1 00:00:00,500 --> 00:00:02,790 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 --> 00:00:17,560 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,560 --> 00:00:20,025 at ocw.mit.edu. 8 00:00:20,025 --> 00:00:20,775 JOANNE STUBBE: Hi. 9 00:00:20,775 --> 00:00:25,190 Today, what we're going to do is focus on yet another cofactor 10 00:00:25,190 --> 00:00:27,050 that you get out of your vitamin bottle. 11 00:00:27,050 --> 00:00:32,189 And today we're going to be looking at vitamin B6. 12 00:00:32,189 --> 00:00:38,240 And vitamin B6 is the cofactor that you use whenever you 13 00:00:38,240 --> 00:00:41,340 want to metabolize amino acids. 14 00:00:41,340 --> 00:00:43,400 Where do you get amino acids? 15 00:00:43,400 --> 00:00:44,750 We all eat proteins. 16 00:00:44,750 --> 00:00:48,170 The proteins get degraded to amino acids. 17 00:00:48,170 --> 00:00:51,750 And we can use the energy released. 18 00:00:51,750 --> 00:00:54,380 And trap that energy to do bio synthesis. 19 00:00:54,380 --> 00:00:58,480 And we use amino acids, convert them into central metabolism, 20 00:00:58,480 --> 00:01:02,030 and use them to make fats, or sugars, depending 21 00:01:02,030 --> 00:01:05,990 on what the environment is telling us we need to do. 22 00:01:05,990 --> 00:01:09,400 So let me introduce you to this cofactor, 23 00:01:09,400 --> 00:01:16,200 the vitamin itself, as you have seen now, over and over again, 24 00:01:16,200 --> 00:01:18,840 is not the actual cofactor. 25 00:01:18,840 --> 00:01:20,410 It's Pyridoxine. 26 00:01:20,410 --> 00:01:22,700 And so that's what you eat out of the vitamin bottle. 27 00:01:22,700 --> 00:01:25,690 And as in the case of all vitamins, inside 28 00:01:25,690 --> 00:01:30,110 the cell it has to be converted into the active form 29 00:01:30,110 --> 00:01:32,270 of the cofactor. 30 00:01:32,270 --> 00:01:34,450 And the cofactor we're going to be talking about, 31 00:01:34,450 --> 00:01:40,580 the active form is Pyridoxal phosphate, which is called PLP. 32 00:01:40,580 --> 00:01:44,560 And the structure of pyridoxal-- there two main structures 33 00:01:44,560 --> 00:01:48,510 of the pyridoxal phosphate cofactor, one of them 34 00:01:48,510 --> 00:01:52,590 is involved in 80% of all the chemistry. 35 00:01:52,590 --> 00:01:54,500 And the one we're going to talk about today, 36 00:01:54,500 --> 00:01:58,360 uses both forms of the cofactor. 37 00:01:58,360 --> 00:02:00,110 That fits into central metabolism. 38 00:02:00,110 --> 00:02:02,290 I'll show you how that fits in a minute. 39 00:02:02,290 --> 00:02:05,260 So pyridoxal phosphate has this structure. 40 00:02:05,260 --> 00:02:09,680 You have a pyridine ring, the pKa. 41 00:02:09,680 --> 00:02:14,290 The pyridine ring is 6 5, so it may or may not be protonated. 42 00:02:14,290 --> 00:02:16,530 And the key part of the cofactor-- one 43 00:02:16,530 --> 00:02:19,240 of the key parts of the cofactor, is this aldehyde. 44 00:02:19,240 --> 00:02:22,770 So pyridoxal means that this is an aldehyde. 45 00:02:22,770 --> 00:02:28,050 The second form of the cofactor is pyridoxamine. 46 00:02:28,050 --> 00:02:31,040 So this aldehyde is somehow-- and we 47 00:02:31,040 --> 00:02:32,900 will look at how this happens-- is 48 00:02:32,900 --> 00:02:37,650 converted from this aldehyde, into an amino group. 49 00:02:37,650 --> 00:02:42,700 And this is called PMP, or pyridoxamine phosphate. 50 00:02:42,700 --> 00:02:45,140 Now the other thing I wanted to show you, 51 00:02:45,140 --> 00:02:46,890 before we move on to look at more 52 00:02:46,890 --> 00:02:50,840 details of pyridoxal phosphate, is-- while this 53 00:02:50,840 --> 00:02:53,360 is the form of the vitamin. 54 00:02:53,360 --> 00:02:57,090 You almost never see this form inside the cell. 55 00:02:57,090 --> 00:03:02,270 It is always bound in the active site of the enzyme. 56 00:03:02,270 --> 00:03:05,290 And it binds in the active site of the enzyme-- 57 00:03:05,290 --> 00:03:09,250 so here's your enzyme-- through the epsilon 58 00:03:09,250 --> 00:03:11,470 amino group of a lysine. 59 00:03:11,470 --> 00:03:15,450 So you never really, when you purify the protein, 60 00:03:15,450 --> 00:03:19,680 you never isolate the aldehyde. 61 00:03:19,680 --> 00:03:21,000 So this is the aldehyde. 62 00:03:21,000 --> 00:03:24,080 And I won't draw out all the rest of the structure, 63 00:03:24,080 --> 00:03:27,420 but what you have is an amino group, 64 00:03:27,420 --> 00:03:32,390 that's attached to a lysine, in the active site of your enzyme. 65 00:03:32,390 --> 00:03:35,370 And what we're going to do is convert this ketone 66 00:03:35,370 --> 00:03:36,600 into an imine. 67 00:03:36,600 --> 00:03:40,010 So this is chemistry you see over and over and over again. 68 00:03:40,010 --> 00:03:42,140 You've seen it already with carbonyl chemistry, 69 00:03:42,140 --> 00:03:45,100 with carbon-carbon bond forming reactions, 70 00:03:45,100 --> 00:03:48,370 with peptide bond hydrolysis, that we've already 71 00:03:48,370 --> 00:03:49,740 talked about. 