40:42.620 align:middle line:90% goes. 00:40:42.620 --> 00:40:48.530 align:middle line:84% Everybody, this is one of the two major redox co-factors 00:40:48.530 --> 00:40:49.340 align:middle line:90% and all of biology. 00:40:49.340 --> 00:40:53.910 align:middle line:90% 00:40:53.910 --> 00:40:58.610 align:middle line:84% Can somebody tell me how this redox reaction goes? 00:40:58.610 --> 00:41:01.550 align:middle line:84% This is one of the vitamins on your bottle, niacin, 00:41:01.550 --> 00:41:05.840 align:middle line:84% which gets metabolized into NAD, NADP. 00:41:05.840 --> 00:41:09.230 align:middle line:90% How does that do a reduction? 00:41:09.230 --> 00:41:10.130 align:middle line:90% Can somebody tell me? 00:41:10.130 --> 00:41:16.696 align:middle line:90% 00:41:16.696 --> 00:41:17.640 align:middle line:90% Yeah. 00:41:17.640 --> 00:41:20.595 align:middle line:84% AUDIENCE: Form an aromatic ring by eliminating the hydride-- 00:41:20.595 --> 00:41:21.810 align:middle line:90% so the hydride attacks the-- 00:41:21.810 --> 00:41:23.268 align:middle line:84% JOANNE STUBBE: Right, so that's it. 00:41:23.268 --> 00:41:25.662 align:middle line:84% And where does the hydride attack? 00:41:25.662 --> 00:41:28.067 align:middle line:90% AUDIENCE: The carbonyl. 00:41:28.067 --> 00:41:29.775 align:middle line:84% JOANNE STUBBE: What part of the carbonyl? 00:41:29.775 --> 00:41:30.770 align:middle line:90% AUDIENCE: The carbon. 00:41:30.770 --> 00:41:31.770 align:middle line:90% JOANNE STUBBE: Yeah, OK. 00:41:31.770 --> 00:41:34.770 align:middle line:84% So this is something that I fight with kids all the time 00:41:34.770 --> 00:41:35.860 align:middle line:90% in 5.07. 00:41:35.860 --> 00:41:38.850 align:middle line:84% It doesn't attack the oxygen. It attacks the carbonyl 00:41:38.850 --> 00:41:42.840 align:middle line:84% because it's polarized delta plus delta minus. 00:41:42.840 --> 00:41:46.920 align:middle line:84% So you have-- this, again, of all the vitamins 00:41:46.920 --> 00:41:49.950 align:middle line:84% on your vitamin bottle, this is the simplest one. 00:41:49.950 --> 00:41:53.640 align:middle line:84% So hydrogen moves with a pair of electrons 00:41:53.640 --> 00:41:58.090 align:middle line:84% that's called the hydride, to do this reduction. 00:41:58.090 --> 00:42:03.300 align:middle line:84% And over here, I think I have the details written out. 00:42:03.300 --> 00:42:07.290 align:middle line:84% So I'm not going to write this out in more detail. 00:42:07.290 --> 00:42:11.070 align:middle line:84% But you generate this intermediate-- 00:42:11.070 --> 00:42:13.780 align:middle line:90% this intermediate-- 00:42:13.780 --> 00:42:17.790 align:middle line:84% this intermediate may-- or the oxygen may or may not 00:42:17.790 --> 00:42:18.570 align:middle line:90% be protonated. 00:42:18.570 --> 00:42:22.440 align:middle line:84% You need to look in the active site of the enzyme. 00:42:22.440 --> 00:42:25.590 align:middle line:84% But then what happens to this intermediate? 00:42:25.590 --> 00:42:28.320 align:middle line:84% This intermediate-- so tetrahedral intermediate's 00:42:28.320 --> 00:42:29.820 align:middle line:90% not very stable. 00:42:29.820 --> 00:42:33.750 align:middle line:84% It can break down to liberate CoA. 00:42:33.750 --> 00:42:34.990 align:middle line:90% And what are you left with? 00:42:34.990 --> 00:42:37.630 align:middle line:90% You're left with an aldehyde. 00:42:37.630 --> 00:42:39.520 align:middle line:90% So that's one reduction. 