1 00:00:00,000 --> 00:00:02,790 SPEAKER 1: The following content is provided under a Creative 2 00:00:02,790 --> 00:00:04,320 Commons license. 3 00:00:04,320 --> 00:00:06,650 Your support will help MIT OpenCourseWare 4 00:00:06,650 --> 00:00:11,010 continue to offer high quality educational resources for free. 5 00:00:11,010 --> 00:00:13,630 To make a donation or view additional materials 6 00:00:13,630 --> 00:00:17,355 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,355 --> 00:00:17,980 at ocw.mit.edu. 8 00:00:20,586 --> 00:00:22,352 PROFESSOR: Good morning everybody. 9 00:00:22,352 --> 00:00:24,060 Today we're going to continue our journey 10 00:00:24,060 --> 00:00:29,610 in looking at the 10 sort of basic types of reactions 11 00:00:29,610 --> 00:00:33,210 that you will encounter when studying basic metabolism. 12 00:00:33,210 --> 00:00:36,470 And they're all found on your vitamin bottle. 13 00:00:36,470 --> 00:00:39,220 So here are the Flintstones vitamins. 14 00:00:39,220 --> 00:00:41,325 I like Flintstones vitamins because you 15 00:00:41,325 --> 00:00:42,450 don't have to swallow them. 16 00:00:42,450 --> 00:00:43,690 You can chew them. 17 00:00:43,690 --> 00:00:46,490 And today what I'm going to start talking about 18 00:00:46,490 --> 00:00:48,200 is redox chemistry. 19 00:00:48,200 --> 00:00:51,350 What are the two redox active cofactors? 20 00:00:51,350 --> 00:00:53,710 And what we will focus on today is 21 00:00:53,710 --> 00:01:02,110 vitamin B2, which is riboflavin, and vitamin B3 which is niacin. 22 00:01:02,110 --> 00:01:08,700 So we're going to talk about oxidation and reduction. 23 00:01:08,700 --> 00:01:11,150 And what the cofactors are that involved 24 00:01:11,150 --> 00:01:15,470 in these transformations in all primary metabolic pathways. 25 00:01:15,470 --> 00:01:18,450 So the first thing I want to do is introduce you 26 00:01:18,450 --> 00:01:21,990 to these two cofactors, the structure of the cofactors. 27 00:01:21,990 --> 00:01:26,700 Then what I'll do is focus on a comparison of their properties. 28 00:01:26,700 --> 00:01:30,050 And then I want to focus on the mechanism 29 00:01:30,050 --> 00:01:32,350 of how these cofactors work. 30 00:01:32,350 --> 00:01:34,470 And once you master these concepts, 31 00:01:34,470 --> 00:01:38,510 you really will encounter them over and over again 32 00:01:38,510 --> 00:01:40,680 throughout your journey in metabolism. 33 00:01:40,680 --> 00:01:42,810 And you'll be familiar with the kinds 34 00:01:42,810 --> 00:01:46,760 of chemical transformations that are occurring. 35 00:01:46,760 --> 00:01:53,320 So the first cofactor comes from niacin. 36 00:01:53,320 --> 00:01:58,310 And it is nicotinamide adenine dinucleotide 37 00:01:58,310 --> 00:01:59,380 in the reduced state. 38 00:01:59,380 --> 00:02:02,480 Or nicotinamide adenine dinucleotide 39 00:02:02,480 --> 00:02:04,820 in the oxidized state. 40 00:02:04,820 --> 00:02:08,940 And as with all cofactors, the vitamins 41 00:02:08,940 --> 00:02:11,130 are a precursor to the cofactor, and they 42 00:02:11,130 --> 00:02:13,130 have to be converted inside the cell 43 00:02:13,130 --> 00:02:15,870 to the active form of the cofactor. 44 00:02:15,870 --> 00:02:20,560 So if you look at NADH-- let me draw that structure for you, 45 00:02:20,560 --> 00:02:24,930 and then I'm going to show you that while the structure is 46 00:02:24,930 --> 00:02:28,250 quite complex, most of it is not volved in the chemical 47 00:02:28,250 --> 00:02:29,240 transformations. 48 00:02:29,240 --> 00:02:34,410 And so we'll put a big R group on most of the cofactor. 49 00:02:34,410 --> 00:02:38,620 And you don't have to memorize the structures of any 50 00:02:38,620 --> 00:02:40,500 of these cofactors. 51 00:02:40,500 --> 00:02:44,580 And that you will be given the structures whenever 52 00:02:44,580 --> 00:02:48,080 you have any kind of exercise to carry out. 53 00:02:48,080 --> 00:02:57,060 So the form of the vitamin is actually a nicotinamide ring. 54 00:02:57,060 --> 00:02:59,080 And then it needs to be metabolized 55 00:02:59,080 --> 00:03:01,070 to the structure I'm drawing now which 56 00:03:01,070 --> 00:03:04,400 has the nicotinamide ring attached 57 00:03:04,400 --> 00:03:13,040 to a ribose ring, which is then attached to ADP, which you're 58 00:03:13,040 --> 00:03:14,390 all familiar with. 59 00:03:14,390 --> 00:03:17,160 And I'm just going to abbreviate the adenosine part 60 00:03:17,160 --> 00:03:19,600 of a molecule with an AD. 61 00:03:19,600 --> 00:03:24,590 So this part of the molecule we're 62 00:03:24,590 --> 00:03:28,280 going to call for the rest of this exercise today 63 00:03:28,280 --> 00:03:31,550 the R group, because it's not involved in the chemistry. 