72 00:03:49,740 --> 00:03:54,140 But what you do is, you're going to convert this aldehyde 73 00:03:54,140 --> 00:03:55,240 into an imine. 74 00:03:55,240 --> 00:03:57,380 You need to go through a tetrahedral intermediate. 75 00:03:57,380 --> 00:04:01,650 And the carbonyl is polarized, delta plus, delta minus. 76 00:04:01,650 --> 00:04:03,330 You spend a lot of time practicing 77 00:04:03,330 --> 00:04:10,430 this kind of chemistry to form a tetrahedral intermediate. 78 00:04:10,430 --> 00:04:12,630 And I'm going to have a proton transfer. 79 00:04:12,630 --> 00:04:17,850 So one of the things that's tricky about this chemistry 80 00:04:17,850 --> 00:04:20,630 is that, if you count the number of protons 81 00:04:20,630 --> 00:04:23,600 they move around a lot in the active site. 82 00:04:23,600 --> 00:04:27,300 And the fact is, that you want to protonate something 83 00:04:27,300 --> 00:04:29,250 to make it into a better leaving group, 84 00:04:29,250 --> 00:04:31,050 you want to deprotonate something 85 00:04:31,050 --> 00:04:33,760 to make it function more like a nucleophile, 86 00:04:33,760 --> 00:04:37,080 or as a base, and nature has figured out 87 00:04:37,080 --> 00:04:40,210 how to orchestrate all the residues in the active site 88 00:04:40,210 --> 00:04:41,020 to do this. 89 00:04:41,020 --> 00:04:44,830 In most cases, we don't understand all of the details. 90 00:04:44,830 --> 00:04:47,020 And so I'm not going to focus on proton transfer, 91 00:04:47,020 --> 00:04:49,860 but you're going to see many proton transfers 92 00:04:49,860 --> 00:04:51,600 within the active site. 93 00:04:51,600 --> 00:04:55,820 So this gives us a tetrahedral intermediate, or transition 94 00:04:55,820 --> 00:04:56,320 stage. 95 00:04:56,320 --> 00:04:58,020 You've seen that over and over again. 96 00:04:58,020 --> 00:05:00,320 And now what will happen is that you want 97 00:05:00,320 --> 00:05:04,710 to lose a molecule of water. 98 00:05:04,710 --> 00:05:09,480 So again, you need to pronate that, and what you form then 99 00:05:09,480 --> 00:05:15,290 is a new carbon doubly bonded to a nitrogen, 100 00:05:15,290 --> 00:05:17,830 rather than doubly bonded to the oxygen, 101 00:05:17,830 --> 00:05:20,040 that's covalently bound to the enzyme. 102 00:05:20,040 --> 00:05:24,150 And so, this is called an imine. 103 00:05:24,150 --> 00:05:27,100 And because it's an imine of an aldehyde, 104 00:05:27,100 --> 00:05:30,970 it's called an aldimine. 105 00:05:30,970 --> 00:05:35,140 So whenever you isolate your enzyme, whenever 106 00:05:35,140 --> 00:05:36,990 you isolate your enzyme, the pyridoxal 107 00:05:36,990 --> 00:05:39,080 is always covalently bound. 108 00:05:39,080 --> 00:05:44,000 OK, so, this bond is chemically easy to hydrolyze, 109 00:05:44,000 --> 00:05:45,780 but it's always covalently bound. 110 00:05:45,780 --> 00:05:46,540 OK. 111 00:05:46,540 --> 00:05:52,520 So what I want to do now, is make a few generalizations 112 00:05:52,520 --> 00:05:57,560 about pyridoxal, but the first thing is, that in all cases, 113 00:05:57,560 --> 00:06:02,580 whenever you metabolize-- I want to metabolize an amino acid. 114 00:06:02,580 --> 00:06:05,670 Pyridoxal phosphate requiring enzyme 115 00:06:05,670 --> 00:06:08,860 is going to be the key player. 116 00:06:08,860 --> 00:06:09,480 OK. 117 00:06:09,480 --> 00:06:11,050 So here's an amino acid. 118 00:06:11,050 --> 00:06:14,870 OK, here's the alpha position, the beta position, 119 00:06:14,870 --> 00:06:16,360 and the gamma position. 120 00:06:16,360 --> 00:06:20,740 Well what's so amazing-- I remember first hearing 121 00:06:20,740 --> 00:06:23,780 about this-- is that you can do chemistry 122 00:06:23,780 --> 00:06:25,690 at all these positions. 123 00:06:25,690 --> 00:06:27,650 And the way nature figures out how 124 00:06:27,650 --> 00:06:30,500 to do this is by orchestrating the active site, 125 00:06:30,500 --> 00:06:35,800 with acid based catalyst sitting around in the right place, 126 00:06:35,800 --> 00:06:38,650 to allow you to do the chemical transformation 127 00:06:38,650 --> 00:06:41,730 that this protein has evolved to do. 128 00:06:41,730 --> 00:06:44,100 So let me to show you what the alpha position, 129 00:06:44,100 --> 00:06:48,760 so this is the alpha position, you can cleave this bond. 130 00:06:48,760 --> 00:06:51,000 I'll show you how this happens. 131 00:06:51,000 --> 00:06:55,290 You can do all this chemistry with a few simple chemical 132 00:06:55,290 --> 00:06:59,010 transformations, takes practice, but once you sort of get 133 00:06:59,010 --> 00:07:01,980 what these transformations are, it's 134 00:07:01,980 --> 00:07:05,320 amazing what you can get this cofactor to help you do. 135 00:07:05,320 --> 00:07:07,030 And I'll explain that in a minute. 136 00:07:07,030 --> 00:07:09,150 So you get cleavage of the carbon carbon bond 137 00:07:09,150 --> 00:07:13,000 that's loss of CO2 that's a decarboxylation reaction. 138 00:07:13,000 --> 00:07:18,240 We don't talk about that in 507, but that kind of reaction 139 00:07:18,240 --> 00:07:21,710 generates all of our neurotransmitters. 