00:42:39.520 --> 00:42:41.410 align:middle line:90% And where do we want to go? 00:42:41.410 --> 00:42:43.270 align:middle line:90% Where we want to go-- 00:42:43.270 --> 00:42:45.400 align:middle line:84% and so I'm not going to draw the whole thing out, 00:42:45.400 --> 00:42:47.440 align:middle line:84% but I'll draw a part of this out-- 00:42:47.440 --> 00:42:53.080 align:middle line:84% so this gives us, then, through a tetrahedral intermediate, 00:42:53.080 --> 00:42:56.040 align:middle line:90% an aldehyde. 00:42:56.040 --> 00:43:00.630 align:middle line:84% And then what can happen to the aldehyde? 00:43:00.630 --> 00:43:03.270 align:middle line:84% We use another molecule of NADPH? 00:43:03.270 --> 00:43:04.530 align:middle line:90% And what happens with that? 00:43:04.530 --> 00:43:05.220 align:middle line:90% The same thing. 00:43:05.220 --> 00:43:08.250 align:middle line:90% You now do a hydride transfer. 00:43:08.250 --> 00:43:17.260 align:middle line:84% And so we need another molecule of NADPH to form the alcohol. 00:43:17.260 --> 00:43:20.650 align:middle line:90% So this is a mevalonic acid. 00:43:20.650 --> 00:43:24.160 align:middle line:84% So this is written out in more detail 00:43:24.160 --> 00:43:28.240 align:middle line:84% here, for those of you who have trouble trouble thinking 00:43:28.240 --> 00:43:28.750 align:middle line:90% about this. 00:43:28.750 --> 00:43:32.730 align:middle line:84% But of all the factors that nature 00:43:32.730 --> 00:43:36.390 align:middle line:84% has evolved to help us expand the repertoire of reactions 00:43:36.390 --> 00:43:38.040 align:middle line:90% in biology, NADPH-- 00:43:38.040 --> 00:43:41.540 align:middle line:90% NADH is the simplest. 00:43:41.540 --> 00:43:43.920 align:middle line:90% Hydride-- it's always hydride. 00:43:43.920 --> 00:43:46.050 align:middle line:90% Flavins, much more complicated. 00:43:46.050 --> 00:43:48.480 align:middle line:84% We'll see some of those-- the chemistry is much more 00:43:48.480 --> 00:43:49.080 align:middle line:90% complicated. 00:43:49.080 --> 00:43:53.450 align:middle line:90% This is really straightforward. 00:43:53.450 --> 00:43:55.700 align:middle line:90% So what do we know about this? 00:43:55.700 --> 00:43:58.030 align:middle line:84% Why are people interested in this? 00:43:58.030 --> 00:44:07.880 align:middle line:84% And this enzyme is called HMG-CoA reductase. 00:44:07.880 --> 00:44:11.630 align:middle line:84% And in your handouts, it's abbreviated-- 00:44:11.630 --> 00:44:14.320 align:middle line:84% I think these things are terrible. 00:44:14.320 --> 00:44:17.240 align:middle line:84% I will give you a list with all the acronyms on them. 00:44:17.240 --> 00:44:19.040 align:middle line:90% I can't remember the acronyms. 00:44:19.040 --> 00:44:20.450 align:middle line:90% And people change them. 00:44:20.450 --> 00:44:25.590 align:middle line:84% And people name things-- enzyme names are extremely difficult. 00:44:25.590 --> 00:44:29.270 align:middle line:84% The older they are, the worse the issue is. 00:44:29.270 --> 00:44:34.310 align:middle line:84% Because do you know what NAD used to be called? 00:44:34.310 --> 00:44:36.010 align:middle line:84% Any of you have a memory of that? 00:44:36.010 --> 00:44:38.083 align:middle line:84% Any of you read the old literature? 00:44:38.083 --> 00:44:41.870 align:middle line:90% 00:44:41.870 --> 00:44:44.310 align:middle line:90% It used to be called DPN-- 00:44:44.310 --> 00:44:47.780 align:middle line:90% dipyridine nucleotide. 