64 00:03:31,550 --> 00:03:33,820 And really, all of the chemistry happens 65 00:03:33,820 --> 00:03:35,530 in this part of the molecule. 66 00:03:35,530 --> 00:03:41,330 This is the business end, and this is the reduced form 67 00:03:41,330 --> 00:03:45,130 of the cofactor, NADH. 68 00:03:45,130 --> 00:03:47,580 So in addition to the reduced form, 69 00:03:47,580 --> 00:03:49,440 since we're doing oxidation and reduction, 70 00:03:49,440 --> 00:03:52,580 you need to know what the oxidized form is. 71 00:03:52,580 --> 00:03:56,410 And the oxidized form is the following 72 00:03:56,410 --> 00:03:59,980 where you need to lose a couple of electrons 73 00:03:59,980 --> 00:04:03,480 and a couple of protons out of this ring 74 00:04:03,480 --> 00:04:06,330 to generate this nicotinamide ring. 75 00:04:09,780 --> 00:04:13,200 And again, this part of the molecule is indicated as R, 76 00:04:13,200 --> 00:04:16,149 and then you retain a positive charge. 77 00:04:16,149 --> 00:04:18,040 So again, this is the part of the molecule 78 00:04:18,040 --> 00:04:19,690 we're going to be focusing on. 79 00:04:19,690 --> 00:04:25,110 And this is the oxidized form of the cofactor. 80 00:04:25,110 --> 00:04:28,200 So what I want to do now is show you 81 00:04:28,200 --> 00:04:32,000 how this compares to the second form of the organic cofactor 82 00:04:32,000 --> 00:04:35,020 that you encounter over and over again in metabolism. 83 00:04:35,020 --> 00:04:41,535 And this is FADH2, or FMNH2. 84 00:04:41,535 --> 00:04:53,273 And that's the reduced form, versus FAD or FMN. 85 00:04:56,440 --> 00:04:58,570 So this is one of the most complicated 86 00:04:58,570 --> 00:05:00,280 of all the cofactors. 87 00:05:00,280 --> 00:05:04,470 And you'll see that it has a very complex ring 88 00:05:04,470 --> 00:05:09,030 structure called an isoalloxazine ring-- which 89 00:05:09,030 --> 00:05:12,010 I'm drawing now-- that most of you 90 00:05:12,010 --> 00:05:17,100 have likely never encountered before. 91 00:05:17,100 --> 00:05:21,360 And when you look at the structure of this molecule, 92 00:05:21,360 --> 00:05:23,950 you probably will have no idea about where 93 00:05:23,950 --> 00:05:25,330 the chemical reactivity is. 94 00:05:25,330 --> 00:05:27,860 And I'll show you where the chemical reactivity is 95 00:05:27,860 --> 00:05:30,995 and how we know that in a few minutes. 96 00:05:34,166 --> 00:05:44,460 So attached to this nitrogen is more complex 97 00:05:44,460 --> 00:05:57,380 structure with a sugar where x can be phosphate. 98 00:05:57,380 --> 00:06:01,870 This is called the flavinmanonucleotide, reduced. 99 00:06:01,870 --> 00:06:03,350 This is the reduced form. 100 00:06:03,350 --> 00:06:11,090 And if x is equal to ADP, this is flavinadninedineucleotide 101 00:06:11,090 --> 00:06:11,670 reduce. 102 00:06:11,670 --> 00:06:18,460 And I want to point out that ADP is present in both NADH 103 00:06:18,460 --> 00:06:24,380 and FADH2, as it is present actually, in coenzyme A 104 00:06:24,380 --> 00:06:27,130 and in ATP. 105 00:06:27,130 --> 00:06:30,740 So ADP plays a central role in almost all the chemical 106 00:06:30,740 --> 00:06:33,390 transformations, the 10 reactions 107 00:06:33,390 --> 00:06:36,450 we actually talk about that are central to all 108 00:06:36,450 --> 00:06:38,180 of primary metabolism. 109 00:06:38,180 --> 00:06:41,990 However in this case, again, this part of the molecule 110 00:06:41,990 --> 00:06:45,530 we're going to call R, is not involved in the chemistry. 111 00:06:45,530 --> 00:06:51,170 So the business end of the molecule is indicated here. 112 00:06:51,170 --> 00:06:56,100 And so this is the business end, in this is the reduced state. 113 00:06:58,860 --> 00:07:02,240 So this cofactor has to be able to toggle 114 00:07:02,240 --> 00:07:05,490 between the reduced state and the oxidized state. 115 00:07:05,490 --> 00:07:10,477 And if you look at the oxidized state-- 116 00:07:10,477 --> 00:07:12,810 so I'm going to indicate this whole part of the molecule 117 00:07:12,810 --> 00:07:21,550 is R-- you could see a change in this part 118 00:07:21,550 --> 00:07:23,800 of the isoalloxazine ring. 119 00:07:23,800 --> 00:07:28,740 That is you've lost two protons and two electrons 120 00:07:28,740 --> 00:07:32,850 to form the oxidized form of this cofactor. 121 00:07:32,850 --> 00:07:35,870 Now I also want to give you the nomenclature 122 00:07:35,870 --> 00:07:37,986 for the isoalloxazine ring because I'm 123 00:07:37,986 --> 00:07:39,860 going to tell you where the chemical reactive 124 00:07:39,860 --> 00:07:40,660 positions are. 125 00:07:40,660 --> 00:07:43,240 And so you need to know the numbering of the ring. 126 00:07:43,240 --> 00:07:49,060 And so this is the N1 position, this is 2, 3, 4. 