140 00:07:21,710 --> 00:07:25,070 You're going to cleave this carbon-hydrogen bond. 141 00:07:25,070 --> 00:07:28,160 Remember, amino acids are in the S configuration-- 142 00:07:28,160 --> 00:07:30,160 but for example in cell wall, in bacteria, 143 00:07:30,160 --> 00:07:33,460 they can be either the S or the R configuration, 144 00:07:33,460 --> 00:07:36,460 so you can cleave that bond, and put the proton back 145 00:07:36,460 --> 00:07:39,650 on the other face, that's a racemization reaction. 146 00:07:39,650 --> 00:07:41,920 You can cleave this carbon-carbon bond 147 00:07:41,920 --> 00:07:44,134 between the alpha and the beta position, 148 00:07:44,134 --> 00:07:46,800 that's a reaction-- remember, we talked about carbon-carbon bond 149 00:07:46,800 --> 00:07:50,920 formation-- this is the reverse aldol reaction. 150 00:07:50,920 --> 00:07:53,480 And the one where we're going to focus on today, which 151 00:07:53,480 --> 00:07:57,690 is the one that fits best into central metabolism, 152 00:07:57,690 --> 00:08:02,160 is what happens to this carbon-nitrogen bond. 153 00:08:02,160 --> 00:08:08,800 So we have an amino group of our amino acid 154 00:08:08,800 --> 00:08:14,600 is going to get converted into a ketone group. 155 00:08:14,600 --> 00:08:18,890 So, this group, is going to get converted into this group. 156 00:08:18,890 --> 00:08:24,465 And to do that, we're going to use the imine of pyrodoxial 157 00:08:24,465 --> 00:08:27,940 phosphate that we have in the active site of the enzyme. 158 00:08:27,940 --> 00:08:30,180 So this is sort of like a carbonyl 159 00:08:30,180 --> 00:08:32,620 and that's going to get converted 160 00:08:32,620 --> 00:08:37,350 into the amine, the pyridoxamine. 161 00:08:37,350 --> 00:08:40,390 So these are the two forms of the cofactor. 162 00:08:40,390 --> 00:08:42,179 So what are we going to be focusing on, is 163 00:08:42,179 --> 00:08:46,180 how this reaction, actually, happens, and this 164 00:08:46,180 --> 00:08:49,850 is the most complicated of all the pyridoxal prostate 165 00:08:49,850 --> 00:08:51,330 dependent reactions. 166 00:08:51,330 --> 00:08:54,290 So that I'm going to come back to this in a minute, 167 00:08:54,290 --> 00:08:59,010 but what I want to do is make a few generalizations 168 00:08:59,010 --> 00:09:03,170 about where you're going to see pyridoxal phosphate chemistry 169 00:09:03,170 --> 00:09:05,240 in primary metabolism. 170 00:09:05,240 --> 00:09:10,660 So what we're going to see is that the TCA cycle, 171 00:09:10,660 --> 00:09:12,980 tricarboxylic acid cycle, or the Krebs cycle, 172 00:09:12,980 --> 00:09:13,980 plays a central role. 173 00:09:13,980 --> 00:09:15,750 It's found in the mitochondria. 174 00:09:15,750 --> 00:09:17,660 You're going to see this over and over again, 175 00:09:17,660 --> 00:09:19,310 over the course of the semester. 176 00:09:19,310 --> 00:09:21,350 Things feed in and out of the cycle. 177 00:09:21,350 --> 00:09:23,690 A cycle means it goes around and around, 178 00:09:23,690 --> 00:09:26,640 and if you remove something from the cycle 179 00:09:26,640 --> 00:09:30,700 and don't put anything back into the cycle, the cycle stops. 180 00:09:30,700 --> 00:09:32,540 And you're in serious trouble. 181 00:09:32,540 --> 00:09:36,450 So one way-- one thing-- one way to feed in and out 182 00:09:36,450 --> 00:09:39,680 of this cycle is through amino acids. 183 00:09:39,680 --> 00:09:41,590 So this reaction, which we're going 184 00:09:41,590 --> 00:09:45,130 to call the transemination, or a transamination, 185 00:09:45,130 --> 00:09:47,520 is metabolism of amino acid, we'll 186 00:09:47,520 --> 00:09:49,060 see into an alpha keto acid. 187 00:09:49,060 --> 00:09:51,660 So if you look at the TCA cycle and we 188 00:09:51,660 --> 00:09:54,760 look at this reaction, what you'll see 189 00:09:54,760 --> 00:10:00,250 is you have this compound, called alpha ketoglutarate. 190 00:10:00,250 --> 00:10:03,020 OK, so alpha ketogluterate and that 191 00:10:03,020 --> 00:10:06,520 going to interconvert with the amino acid 192 00:10:06,520 --> 00:10:09,100 and so this ketone is going to be converted 193 00:10:09,100 --> 00:10:13,400 into an amino group, and so we're 194 00:10:13,400 --> 00:10:17,180 having the amino group converted into a ketone group. 195 00:10:17,180 --> 00:10:20,910 And pyridoxial is going to be converted into pyridoxamine. 196 00:10:20,910 --> 00:10:25,150 So we're going to have PLP converted into PMP. 197 00:10:25,150 --> 00:10:27,420 I'm going to show you how that works. 198 00:10:27,420 --> 00:10:31,510 So, if you feed in glutamate from your diet, 199 00:10:31,510 --> 00:10:34,550 it-- by this pyridoxal phosphate dependent reaction-- 200 00:10:34,550 --> 00:10:36,300 can feed into the Krebs cycle. 201 00:10:36,300 --> 00:10:38,900 If you want to make amino acids, on the other hand, 202 00:10:38,900 --> 00:10:41,330 you can suck some of the alpha keto acid 203 00:10:41,330 --> 00:10:43,980 out, and convert it into amino acids. 