00:44:47.780 --> 00:44:52.670 align:middle line:84% So this is pyridine, and that's where they got the name from. 00:44:52.670 --> 00:44:55.040 align:middle line:84% And I used to teach with somebody, [INAUDIBLE] 00:44:55.040 --> 00:44:58.010 align:middle line:84% a long time ago, and everything was DPN. 00:44:58.010 --> 00:44:59.780 align:middle line:84% So anyhow, if you've read the literature, 00:44:59.780 --> 00:45:02.030 align:middle line:84% nothing will be called in NAD, NADH. 00:45:02.030 --> 00:45:04.190 align:middle line:84% And in fact, a lot of the seminal experiments 00:45:04.190 --> 00:45:08.540 align:middle line:84% that elucidate the pathways came out of the old literature. 00:45:08.540 --> 00:45:10.760 align:middle line:84% So why is this protein interesting? 00:45:10.760 --> 00:45:13.400 align:middle line:84% So we're going to spend a little bit of time on this protein. 00:45:13.400 --> 00:45:18.170 align:middle line:84% People have spent a huge amount of time looking 00:45:18.170 --> 00:45:19.820 align:middle line:90% at this protein in detail. 00:45:19.820 --> 00:45:22.286 align:middle line:90% Does anybody know why? 00:45:22.286 --> 00:45:24.745 align:middle line:84% AUDIENCE: [INAUDIBLE] people target it-- like statins 00:45:24.745 --> 00:45:26.240 align:middle line:90% target it, or cholesterol. 00:45:26.240 --> 00:45:28.790 align:middle line:84% JOANNE STUBBE: So the key thing in this system 00:45:28.790 --> 00:45:35.600 align:middle line:84% is it's the rate limiting step in cholesterol biosynthesis. 00:45:35.600 --> 00:45:38.330 align:middle line:90% 00:45:38.330 --> 00:45:45.290 align:middle line:84% And it's the target of, I would say, 00:45:45.290 --> 00:45:50.920 align:middle line:84% a wonder drug-- the wonder drugs of the statins. 00:45:50.920 --> 00:45:53.890 align:middle line:84% So people really care about the detailed mechanism. 00:45:53.890 --> 00:45:56.560 align:middle line:84% We don't care about the detailed mechanism. 00:45:56.560 --> 00:45:59.950 align:middle line:84% We do care that hydride attacks the carbonyl, 00:45:59.950 --> 00:46:02.650 align:middle line:84% and it attacks the carbon and not the oxygen. 00:46:02.650 --> 00:46:04.810 align:middle line:84% But the details, if you're interested in that, 00:46:04.810 --> 00:46:07.540 align:middle line:84% you can go read about this in the reference. 00:46:07.540 --> 00:46:09.550 align:middle line:84% A lot of people have focused a lot of energy 00:46:09.550 --> 00:46:14.050 align:middle line:84% on this, trying to make better statin inhibitors. 00:46:14.050 --> 00:46:15.910 align:middle line:90% So what do we know about this? 00:46:15.910 --> 00:46:18.670 align:middle line:84% And there's a few things I want to say about this. 00:46:18.670 --> 00:46:20.770 align:middle line:84% And so if we look at the protein, 00:46:20.770 --> 00:46:26.620 align:middle line:84% we're going to come back to this in lecture 3. 00:46:26.620 --> 00:46:29.790 align:middle line:84% So this is important to remember. 00:46:29.790 --> 00:46:31.280 align:middle line:90% So this is the protein. 00:46:31.280 --> 00:46:33.370 align:middle line:90% I'm going to use this cartoon. 00:46:33.370 --> 00:46:36.090 align:middle line:84% And Liz used these cartoons as well. 00:46:36.090 --> 00:46:39.770 align:middle line:84% But what we're going to see is the protein 00:46:39.770 --> 00:46:42.120 align:middle line:90% has eight of these things-- 00:46:42.120 --> 00:46:43.217 align:middle line:90% eight. 00:46:43.217 --> 00:46:44.300 align:middle line:90% Each one of these things-- 00:46:44.300 --> 00:46:48.280 align:middle line:90% 00:46:48.280 --> 00:46:51.030 align:middle line:84% OK, [INAUDIBLE],, and we need three more. 00:46:51.030 --> 00:46:52.930 align:middle line:90% I'm not going to fit this. 00:46:52.930 --> 00:46:55.440 align:middle line:84% So it has a transmembrane helices. 