127 00:07:49,060 --> 00:07:54,470 This carbon between these two positions is called the C4A. 128 00:07:54,470 --> 00:07:57,130 This is carbon C4. 129 00:07:57,130 --> 00:08:00,350 And this is the N5 position. 130 00:08:00,350 --> 00:08:04,270 And we'll see that C4 and N5 become important 131 00:08:04,270 --> 00:08:08,450 in the overall chemistry of the transformation. 132 00:08:08,450 --> 00:08:10,560 So these are the two cofactors. 133 00:08:10,560 --> 00:08:14,260 Again, this one is considerably more complex than this one. 134 00:08:14,260 --> 00:08:16,070 But what I want to do, and you'll 135 00:08:16,070 --> 00:08:19,550 see both of these cofactors used over and over again in break 136 00:08:19,550 --> 00:08:20,960 down or biosynthesis. 137 00:08:20,960 --> 00:08:26,120 The sugars breakdown or biosynthesis of fatty acids. 138 00:08:26,120 --> 00:08:28,330 The question is when are they used 139 00:08:28,330 --> 00:08:32,289 and what is the distinction between these cofactors? 140 00:08:32,289 --> 00:08:37,820 So what I want to do is compare the properties of these two 141 00:08:37,820 --> 00:08:41,780 cofactors and then specifically give you 142 00:08:41,780 --> 00:08:45,560 an example of how each of these cofactors work. 143 00:08:45,560 --> 00:08:51,160 So on one side I'll have NAD/NADH, 144 00:08:51,160 --> 00:08:55,210 and the other side will have FAD, again, the oxidized form, 145 00:08:55,210 --> 00:08:58,650 and FADH2, the reduced form. 146 00:08:58,650 --> 00:09:02,900 So we have NAD/NADH FAD/FADH2. 147 00:09:02,900 --> 00:09:05,460 And I want to compare these two cofactors. 148 00:09:05,460 --> 00:09:10,520 And the first thing I want to compare is the chemistry. 149 00:09:10,520 --> 00:09:14,030 And what we will see is NAD/NADH always 150 00:09:14,030 --> 00:09:18,820 does 2 electron chemistry all of the time. 151 00:09:18,820 --> 00:09:26,550 On the other hand, FAD/FADH2 can do either 1 electron chemistry 152 00:09:26,550 --> 00:09:29,420 or 2 electron chemistry. 153 00:09:29,420 --> 00:09:34,240 And in fact, function of flavins inside the cell 154 00:09:34,240 --> 00:09:37,520 is the major mediator between something 155 00:09:37,520 --> 00:09:41,440 that can donate 2 electrons and can something that can 156 00:09:41,440 --> 00:09:43,720 accept only a single electron. 157 00:09:43,720 --> 00:09:46,040 So something they could donate 2 electrons 158 00:09:46,040 --> 00:09:49,190 would be NAD/NADH, which I'll show you in a minute, 159 00:09:49,190 --> 00:09:52,600 and something that can accept only a single electron, 160 00:09:52,600 --> 00:09:53,980 or like metals. 161 00:09:53,980 --> 00:09:55,640 Like, if you think back to hemoglobin, 162 00:09:55,640 --> 00:10:01,910 and you think about the ion 3 state going to the ion 2 state. 163 00:10:01,910 --> 00:10:04,422 So there's a big distinction between the two. 164 00:10:04,422 --> 00:10:09,040 The second thing is since were doing oxidation and reduction, 165 00:10:09,040 --> 00:10:13,400 we need to think about the reduction potentials. 166 00:10:13,400 --> 00:10:15,350 And if you think about the reduction 167 00:10:15,350 --> 00:10:22,100 potential of NAD NADH, it turns out to be minus 320 millivolts. 168 00:10:22,100 --> 00:10:25,400 And what you need to recall from freshman chemistry 169 00:10:25,400 --> 00:10:29,350 is that the more positive the number, 170 00:10:29,350 --> 00:10:31,630 the easier it is to reduce. 171 00:10:31,630 --> 00:10:34,790 So the fact that this is minus 320 millivolts 172 00:10:34,790 --> 00:10:39,000 tells you that NAD likes to be in the oxidized state, the NAD 173 00:10:39,000 --> 00:10:40,410 state. 174 00:10:40,410 --> 00:10:45,220 On the other hand, FAD/FADH2 can vary 175 00:10:45,220 --> 00:10:52,920 between minus 450 millivolts to plus 150 millivolts. 176 00:10:52,920 --> 00:10:55,330 So this is a huge difference in reduction potential, 177 00:10:55,330 --> 00:10:57,270 600 millivolts. 178 00:10:57,270 --> 00:10:59,060 So why is that true? 179 00:10:59,060 --> 00:11:02,600 It turns out that if you look at the hundreds 180 00:11:02,600 --> 00:11:05,640 of enzyme structures that we now have-- we've 181 00:11:05,640 --> 00:11:08,480 gone through a structural revolution in the last 15 182 00:11:08,480 --> 00:11:15,370 years-- all of the structures where NAD/NADH bind are 183 00:11:15,370 --> 00:11:17,270 homologous to each other. 184 00:11:17,270 --> 00:11:19,390 And in fact, these structures don't 185 00:11:19,390 --> 00:11:22,470 have anything designed into the structure where they 186 00:11:22,470 --> 00:11:24,360 perturb the redox potential. 