204 00:10:43,980 --> 00:10:46,400 And you have to have a way of controlling whether you 205 00:10:46,400 --> 00:10:49,690 feed in, or you, actually, remove your metabolites 206 00:10:49,690 --> 00:10:50,880 from the cycle. 207 00:10:50,880 --> 00:10:56,000 If you come up here and look at oxaloacetic and aspartic acid-- 208 00:10:56,000 --> 00:11:05,050 So oxaloacetic acid is also an alpha keto acid, OK. 209 00:11:05,050 --> 00:11:10,970 And the amino acid-- can you see? 210 00:11:10,970 --> 00:11:16,610 I'm probably too close to the edge-- OK, 211 00:11:16,610 --> 00:11:18,530 and so here we have the amino acid. 212 00:11:18,530 --> 00:11:22,640 So here we have amino acid alpha keto acid, amino acid alpha 213 00:11:22,640 --> 00:11:23,850 keto acid. 214 00:11:23,850 --> 00:11:27,290 If you go further up and go pyruvate feeds 215 00:11:27,290 --> 00:11:29,540 into the TCA cycle-- pyruvate comes 216 00:11:29,540 --> 00:11:31,870 from the glycolysis pathway, which 217 00:11:31,870 --> 00:11:39,130 again is breakdown of sugars, pyruvate is an alpha keto acid. 218 00:11:39,130 --> 00:11:42,330 So this is a CH3, so this is the simplest, 219 00:11:42,330 --> 00:11:45,880 and this can get interconverted into alanine. 220 00:11:48,450 --> 00:11:54,170 So what you see is this same theme-- amino group ketone, 221 00:11:54,170 --> 00:11:55,790 amino group ketone. 222 00:11:55,790 --> 00:11:57,330 I'm being sloppy, here. 223 00:11:57,330 --> 00:11:58,980 Most amino groups are protonated, 224 00:11:58,980 --> 00:12:01,580 because the pKa, you should remember, is around 9. 225 00:12:01,580 --> 00:12:03,500 So they're, mostly, protonated in solution. 226 00:12:03,500 --> 00:12:05,760 I should protonate this over here, as well. 227 00:12:05,760 --> 00:12:09,000 And so you have a ketone group and amino group. 228 00:12:09,000 --> 00:12:13,410 So this is called anaplerotic pathways 229 00:12:13,410 --> 00:12:17,950 where things can feed in and feed out of central metabolism. 230 00:12:17,950 --> 00:12:20,610 And this is the only time, during the course of 507, 231 00:12:20,610 --> 00:12:23,410 that you're introduced to amino acids, 232 00:12:23,410 --> 00:12:24,980 and how they're metabolized. 233 00:12:24,980 --> 00:12:26,540 So what I want to do now is then, 234 00:12:26,540 --> 00:12:30,970 briefly, show you how this transformation, actually, 235 00:12:30,970 --> 00:12:31,690 works. 236 00:12:31,690 --> 00:12:32,790 OK. 237 00:12:32,790 --> 00:12:35,180 So I'm going to give you some general rules. 238 00:12:35,180 --> 00:12:40,090 So the transformation I just showed you-- 239 00:12:40,090 --> 00:12:43,430 an amino acid into an alpha keto acid-- looks to be complicated. 240 00:12:43,430 --> 00:12:46,320 And, I told you, the cofactor changes its structure 241 00:12:46,320 --> 00:12:50,610 from an imine into an amino group. 242 00:12:50,610 --> 00:12:52,360 And the question is, how does that happen? 243 00:12:52,360 --> 00:12:56,960 So I want to show you a bunch of simple, basic rules 244 00:12:56,960 --> 00:13:01,000 that allow you to think about all pyridoxal phosphate 245 00:13:01,000 --> 00:13:02,180 requiring chemistry. 246 00:13:02,180 --> 00:13:03,894 So I'm going to write down the rules, 247 00:13:03,894 --> 00:13:05,310 and then I'm going to show you how 248 00:13:05,310 --> 00:13:07,230 it works on the reactions we were just 249 00:13:07,230 --> 00:13:09,430 looking at this these transeminations, 250 00:13:09,430 --> 00:13:11,960 or transamination reactions. 251 00:13:11,960 --> 00:13:17,660 So how do we think about the mechanism of these PLP enzymes? 252 00:13:17,660 --> 00:13:23,960 So the first thing is-- the first step 253 00:13:23,960 --> 00:13:28,920 is-- so you're going to start out with an imine bound 254 00:13:28,920 --> 00:13:33,890 to the active site of your pyridoxal phosphate 255 00:13:33,890 --> 00:13:35,020 and an amino acid. 256 00:13:35,020 --> 00:13:38,592 I'll abbreviate it aa. 257 00:13:38,592 --> 00:13:40,550 And the first thing you do is, you're going to, 258 00:13:40,550 --> 00:13:46,670 remove this imine and form a new imine with the amino acid. 259 00:13:46,670 --> 00:13:50,120 So that's called a transimination reaction, OK. 260 00:13:50,120 --> 00:13:59,420 So what we're going to do is form a new imine. 261 00:13:59,420 --> 00:14:01,964 And so the imine from the amino acid, 262 00:14:01,964 --> 00:14:03,380 and I'll show you how this happen, 263 00:14:03,380 --> 00:14:05,480 is going to switch with this one. 264 00:14:05,480 --> 00:14:09,360 And so what you're left with, in the active site, 265 00:14:09,360 --> 00:14:12,160 is the amino group of the lysine. 266 00:14:12,160 --> 00:14:12,790 OK. 267 00:14:12,790 --> 00:14:15,940 So this lysine-- nature has figured out 268 00:14:15,940 --> 00:14:20,220 how to minimize the numbers of acid and base 269 00:14:20,220 --> 00:14:23,920 groups constrained in the region, the active site, where 270 00:14:23,920 --> 00:14:26,790 all the chemistry happens. 