00:46:55.440 --> 00:47:00.860 align:middle line:90% 00:47:00.860 --> 00:47:06.545 align:middle line:84% And the protein itself is, again, 888 amino acids. 00:47:06.545 --> 00:47:09.640 align:middle line:90% 00:47:09.640 --> 00:47:11.410 align:middle line:84% And what's interesting about this, 00:47:11.410 --> 00:47:15.070 align:middle line:84% if you have this many transmembrane helices, where's 00:47:15.070 --> 00:47:16.650 align:middle line:90% the protein going to be located? 00:47:16.650 --> 00:47:19.150 align:middle line:84% It's going to be located in a membrane. 00:47:19.150 --> 00:47:28.380 align:middle line:84% So these five are called the sterile sensor domain. 00:47:28.380 --> 00:47:33.660 align:middle line:84% HMG-CoA reductase is going to be a key player in regulation 00:47:33.660 --> 00:47:35.670 align:middle line:90% of cholesterol levels. 00:47:35.670 --> 00:47:39.780 align:middle line:84% And it exists-- it's found, this protein 00:47:39.780 --> 00:47:41.400 align:middle line:90% is found in the ER membrane. 00:47:41.400 --> 00:47:46.260 align:middle line:90% 00:47:46.260 --> 00:47:56.950 align:middle line:84% And SSD is the sterile sensor domain. 00:47:56.950 --> 00:47:59.070 align:middle line:84% And we're to come back to this when we start 00:47:59.070 --> 00:48:00.390 align:middle line:90% talking about homeostasis. 00:48:00.390 --> 00:48:04.050 align:middle line:84% We're going to see that there are other proteins that also 00:48:04.050 --> 00:48:08.130 align:middle line:84% have transmembrane helices that somehow bind and sense 00:48:08.130 --> 00:48:09.870 align:middle line:84% cholesterol that are going to help us 00:48:09.870 --> 00:48:12.360 align:middle line:90% control cholesterol levels. 00:48:12.360 --> 00:48:14.580 align:middle line:84% Now, what's really interesting about this-- 00:48:14.580 --> 00:48:16.130 align:middle line:90% so the protein is huge. 00:48:16.130 --> 00:48:17.730 align:middle line:90% It's stuck in membrane. 00:48:17.730 --> 00:48:19.740 align:middle line:84% What's really interesting is that you 00:48:19.740 --> 00:48:22.530 align:middle line:84% can cut the protein in half, about in half. 00:48:22.530 --> 00:48:24.030 align:middle line:84% That's what you're looking at there. 00:48:24.030 --> 00:48:25.488 align:middle line:84% You have a soluble protein, they're 00:48:25.488 --> 00:48:28.470 align:middle line:84% much easier to crystallize than membrane proteins. 00:48:28.470 --> 00:48:33.150 align:middle line:84% And it turns out, if you cut the protein in half, this guy, 00:48:33.150 --> 00:48:38.062 align:middle line:90% if you cut, is active. 00:48:38.062 --> 00:48:38.770 align:middle line:90% And it's soluble. 00:48:38.770 --> 00:48:41.540 align:middle line:90% 00:48:41.540 --> 00:48:44.660 align:middle line:84% And the activity is the same as the protein 00:48:44.660 --> 00:48:45.920 align:middle line:90% bound to the membrane. 00:48:45.920 --> 00:48:47.450 align:middle line:90% So it has very high activity. 00:48:47.450 --> 00:48:49.910 align:middle line:84% So it's like you have two separate domains. 00:48:49.910 --> 00:48:52.850 align:middle line:84% Furthermore, if you cut this in half, 00:48:52.850 --> 00:48:55.910 align:middle line:84% you can still target this to the ER membrane, 00:48:55.910 --> 00:48:58.470 align:middle line:84% and you can still sense cholesterol. 00:48:58.470 --> 00:49:00.950 align:middle line:84% So somehow these two things have come together. 00:49:00.950 --> 00:49:03.110 align:middle line:84% They have two really independent activities. 00:49:03.110 --> 00:49:05.060 align:middle line:84% But we're going to see, they work together 00:49:05.060 --> 00:49:08.540 align:middle line:90% to control cholesterol levels. 