187 00:11:24,360 --> 00:11:26,940 The redox potential remains the same, 188 00:11:26,940 --> 00:11:31,090 regardless of what enzyme time it's bound to. 189 00:11:31,090 --> 00:11:34,320 On the other hand, flavins don't maintain 190 00:11:34,320 --> 00:11:35,930 a single type of binding site. 191 00:11:35,930 --> 00:11:38,970 They have five or six different kinds of binding sites. 192 00:11:38,970 --> 00:11:41,290 And that's because the flavin chemistry, 193 00:11:41,290 --> 00:11:44,930 as you can see from this isoalloxazine ring I introduced 194 00:11:44,930 --> 00:11:50,340 to you just a minute ago, does much more diverse chemistry. 195 00:11:50,340 --> 00:11:58,320 And in fact, the active site can tune the redox potential 196 00:11:58,320 --> 00:12:00,470 so it facilitates either 2 electron 197 00:12:00,470 --> 00:12:02,790 chemistry or 1 electron chemistry 198 00:12:02,790 --> 00:12:06,440 or a combination of the two. 199 00:12:06,440 --> 00:12:09,360 So the next thing that's really important in thinking 200 00:12:09,360 --> 00:12:12,220 about the two cofactors and their differences 201 00:12:12,220 --> 00:12:18,710 is that NAD/NADH can act as a substrate. 202 00:12:18,710 --> 00:12:23,130 That is, NAD/NADH is any D, for example 203 00:12:23,130 --> 00:12:26,180 would act as an oxidant of some second substrate. 204 00:12:26,180 --> 00:12:27,660 Every time you have an oxidant, you 205 00:12:27,660 --> 00:12:31,190 have to have something that's going to be oxidized. 206 00:12:31,190 --> 00:12:35,130 And during turn over of oxidized reduced states, 207 00:12:35,130 --> 00:12:39,240 the substrates and products must come on and off the enzyme. 208 00:12:39,240 --> 00:12:42,900 So they dissociate in every single turn over. 209 00:12:42,900 --> 00:12:47,860 FAD/FADH2 on the other hand, is always 210 00:12:47,860 --> 00:12:57,350 tightly bound or covalently bound to the substrate. 211 00:12:57,350 --> 00:13:02,860 And so that means that if FAD gets reduced to FADH2, 212 00:13:02,860 --> 00:13:04,340 it doesn't disassociate. 213 00:13:04,340 --> 00:13:08,170 So you need to have a way in the active site of the enzyme 214 00:13:08,170 --> 00:13:12,042 where the FADH2 can be reoxidized. 215 00:13:12,042 --> 00:13:14,390 And again, that's quite distinct from NAD/NADH. 216 00:13:17,514 --> 00:13:19,680 The fourth thing I want to talk about just briefly-- 217 00:13:19,680 --> 00:13:21,138 and this is what I'm going to focus 218 00:13:21,138 --> 00:13:22,760 the rest of this little section on-- 219 00:13:22,760 --> 00:13:27,730 is the mechanisms by which these cofactors work. 220 00:13:27,730 --> 00:13:30,470 And so out of all of the cofactors 221 00:13:30,470 --> 00:13:33,500 you're going to be introduced to in this little this section 222 00:13:33,500 --> 00:13:36,870 in general on the 10 basic reactions, 223 00:13:36,870 --> 00:13:38,680 this is probably the simplest. 224 00:13:38,680 --> 00:13:45,250 And NAD/NADH involves hydride transfer and proton transfer. 225 00:13:45,250 --> 00:13:48,920 So hydride is a hydrogen with a pair of electrons and a proton. 226 00:13:48,920 --> 00:13:51,640 And we'll see that in a minute. 227 00:13:51,640 --> 00:13:58,570 With FAD/FADH2 the chemistry is considerably more complicated. 228 00:13:58,570 --> 00:14:02,070 But what I will show you is that it can also 229 00:14:02,070 --> 00:14:04,160 do hydride transfer. 230 00:14:04,160 --> 00:14:06,360 And when it does hydride transfer, 231 00:14:06,360 --> 00:14:09,760 it does it to the N5 position. 232 00:14:09,760 --> 00:14:15,740 So if we go back for a second and look 233 00:14:15,740 --> 00:14:18,270 at the flavin in the nomenclature, 234 00:14:18,270 --> 00:14:20,270 this is the N5 position. 235 00:14:20,270 --> 00:14:23,350 I also pointed out you can do C4A chemistry, which 236 00:14:23,350 --> 00:14:24,910 is in your lexicon, but we're not 237 00:14:24,910 --> 00:14:27,990 going to discuss further today. 238 00:14:27,990 --> 00:14:31,140 So you can do N5 chemistry by hydride transfer. 239 00:14:31,140 --> 00:14:34,500 And you can also do C4A chemistry, 240 00:14:34,500 --> 00:14:37,450 which you will see later on when you 241 00:14:37,450 --> 00:14:41,350 start talking about primary metabolic pathways. 242 00:14:41,350 --> 00:14:45,750 So what I want to do now is focus on a specific example 243 00:14:45,750 --> 00:14:48,350 and show you what the basic chemistry is 244 00:14:48,350 --> 00:14:51,840 with these two systems focusing on hydride transfer 245 00:14:51,840 --> 00:14:52,900 of chemistry. 246 00:14:52,900 --> 00:14:57,990 So an example I've chosen to briefly look at 247 00:14:57,990 --> 00:15:03,090 is what I would call an ending to the glycolysis pathway. 