271 00:14:26,790 --> 00:14:32,320 And she uses this lysine, which initially is holding covalently 272 00:14:32,320 --> 00:14:34,080 the cofactor, in the active site. 273 00:14:34,080 --> 00:14:38,850 She then uses this lysine to do general acid, general base 274 00:14:38,850 --> 00:14:39,580 catalysis. 275 00:14:39,580 --> 00:14:42,150 And she uses it over and over and over again. 276 00:14:42,150 --> 00:14:45,840 Now every pyridoxal enzyme is distinct 277 00:14:45,840 --> 00:14:50,230 and has additional groups in the active site. 278 00:14:50,230 --> 00:14:52,095 But we know a lot about this chemistry. 279 00:14:52,095 --> 00:14:53,470 We even know what the groups are, 280 00:14:53,470 --> 00:14:56,070 but we're going to look and talk about generalizations. 281 00:14:56,070 --> 00:14:57,610 So the first thing, we have to do 282 00:14:57,610 --> 00:15:00,960 is, we need to free up our general acid base 283 00:15:00,960 --> 00:15:05,150 catalyst, lysine, and we need to covalently bind 284 00:15:05,150 --> 00:15:08,380 the amino acid, which is a substrate, into pyridoxal. 285 00:15:08,380 --> 00:15:09,902 So that's the first step. 286 00:15:09,902 --> 00:15:11,860 And I'm going to come back to this in a minute. 287 00:15:11,860 --> 00:15:14,190 The second step in all of these reactions 288 00:15:14,190 --> 00:15:20,990 is-- all amino acids have an alpha hydrogen, 289 00:15:20,990 --> 00:15:24,660 that alpha hydrogen has a very high pKa. 290 00:15:24,660 --> 00:15:28,190 It's very hard for a normal amino acid side chain, 291 00:15:28,190 --> 00:15:30,420 in the active site, to remove that proton, 292 00:15:30,420 --> 00:15:32,770 because it's not acidic enough. 293 00:15:32,770 --> 00:15:35,490 So what I'm going to show you is pyridoxal 294 00:15:35,490 --> 00:15:38,580 increases the acidity of that alpha hydrogen, 295 00:15:38,580 --> 00:15:41,170 making it easier to do the chemistry. 296 00:15:41,170 --> 00:15:44,640 So the second step in almost all pyridoxal reactions, 297 00:15:44,640 --> 00:15:50,750 is removal of the alpha hydrogen. 298 00:15:50,750 --> 00:15:57,350 And we can do that, because PLP makes the hydrogen more acidic. 299 00:15:57,350 --> 00:16:00,920 And I'll show you why that's true in a minute. 300 00:16:00,920 --> 00:16:04,540 And then, the third thing is, once you remove that hydrogen, 301 00:16:04,540 --> 00:16:07,480 then you get a look at the chemistry you want to catalyze. 302 00:16:07,480 --> 00:16:10,350 And we're going to be talking about this transimination 303 00:16:10,350 --> 00:16:11,950 transamination reactions. 304 00:16:11,950 --> 00:16:14,640 But remember I told-- you can do all this chemistry at alpha, 305 00:16:14,640 --> 00:16:15,870 beta, gamma. 306 00:16:15,870 --> 00:16:19,340 So you need to assess what the substrate is, 307 00:16:19,340 --> 00:16:22,380 and what the product is, and then, within the active site, 308 00:16:22,380 --> 00:16:25,090 you're going to have to do a lot of manipulation 309 00:16:25,090 --> 00:16:27,810 with acid and base catalysts to get you 310 00:16:27,810 --> 00:16:31,970 into the final stage, where you can release the product you 311 00:16:31,970 --> 00:16:33,040 want to release. 312 00:16:33,040 --> 00:16:35,650 So the last step in this reaction-- 313 00:16:35,650 --> 00:16:39,940 in all pyridoxal reactions-- so we do some chemistry in here. 314 00:16:39,940 --> 00:16:41,760 And I'll show you what the chemistry is 315 00:16:41,760 --> 00:16:47,240 with transamination reactions. 316 00:16:47,240 --> 00:16:50,580 The word transimination reactions-- now 317 00:16:50,580 --> 00:16:55,500 I keep saying transamination and transimination. 318 00:16:55,500 --> 00:16:58,300 That's because most textbooks call it transamination, 319 00:16:58,300 --> 00:17:01,050 because they think about pyridoxal as the aldehyde. 320 00:17:01,050 --> 00:17:04,140 But, in reality, pyridoxal is always in the imine, 321 00:17:04,140 --> 00:17:05,760 covalently bound. 322 00:17:05,760 --> 00:17:09,079 And so that's why it's that transimination, rather than 323 00:17:09,079 --> 00:17:10,960 a transamination. 324 00:17:10,960 --> 00:17:15,950 So the last step then, is, hydrolysis. 325 00:17:15,950 --> 00:17:22,450 And I'll show you how that happens, or transimination 326 00:17:22,450 --> 00:17:27,710 to reform this structure. 327 00:17:27,710 --> 00:17:30,410 So we get ourselves back to where we started. 328 00:17:30,410 --> 00:17:33,160 So there's three simple steps, and in the middle, 329 00:17:33,160 --> 00:17:35,170 depending on what the reaction is, you have 330 00:17:35,170 --> 00:17:37,250 to do additional manipulations. 331 00:17:37,250 --> 00:17:41,010 But all pyridoxal enzymes go through these three, 332 00:17:41,010 --> 00:17:42,260 general steps. 333 00:17:42,260 --> 00:17:44,730 The first step, I told you, in all these reactions 334 00:17:44,730 --> 00:17:46,730 is transimination. 335 00:17:46,730 --> 00:17:47,260 OK. 336 00:17:47,260 --> 00:17:53,080 So here's our Schiff-base, and it's your pyridoxal. 