00:49:08.540 --> 00:49:09.930 align:middle line:90% So what I want to do-- 00:49:09.930 --> 00:49:10.770 align:middle line:90% how am I doing? 00:49:10.770 --> 00:49:14.000 align:middle line:90% Oh, see, time goes by too fast. 00:49:14.000 --> 00:49:15.890 align:middle line:84% Isn't time going by too fast for you? 00:49:15.890 --> 00:49:18.880 align:middle line:90% Anyhow, I want to show you-- 00:49:18.880 --> 00:49:20.840 align:middle line:84% and we'll come back to it next time-- 00:49:20.840 --> 00:49:27.080 align:middle line:84% is that the statins are the target of HMG-CoA reductase. 00:49:27.080 --> 00:49:29.300 align:middle line:84% I mean, this is like an amazing thing. 00:49:29.300 --> 00:49:34.850 align:middle line:84% Cholesterol biosynthesis was only elucidated in 1955. 00:49:34.850 --> 00:49:38.660 align:middle line:84% And it turns out this guy, Endo, was the first one 00:49:38.660 --> 00:49:42.680 align:middle line:84% to discover a natural product that somehow could 00:49:42.680 --> 00:49:45.770 align:middle line:90% lower cholesterol in 1976. 00:49:45.770 --> 00:49:49.040 align:middle line:84% And actually, when I was a young person, 00:49:49.040 --> 00:49:50.930 align:middle line:84% Al Alberts used to work at Merck. 00:49:50.930 --> 00:49:53.060 align:middle line:84% I used to consult for Merck back in those days. 00:49:53.060 --> 00:49:55.850 align:middle line:84% It was incredibly exciting times, 00:49:55.850 --> 00:49:58.880 align:middle line:84% because he discovered really sort 00:49:58.880 --> 00:50:02.490 align:middle line:84% of the first real statin that worked, that wasn't toxic-- 00:50:02.490 --> 00:50:04.250 align:middle line:90% lovastatin. 00:50:04.250 --> 00:50:07.070 align:middle line:84% And really, within a period of only seven years, 00:50:07.070 --> 00:50:08.870 align:middle line:90% this was approved by the FDA. 00:50:08.870 --> 00:50:10.580 align:middle line:84% So that's an amazing observation. 00:50:10.580 --> 00:50:14.090 align:middle line:84% People are still gobbling down statins everywhere. 00:50:14.090 --> 00:50:18.596 align:middle line:84% There are issues with them, but it makes $30 billion 00:50:18.596 --> 00:50:21.440 align:middle line:90% for the companies that own this. 00:50:21.440 --> 00:50:26.110 align:middle line:84% So you now might have heard of Lipitor or Crestor-- 00:50:26.110 --> 00:50:29.010 align:middle line:90% anyhow, they are there. 00:50:29.010 --> 00:50:31.340 align:middle line:90% And it really is a wonder drug. 00:50:31.340 --> 00:50:34.760 align:middle line:84% And it works, we're going to see next time. 00:50:34.760 --> 00:50:37.400 align:middle line:84% Because it looks like the substrate 00:50:37.400 --> 00:50:41.150 align:middle line:90% hydroxymethylglutaryl-CoA-- 00:50:41.150 --> 00:50:44.180 align:middle line:84% So that it acts as a competitive inhibitor 00:50:44.180 --> 00:50:48.620 align:middle line:84% for binding to the active site of HMG-CoA reductase, 00:50:48.620 --> 00:50:51.200 align:middle line:84% and prevents the reduction process. 00:50:51.200 --> 00:50:53.330 align:middle line:84% And we'll come back next time and look 00:50:53.330 --> 00:50:55.157 align:middle line:90% at a little bit at the details. 00:50:55.157 --> 00:50:57.740 align:middle line:84% We're not going to spend a lot of time looking at the details, 00:50:57.740 --> 00:51:02.300 align:middle line:84% but then finish on to get to IPP and dimethyl APP, 00:51:02.300 --> 00:51:06.550 align:middle line:84% the building blocks we're after to make all terpenes. 00:51:06.550 --> 00:51:08.100 align:middle line:90% OK. 00:51:08.100 --> 00:51:27.161 align:middle line:90%