248 00:15:03,090 --> 00:15:05,020 Glycolosis, as you already know, is 249 00:15:05,020 --> 00:15:09,640 breakdown of sugar under anaerobic conditions. 250 00:15:09,640 --> 00:15:11,820 So under aerobic conditions, what 251 00:15:11,820 --> 00:15:14,930 happens is when all this energy is given off 252 00:15:14,930 --> 00:15:16,940 when you break down your sugar, it's 253 00:15:16,940 --> 00:15:19,040 stored in chemical bonds, NADH. 254 00:15:19,040 --> 00:15:23,280 The NADH then goes into the respiratory chain. 255 00:15:23,280 --> 00:15:26,050 And the respiratory chain ultimately 256 00:15:26,050 --> 00:15:29,330 transfers this energy to convert oxygen 257 00:15:29,330 --> 00:15:32,330 into water, which is really releases 258 00:15:32,330 --> 00:15:35,810 a huge amount of energy, which is then 259 00:15:35,810 --> 00:15:40,920 trapped to make the energy currency of the cell, ATP. 260 00:15:40,920 --> 00:15:43,180 So we're under anaerobic conditions. 261 00:15:43,180 --> 00:15:46,530 And we need to be able to recycle and keep. 262 00:15:46,530 --> 00:15:48,930 Because the NAD/NADH comes off and on 263 00:15:48,930 --> 00:15:51,220 the enzyme at every single turnover, 264 00:15:51,220 --> 00:15:57,110 you really need to be able to recycle the reduced form back 265 00:15:57,110 --> 00:15:58,180 to the oxidized form. 266 00:15:58,180 --> 00:16:01,870 So glycolysis can actually continue. 267 00:16:01,870 --> 00:16:05,880 So what happens at the ending, one anaerobic ending 268 00:16:05,880 --> 00:16:11,190 of glycolysis, is you need a proton plus NADH, 269 00:16:11,190 --> 00:16:12,950 so that's the reduced form that's 270 00:16:12,950 --> 00:16:17,520 generated during glycolosis, plus the molecule called 271 00:16:17,520 --> 00:16:18,260 pyruvate. 272 00:16:18,260 --> 00:16:21,730 This is a molecule you're going to see over and over again 273 00:16:21,730 --> 00:16:24,270 over the course of the semester whose structure you 274 00:16:24,270 --> 00:16:25,320 need to remember. 275 00:16:25,320 --> 00:16:31,290 So this is the reduced state and this is the oxidized state. 276 00:16:31,290 --> 00:16:36,950 And NADH is going to reduce this ketone to an alcohol. 277 00:16:36,950 --> 00:16:39,225 And it itself becomes oxidized. 278 00:16:45,960 --> 00:16:50,090 NADH loses a hydron and a proton to form NAD, 279 00:16:50,090 --> 00:16:57,200 and you generate lactic acid where 280 00:16:57,200 --> 00:17:01,110 this hydrogen from the NAD as a hydride 281 00:17:01,110 --> 00:17:06,800 is going to get transferred to the carbon of the carbonyl. 282 00:17:06,800 --> 00:17:11,560 And this proton in some way comes from solution, 283 00:17:11,560 --> 00:17:15,550 and has picked up a proton from the active site of the enzyme. 284 00:17:15,550 --> 00:17:18,930 Now another point I wanted to make, 285 00:17:18,930 --> 00:17:21,770 which you will encounter again over the course of thinking 286 00:17:21,770 --> 00:17:26,130 about coupling reactions in metabolism 287 00:17:26,130 --> 00:17:31,860 to make them favorable, is that with NAD/NADH cofactor, 288 00:17:31,860 --> 00:17:34,500 you always have to think about the proton. 289 00:17:34,500 --> 00:17:37,650 So you need a proton to have a balanced equation. 290 00:17:37,650 --> 00:17:43,120 That means that if you're at pH 6 versus pH 8, 291 00:17:43,120 --> 00:17:46,630 the concentration of the species changes dramatically. 292 00:17:46,630 --> 00:17:48,510 And that can affect where the equilibrium 293 00:17:48,510 --> 00:17:50,550 of the overall reaction lies. 294 00:17:50,550 --> 00:17:53,800 So you need to keep in the back of your mind there's 295 00:17:53,800 --> 00:17:58,210 a proton with NAD/NADH systems. 296 00:17:58,210 --> 00:18:02,050 And so this is NAD, but it turns out 297 00:18:02,050 --> 00:18:08,620 that flavins can also catalyze the same type of reaction. 298 00:18:08,620 --> 00:18:13,960 So in the case of this particular enzyme, 299 00:18:13,960 --> 00:18:15,570 it's found in eukaryotic systems, 300 00:18:15,570 --> 00:18:18,200 it's found also in prokaryotic systems. 301 00:18:18,200 --> 00:18:20,410 But in the case of the flavin dependent enzymes, 302 00:18:20,410 --> 00:18:23,130 it's only found in bacterial systems. 303 00:18:23,130 --> 00:18:26,742 And so their reaction goes more favorably in the direction 304 00:18:26,742 --> 00:18:28,200 I'm writing it now, but again, it's 305 00:18:28,200 --> 00:18:32,490 an equilibrium reaction where you take lactate-- so this 306 00:18:32,490 --> 00:18:35,050 is now the reduced state. 307 00:18:35,050 --> 00:18:38,470 And so you now have FMN. 308 00:18:41,010 --> 00:18:43,730 So this is the form of the cofactor that's 309 00:18:43,730 --> 00:18:45,800 used with this specific enzyme, but the chemistry 310 00:18:45,800 --> 00:18:49,170 is same with FMN and FAD. 311 00:18:49,170 --> 00:18:50,820 And so this needs to get oxidized. 