337 00:17:53,080 --> 00:17:56,480 It's covalently bound to the lysine in the active site. 338 00:17:56,480 --> 00:17:59,640 Now one thing that students often find confusing 339 00:17:59,640 --> 00:18:02,030 is the protonation state of this imine. 340 00:18:02,030 --> 00:18:05,630 And that's because it's right around neutral pH, pH 7. 341 00:18:05,630 --> 00:18:08,640 So depending on what's in the active site, 342 00:18:08,640 --> 00:18:11,370 it could be protonated, or not protonated, 343 00:18:11,370 --> 00:18:14,540 if it's protonated of course it enhances reactivity 344 00:18:14,540 --> 00:18:15,540 for nucleophilic attack. 345 00:18:15,540 --> 00:18:17,330 So you want it to be protonated. 346 00:18:17,330 --> 00:18:20,000 So the active site is going to manipulate itself 347 00:18:20,000 --> 00:18:22,370 to put in the protonated state. 348 00:18:22,370 --> 00:18:26,180 So here you have an amino acid, and here we 349 00:18:26,180 --> 00:18:30,490 have a protonated imine, and so this is the nucleophile, 350 00:18:30,490 --> 00:18:33,510 and it can attack the carbon of the imine 351 00:18:33,510 --> 00:18:36,157 to form a tetrahedral adduct. 352 00:18:36,157 --> 00:18:37,490 So that's what we're doing here. 353 00:18:37,490 --> 00:18:42,140 So, this, is going to attack, this, 354 00:18:42,140 --> 00:18:44,330 to form this tetrahedral adduct. 355 00:18:44,330 --> 00:18:45,980 And, you've seen again, the tetrahedral 356 00:18:45,980 --> 00:18:48,530 chemistry over again, when I showed you 357 00:18:48,530 --> 00:18:51,160 how you formed this imine in the first place. 358 00:18:51,160 --> 00:18:54,510 So tetrahedral chemistry, tetrahedral intermediates, 359 00:18:54,510 --> 00:18:57,720 transition states, which collapse to form back 360 00:18:57,720 --> 00:19:01,010 imines, or carbonyls, happens over and over, and over again 361 00:19:01,010 --> 00:19:03,160 in pyridoxal chemistry. 362 00:19:03,160 --> 00:19:07,680 So now what happens is we have this tetrahedral intermediate, 363 00:19:07,680 --> 00:19:11,450 or transition state-- I have it in parentheses, because it's 364 00:19:11,450 --> 00:19:14,430 a high energy intermediate, it doesn't sit around, 365 00:19:14,430 --> 00:19:15,370 and let us look at it. 366 00:19:15,370 --> 00:19:16,953 Most of the time you can never see it. 367 00:19:16,953 --> 00:19:19,530 It's very high on a reaction coordinate. 368 00:19:19,530 --> 00:19:23,260 This, collapses then, and when it collapses, 369 00:19:23,260 --> 00:19:24,990 what do you generate? 370 00:19:24,990 --> 00:19:28,510 You generate the lysine in the active site. 371 00:19:28,510 --> 00:19:31,010 So now we have generated a residue 372 00:19:31,010 --> 00:19:32,560 in the active site that can function 373 00:19:32,560 --> 00:19:34,370 as a general acid, or general base, 374 00:19:34,370 --> 00:19:39,180 catalysis through the rest of the chemical transformations. 375 00:19:39,180 --> 00:19:42,930 And what've we done, is we've converted this imine 376 00:19:42,930 --> 00:19:47,690 with lysine, now to an imine of the amino acid. 377 00:19:47,690 --> 00:19:53,530 So that's what transimination is, one imine to another imine. 378 00:19:53,530 --> 00:19:56,250 The imine that's covalently bound 379 00:19:56,250 --> 00:19:59,580 to the protein through pyridoxal, to an amino acid 380 00:19:59,580 --> 00:20:00,160 imine. 381 00:20:00,160 --> 00:20:02,340 OK, so, that's the first step. 382 00:20:02,340 --> 00:20:05,490 The second step is that ultimately, 383 00:20:05,490 --> 00:20:07,790 in almost all pyridoxal reactions, you 384 00:20:07,790 --> 00:20:10,880 want to remove this alpha hydrogen. 385 00:20:10,880 --> 00:20:14,960 And that alpha hydrogen, again, is extremely non-acidic 386 00:20:14,960 --> 00:20:20,580 but by complexing the amino acid to pyridoxal-- 387 00:20:20,580 --> 00:20:22,720 this is what the function of the cofactor 388 00:20:22,720 --> 00:20:28,060 is-- you are enhancing the acidity of this alpha hydrogen. 389 00:20:28,060 --> 00:20:31,150 You're making it easier for a group in the active site, 390 00:20:31,150 --> 00:20:34,020 a general base catalyst, like lysine, 391 00:20:34,020 --> 00:20:42,960 can now pull off this proton to generate this intermediate. 392 00:20:42,960 --> 00:20:46,130 Now why is this hydrogen more acidic? 393 00:20:46,130 --> 00:20:48,330 Well, if you look at the structure, 394 00:20:48,330 --> 00:20:51,480 you can draw all kinds of resonance structures 395 00:20:51,480 --> 00:20:54,730 which shows that this carbanion is more stabilized, 396 00:20:54,730 --> 00:20:58,210 because it's attached to the pyridoxal cofactor. 397 00:20:58,210 --> 00:21:02,520 So if you look at this, you can draw this resonance structure, 398 00:21:02,520 --> 00:21:04,990 which is shown here, and you can draw 20 other resonance 399 00:21:04,990 --> 00:21:06,220 structures. 400 00:21:06,220 --> 00:21:10,220 OK, so, the key here is you can remove the alpha hydrogen, 401 00:21:10,220 --> 00:21:14,020 because you're able to delocalize 402 00:21:14,020 --> 00:21:19,560 these unpaired electrons on this carbon over the entire system. 403 00:21:19,560 --> 00:21:22,080 So let me show you what that looks like. 404 00:21:22,080 --> 00:21:27,750 So if I draw-- this is called Dunathan's hypothesis-- so 405 00:21:27,750 --> 00:21:36,170 here's our pyridine ring, here's our imine, 406 00:21:36,170 --> 00:21:39,730 and here's our amino acid. 407 00:21:39,730 --> 00:21:42,170 Here's the carboxal-- here's the side chain in our group 408 00:21:42,170 --> 00:21:44,410 and here's a carboxylate, OK. 409 00:21:44,410 --> 00:21:47,650 So the idea is, you have like a benzene ring-- if you haven't 410 00:21:47,650 --> 00:21:51,150 seen pyridine rings-- but you have a pi cloud that 411 00:21:51,150 --> 00:21:54,470 delocalizes-- where these electrons are completely 412 00:21:54,470 --> 00:21:56,930 delocalized over the aromatic ring-- but here, 413 00:21:56,930 --> 00:22:01,040 you also have a pi cloud and these things are close 414 00:22:01,040 --> 00:22:02,890 enough so you can delocalize. 415 00:22:02,890 --> 00:22:06,040 Now what you're going to do-- and the way nature decides 416 00:22:06,040 --> 00:22:10,480 what chemistry happens is since we want to cleave, in this case 417 00:22:10,480 --> 00:22:12,960 the carbon-hydrogen bond, she places 418 00:22:12,960 --> 00:22:17,330 that carbon-hydrogen bond-- by complexing the carboxylate 419 00:22:17,330 --> 00:22:19,950 and complexing whatever the R group is-- 420 00:22:19,950 --> 00:22:22,440 she places that carbon-hydrogen bond 421 00:22:22,440 --> 00:22:27,930 perpendicular to this plane of the pi aldimine system. 422 00:22:27,930 --> 00:22:34,530 So what you're doing then is the lysine, that we just liberated 423 00:22:34,530 --> 00:22:37,250 through doing the transimination reaction, 424 00:22:37,250 --> 00:22:41,060 you now generate an empty P orbital 425 00:22:41,060 --> 00:22:46,010 with unpaired electrons in it, generate the carbanion 426 00:22:46,010 --> 00:22:54,440 and now this system can completely delocalize. 427 00:22:54,440 --> 00:22:58,960 So, again, this pyridine ring is planar. 428 00:22:58,960 --> 00:23:03,630 So you can, completely, delocalize the electrons 429 00:23:03,630 --> 00:23:04,810 over this whole system. 430 00:23:04,810 --> 00:23:07,220 This is a pi cloud. 431 00:23:07,220 --> 00:23:12,250 And now this is already set up so that it can delocalize over 432 00:23:12,250 --> 00:23:13,400 this whole system. 433 00:23:13,400 --> 00:23:16,070 So what you've done then is because of the ability 434 00:23:16,070 --> 00:23:18,410 to stabilize this carbanion you're 435 00:23:18,410 --> 00:23:20,810 making that hydrogen more acidic. 436 00:23:20,810 --> 00:23:24,355 So if you wanted to say, for example, cleave-- remember, 437 00:23:24,355 --> 00:23:25,730 I told you at the very beginning, 438 00:23:25,730 --> 00:23:29,380 you might be able to decarboxylate-- that enzyme, 439 00:23:29,380 --> 00:23:32,490 would place that CO2 perpendicular 440 00:23:32,490 --> 00:23:34,800 to the plane of the pi aldimine system 441 00:23:34,800 --> 00:23:38,840 and use the same strategy to stabilize the resulting 442 00:23:38,840 --> 00:23:40,700 carbanion intermediate. 443 00:23:40,700 --> 00:23:44,550 We're not going to talk about that chemistry in 507. 444 00:23:44,550 --> 00:23:47,780 So what you've done then-- what the beauty of pyridoxal 445 00:23:47,780 --> 00:23:50,600 is that she's increased this acidity, 446 00:23:50,600 --> 00:23:52,800 and allows you a great deal of flexibility. 447 00:23:52,800 --> 00:23:56,930 Because once you generate that carbanion-- those of you who've 448 00:23:56,930 --> 00:24:00,130 had 513-- you'll, immediately, recognize if you have a leaving 449 00:24:00,130 --> 00:24:03,380 group on a carbon adjacent to this carbon, 450 00:24:03,380 --> 00:24:05,010 you can do an elimination reaction. 451 00:24:05,010 --> 00:24:09,120 So it sets up a whole series of transformations. 452 00:24:09,120 --> 00:24:13,620 For today, were only focusing on the transimination reaction. 453 00:24:13,620 --> 00:24:17,340 How do we convert this to an alpha keto 454 00:24:17,340 --> 00:24:21,550 acid, and the pyridoxal to pyridoxamine? 455 00:24:21,550 --> 00:24:23,350 That's the question we're asking. 456 00:24:23,350 --> 00:24:26,510 So we do this chemistry. 457 00:24:26,510 --> 00:24:29,380 And now what we need to do, we can use this resonance 458 00:24:29,380 --> 00:24:32,090 structure, we want to ask the question, what is the product? 459 00:24:32,090 --> 00:24:34,820 Well, we want to get to an alpha keto acid. 460 00:24:34,820 --> 00:24:37,660 OK, and if you look at this structure, 461 00:24:37,660 --> 00:24:42,130 this molecule is an imine of an alpha keto acid. 462 00:24:42,130 --> 00:24:45,600 So this is, exactly, the state we want to be in, 463 00:24:45,600 --> 00:24:47,210 but then we have all of this-- we 464 00:24:47,210 --> 00:24:49,450 have this reactive intermediate here. 465 00:24:49,450 --> 00:24:53,060 So what we want to do is protonate some place here 466 00:24:53,060 --> 00:24:55,190 to generate this state. 