312 00:18:50,820 --> 00:18:56,050 And it gets oxidized to form pyruvate. 313 00:18:56,050 --> 00:19:02,040 And the FMN gets reduced to form FMNH2. 314 00:19:02,040 --> 00:19:05,940 So both of these are capable of catalyzing the same redox 315 00:19:05,940 --> 00:19:06,540 chemistry. 316 00:19:06,540 --> 00:19:09,050 And this is consistent with what I've 317 00:19:09,050 --> 00:19:11,300 told you before about the redox potential. 318 00:19:11,300 --> 00:19:15,100 So NAD/NADH is always in this region. 319 00:19:15,100 --> 00:19:19,010 But you can see that FAD/FADH2 also 320 00:19:19,010 --> 00:19:20,940 covers that region as well. 321 00:19:20,940 --> 00:19:25,130 So many of the chemistries between these two cofactors 322 00:19:25,130 --> 00:19:26,900 can be overlapping. 323 00:19:26,900 --> 00:19:32,270 And you'll see this in for example, fatty acid oxidation 324 00:19:32,270 --> 00:19:34,470 and fatty acid biosynthesis. 325 00:19:34,470 --> 00:19:38,840 And it really is not clear at all which one of the redox 326 00:19:38,840 --> 00:19:40,080 chemistry you could use. 327 00:19:40,080 --> 00:19:43,910 Nature, in fact could use different cofactors 328 00:19:43,910 --> 00:19:45,420 in different organisms. 329 00:19:45,420 --> 00:19:48,350 But we know what the cofactors are in human systems 330 00:19:48,350 --> 00:19:50,360 because we purified all the enzymes 331 00:19:50,360 --> 00:19:53,470 and study the cofactor requirements. 332 00:19:53,470 --> 00:19:57,020 So this is the reaction I want to focus on. 333 00:19:57,020 --> 00:19:59,470 And what I forgot to tell you to maybe make 334 00:19:59,470 --> 00:20:01,980 this more relevant to some of you 335 00:20:01,980 --> 00:20:04,310 I guess, since this is what you do when you go 336 00:20:04,310 --> 00:20:09,750 to college nowadays, is most of you have heard about yeast, 337 00:20:09,750 --> 00:20:12,850 that under anaerobic conditions make you a friend, 338 00:20:12,850 --> 00:20:17,100 to make you happy and cheery when 339 00:20:17,100 --> 00:20:19,600 you're having tough times at MIT, 340 00:20:19,600 --> 00:20:21,110 you want to drink some ethanol. 341 00:20:21,110 --> 00:20:23,420 Yeast are able to convert glucose 342 00:20:23,420 --> 00:20:26,810 all the way down under anaerobic conditions into ethanol, 343 00:20:26,810 --> 00:20:31,470 and in fact uses the cofactor NADH to in two steps, 344 00:20:31,470 --> 00:20:35,270 convert pyruvate into a molecule called acid aldehyde. 345 00:20:35,270 --> 00:20:38,490 And then the NADH converts the acid aldehyde 346 00:20:38,490 --> 00:20:44,740 into ethanol, which is the major ingredient of beer and wine. 347 00:20:44,740 --> 00:20:47,890 So what I want to do now is very briefly then 348 00:20:47,890 --> 00:20:54,190 focus on the chemistry of how NADH actually works. 349 00:20:54,190 --> 00:20:56,820 So if we're now going to look at the chemistry of this 350 00:20:56,820 --> 00:21:02,560 and the mechanism, let's start with pyruvate. 351 00:21:02,560 --> 00:21:04,190 And you need to go back and you need 352 00:21:04,190 --> 00:21:06,030 to think about the carbonyl chemistry you've 353 00:21:06,030 --> 00:21:08,930 been introduced to when talking about the claisen reaction 354 00:21:08,930 --> 00:21:11,070 and the aldol reaction. 355 00:21:11,070 --> 00:21:14,560 And we spent a lot of time on carbonyl chemistry 356 00:21:14,560 --> 00:21:18,930 because it's central to everything in biochemistry. 357 00:21:18,930 --> 00:21:23,000 And so you remember that a carbonyl 358 00:21:23,000 --> 00:21:26,730 is polarized where the electrons like to result on the oxygen, 359 00:21:26,730 --> 00:21:29,750 leaving a deficiency of electrons 360 00:21:29,750 --> 00:21:32,050 on the carbon of the carbonyl. 361 00:21:32,050 --> 00:21:35,130 And so one can draw a resonant structure. 362 00:21:35,130 --> 00:21:36,950 This is the predominate structure, 363 00:21:36,950 --> 00:21:41,390 but these resonance structures often tell you something 364 00:21:41,390 --> 00:21:46,640 about where the molecule is most reactive. 365 00:21:46,640 --> 00:21:50,790 And so what we've done is oxygen is electro negative 366 00:21:50,790 --> 00:21:54,400 and likes to can accommodate electrons in its orbitals. 367 00:21:54,400 --> 00:21:56,210 So it forms a resonance structure 368 00:21:56,210 --> 00:21:58,660 like this leaving behind electron deficiency 369 00:21:58,660 --> 00:22:00,040 at the carbon. 370 00:22:00,040 --> 00:22:05,240 And so we want to reduce this carbonyl to an alcohol. 371 00:22:05,240 --> 00:22:13,020 And this is done with the reduced form of the cofactor. 372 00:22:13,020 --> 00:22:15,830 So again, in every redox chemistry, 373 00:22:15,830 --> 00:22:19,880 this is the oxidized form, this is the reduced form. 374 00:22:19,880 --> 00:22:22,670 And so now the question is, how does this chemistry happen? 375 00:22:22,670 --> 00:22:26,250 So let me point out here-- and this 376 00:22:26,250 --> 00:22:28,540 is something you will hear about over and over again 377 00:22:28,540 --> 00:22:34,270 in basic metabolism-- but these two hydrogens-- and this 378 00:22:34,270 --> 00:22:38,430 is the 1, 2, 3, 4 position of the nicotinamide ring are not 379 00:22:38,430 --> 00:22:39,010 equivalent. 380 00:22:39,010 --> 00:22:41,020 And why aren't they equivalent? 381 00:22:41,020 --> 00:22:43,530 They're not equivalent because all of the chemistry 382 00:22:43,530 --> 00:22:48,900 happens in the active site of an enzyme that is chiral. 383 00:22:48,900 --> 00:22:51,460 So these hydrogens are prochiral. 384 00:22:51,460 --> 00:22:54,530 And because they sit in a chiral environment, 385 00:22:54,530 --> 00:22:57,510 that makes these hydrogens act like they're chiral. 386 00:22:57,510 --> 00:23:01,560 So all chemistry with NADH is stereo specific. 387 00:23:01,560 --> 00:23:05,430 Depending on the active site, only one of these two protons 388 00:23:05,430 --> 00:23:09,190 is involved in hydride transfer. 389 00:23:09,190 --> 00:23:11,540 So if you look at the chemistry now, 390 00:23:11,540 --> 00:23:15,750 we're set up to deliver a proton with a pair of electrons, 391 00:23:15,750 --> 00:23:21,230 that's a hydride, to this carbon of the carbonyl. 392 00:23:21,230 --> 00:23:25,330 See we have a hydride transfer. 393 00:23:25,330 --> 00:23:31,000 And now we have the electron density sitting on the oxygen. 394 00:23:31,000 --> 00:23:34,260 And so likely then in the active site of the enzyme 395 00:23:34,260 --> 00:23:36,890 you have a general acid catalyst which is 396 00:23:36,890 --> 00:23:39,360 able to then donate a proton. 397 00:23:39,360 --> 00:23:42,230 And remember I told you that NAD/NADH 398 00:23:42,230 --> 00:23:46,100 involves hydride transfer and proton transfer. 399 00:23:46,100 --> 00:23:51,910 So what you generate then is OH, where 400 00:23:51,910 --> 00:23:55,260 this hydrogen comes from a general acid catalyst 401 00:23:55,260 --> 00:23:57,670 in the active site. 402 00:23:57,670 --> 00:24:04,260 And you have the hydride transfer coming from the NADH 403 00:24:04,260 --> 00:24:06,610 to generate lactic acid. 404 00:24:06,610 --> 00:24:14,750 And now what you've generated is the oxidized form of the NAD. 405 00:24:14,750 --> 00:24:21,060 So reduced form oxidized, reduced form oxidized. 406 00:24:21,060 --> 00:24:26,040 So this is the general reaction and all NAD/NADH enzymes 407 00:24:26,040 --> 00:24:30,890 involves this simple type of [INAUDIBLE], protons, hydrides, 408 00:24:30,890 --> 00:24:32,650 and protons. 409 00:24:32,650 --> 00:24:35,940 Now what I want to do is show you 410 00:24:35,940 --> 00:24:37,960 one of the many types of reactions 411 00:24:37,960 --> 00:24:39,820 that flavins can undergo. 412 00:24:39,820 --> 00:24:44,080 And again we're focusing on hydride transfer reactions. 413 00:24:44,080 --> 00:24:49,440 And a recall for you that again, the redox 414 00:24:49,440 --> 00:24:54,320 potentials of the flavins in the NAD/NADH overlap. 415 00:24:54,320 --> 00:24:56,430 So this is always minus 320. 416 00:24:56,430 --> 00:24:59,790 But this has a span of redox potentials. 417 00:24:59,790 --> 00:25:01,330 And in fact, what you will see when 418 00:25:01,330 --> 00:25:11,330 you look at basic metabolism is often FAD is reduced by NADH. 419 00:25:11,330 --> 00:25:15,390 And so these two partner up to do chemistry. 420 00:25:15,390 --> 00:25:18,370 And so what I want to do now is show you the chemistry 421 00:25:18,370 --> 00:25:22,260 with the flavin and NADH. 422 00:25:22,260 --> 00:25:35,590 If we go back and we look at our oxidized form of the flavin, 423 00:25:35,590 --> 00:25:38,790 and again, I'm not going to draw out all the structure. 424 00:25:38,790 --> 00:25:41,050 And remember there's a lot of spinach here 425 00:25:41,050 --> 00:25:43,210 and there's also some spinach here. 426 00:25:43,210 --> 00:25:45,770 This is the business end of the molecule. 427 00:25:45,770 --> 00:25:49,220 And remember I told you the business end of the molecule 428 00:25:49,220 --> 00:25:52,850 is the N5 and the C4A position. 429 00:25:52,850 --> 00:25:55,940 And so now we have our NAD. 430 00:25:59,770 --> 00:26:01,170 NADH, sorry. 431 00:26:01,170 --> 00:26:05,850 So this is the oxidized form, this is the reduced form. 432 00:26:05,850 --> 00:26:10,640 And so NAD/NADH goes through a hydride proton transfers, 433 00:26:10,640 --> 00:26:12,406 does it stereo specifically. 434 00:26:12,406 --> 00:26:14,280 And the question is, where does the chemistry 435 00:26:14,280 --> 00:26:16,510 happen with the flavin? 436 00:26:16,510 --> 00:26:20,950 And the chemistry happens at stereo specifically. 437 00:26:20,950 --> 00:26:22,610 So each enzyme will be different. 438 00:26:22,610 --> 00:26:24,580 You can figure out what the stereo chemistry 439 00:26:24,580 --> 00:26:28,070 is at the N5 position. 440 00:26:28,070 --> 00:26:30,510 So here is again, the N5 position. 441 00:26:30,510 --> 00:26:36,530 So you have hydride transfer to the N5 position. 442 00:26:36,530 --> 00:26:41,130 And now you can pick up a proton or not at this position. 443 00:26:41,130 --> 00:26:42,440 I'm going to show you the PKA. 444 00:26:42,440 --> 00:26:45,200 If you go back and you look at the handout in the lexicon, 445 00:26:45,200 --> 00:26:47,010 you'll see the PKA of this nitrogen 446 00:26:47,010 --> 00:26:50,080 is under physiological conditions, about 7. 447 00:26:50,080 --> 00:26:53,370 So it can be proteinated or not proteinated. 448 00:26:53,370 --> 00:27:04,760 And so what then happens is you end up with the reduced form 449 00:27:04,760 --> 00:27:11,140 where this hydrogen is transferred to the N5 position, 450 00:27:11,140 --> 00:27:15,210 and you could pick up a hydrogen here, from AH leaving you 451 00:27:15,210 --> 00:27:22,910 with A. So this proton, this may or may not 452 00:27:22,910 --> 00:27:28,510 be proteinated depending on the active site of the enzyme. 453 00:27:28,510 --> 00:27:30,760 And so then what you also have here 454 00:27:30,760 --> 00:27:35,650 is the oxidized form of NADH. 455 00:27:35,650 --> 00:27:37,930 Now those of you who were paying attention 456 00:27:37,930 --> 00:27:42,810 to what I was just saying with the reduction of pyruvate 457 00:27:42,810 --> 00:27:47,690 to lactate, I just went through this whole big spiel 458 00:27:47,690 --> 00:27:51,680 about how you wanted to take advantage of the fact 459 00:27:51,680 --> 00:27:54,700 that carbonyl is a polarized delta plus delta minus, 460 00:27:54,700 --> 00:27:57,110 and that the nucleophile, this case the hydride, 461 00:27:57,110 --> 00:28:01,290 is always being transferred to the electron deficient carbon, 462 00:28:01,290 --> 00:28:04,610 not to the oxygen that already has a lot of electron density. 463 00:28:04,610 --> 00:28:06,940 So you might be asking yourself well, 464 00:28:06,940 --> 00:28:13,320 why doesn't that hydride get transferred to this position? 465 00:28:13,320 --> 00:28:16,530 This is a position, if you look at this, 466 00:28:16,530 --> 00:28:20,740 and you take it out of context of the isoalloxazine ring. 467 00:28:20,740 --> 00:28:23,870 This looks remarkably similar to this carbonyl. 468 00:28:23,870 --> 00:28:28,702 And in fact, this is where your chemical intuition falls apart. 469 00:28:28,702 --> 00:28:30,160 And you need to know that you don't 470 00:28:30,160 --> 00:28:32,200 have chemical intuition about those people that 471 00:28:32,200 --> 00:28:36,500 spent 40 years studying isoalloxazine rings. 472 00:28:36,500 --> 00:28:40,100 And this is a complex heterocyclical molecule 473 00:28:40,100 --> 00:28:41,850 where I think most chemists, when 474 00:28:41,850 --> 00:28:43,310 looking at this for the first time, 475 00:28:43,310 --> 00:28:47,470 had no clue as to what the reactive positions were 476 00:28:47,470 --> 00:28:48,860 and what the chemistry was. 477 00:28:48,860 --> 00:28:52,680 And they spent, in the 1970s and 1980s, 478 00:28:52,680 --> 00:28:56,680 spent a decade figuring out the chemistry of flavins. 479 00:28:56,680 --> 00:29:00,360 And so I'm telling you what the answer is. 480 00:29:00,360 --> 00:29:02,920 There is experimental evidence that supports this 481 00:29:02,920 --> 00:29:05,460 that in the case of NADH, hydride 482 00:29:05,460 --> 00:29:08,690 is transferred to the N5 position 483 00:29:08,690 --> 00:29:15,360 to give you the reduced form and the oxidized form of NAD. 484 00:29:15,360 --> 00:29:18,600 So I hope I've gotten you excited 485 00:29:18,600 --> 00:29:22,440 interested about the two redox active cofactors that you're 486 00:29:22,440 --> 00:29:25,930 going to encounter over and over again during your journey 487 00:29:25,930 --> 00:29:30,120 through primary metabolic pathways, FAD and NAD. 488 00:29:30,120 --> 00:29:33,920 And I hope I've taught you something 489 00:29:33,920 --> 00:29:38,420 about the chemistry of how these two cofactors work 490 00:29:38,420 --> 00:29:40,420 and you'll get plenty of practice 491 00:29:40,420 --> 00:29:43,850 with these cofactors over the rest of the semester.