467 00:24:55,190 --> 00:24:58,460 So that the last step in all pyridoxal reactions 468 00:24:58,460 --> 00:24:59,950 is hydrolysis. 469 00:24:59,950 --> 00:25:02,330 And now we're set up with the hydrolysis reaction 470 00:25:02,330 --> 00:25:04,540 to generate the alpha keto acid. 471 00:25:04,540 --> 00:25:07,010 So, we then want to ask the question, 472 00:25:07,010 --> 00:25:08,140 where can we protonate? 473 00:25:08,140 --> 00:25:10,260 And, so, again, we have this lysine 474 00:25:10,260 --> 00:25:13,390 which we now have used to pull off the alpha 475 00:25:13,390 --> 00:25:15,250 hydrogen. It's now protonated. 476 00:25:15,250 --> 00:25:17,790 So now instead of being a general based catalyst, 477 00:25:17,790 --> 00:25:21,740 here, it's functioning as a general acid catalyst. 478 00:25:21,740 --> 00:25:24,700 And so now what can happen is you can pick up 479 00:25:24,700 --> 00:25:30,350 a proton from this lysine, it's supplying it with that proton, 480 00:25:30,350 --> 00:25:34,040 to generate this structure and regenerate 481 00:25:34,040 --> 00:25:38,240 lysine that can function now as a general base catalyst. 482 00:25:38,240 --> 00:25:41,870 So it's toggeling between general acid and general base 483 00:25:41,870 --> 00:25:43,140 catalysis. 484 00:25:43,140 --> 00:25:45,580 And now remember, what we want to get in the end 485 00:25:45,580 --> 00:25:49,560 is this pyridoxamine, and we want this alpha keto acid, 486 00:25:49,560 --> 00:25:54,770 and now we're set up to rapidly go to an alpha keto acid. 487 00:25:54,770 --> 00:25:56,760 Where have you seen chemistry like this before? 488 00:25:56,760 --> 00:25:59,000 You've seen it in the aldolase reaction 489 00:25:59,000 --> 00:26:01,400 that we talked about with carbon-carbon bond forming 490 00:26:01,400 --> 00:26:02,670 reactions. 491 00:26:02,670 --> 00:26:06,860 So the last step in all PLP dependent transaminations 492 00:26:06,860 --> 00:26:08,490 is hydrolysis. 493 00:26:08,490 --> 00:26:13,070 So here we have the lysine acting as a general base 494 00:26:13,070 --> 00:26:16,840 to activate water for a nucleophilic attack 495 00:26:16,840 --> 00:26:21,960 on this imine, which is activated to have water add, 496 00:26:21,960 --> 00:26:25,570 and now you form again, your tetrahedral transition state. 497 00:26:25,570 --> 00:26:28,960 I have all of these unstable species in brackets. 498 00:26:28,960 --> 00:26:31,680 We really-- sometimes we see them, sometimes we don't. 499 00:26:31,680 --> 00:26:33,950 But you have to work hard to see them. 500 00:26:33,950 --> 00:26:36,740 And now this simply, the tetrahedral adduct, 501 00:26:36,740 --> 00:26:43,590 collapses to form pyridoxamine and forms the alpha keto acid. 502 00:26:43,590 --> 00:26:45,660 So that's where we wanted to get. 503 00:26:45,660 --> 00:26:51,010 But now what happens, is we're in the form of the cofactor-- 504 00:26:51,010 --> 00:26:54,570 instead of being in the aldehyde form we're in the imine form. 505 00:26:54,570 --> 00:26:57,530 So what we want to do now is reverse 506 00:26:57,530 --> 00:27:02,590 this reaction using a different alpha keto acid, 507 00:27:02,590 --> 00:27:06,280 and we will generate a different amino acid. 508 00:27:06,280 --> 00:27:09,760 So now what can happen-- so in their pyridoxamine form, 509 00:27:09,760 --> 00:27:13,360 we then can bind a different alpha keto acid-- 510 00:27:13,360 --> 00:27:15,500 remember, in the first slide, I showed 511 00:27:15,500 --> 00:27:17,760 you three different alpha keto acids-- 512 00:27:17,760 --> 00:27:19,960 oxaloacetic acid, alpha-ketoglutarate, 513 00:27:19,960 --> 00:27:23,770 pyruvate-- you can reverse this whole process. 514 00:27:23,770 --> 00:27:36,030 And, in the end, what you end up with is a different amino acid 515 00:27:36,030 --> 00:27:40,190 and you regenerate your imine of pyridoxal. 516 00:27:40,190 --> 00:27:44,230 So out of all-- the only pyridoxal phosphate 517 00:27:44,230 --> 00:27:47,300 requiring enzyme that goes from the aldehyde, 518 00:27:47,300 --> 00:27:49,290 or imine, to the pyridoxamine are 519 00:27:49,290 --> 00:27:51,470 these transamination reactions. 520 00:27:51,470 --> 00:27:53,140 And, so, this is the most complicated. 521 00:27:53,140 --> 00:27:54,870 Normally, at the end of your reaction 522 00:27:54,870 --> 00:27:57,550 you wind up in this state. 523 00:27:57,550 --> 00:28:00,930 And this probably seems extremely confusing to most 524 00:28:00,930 --> 00:28:04,830 of you, but after you solve three, or four, problems 525 00:28:04,830 --> 00:28:07,850 where you have to look at the actual transformations, 526 00:28:07,850 --> 00:28:12,530 and think about this tetrahedral chemistry, imines, 527 00:28:12,530 --> 00:28:15,670 and amino groups, and alpha keto groups, you will, I think, 528 00:28:15,670 --> 00:28:19,340 be able to actually easily see how 529 00:28:19,340 --> 00:28:24,580 ingenious nature has been to actually design 530 00:28:24,580 --> 00:28:25,750 pyridoxal phosphate. 531 00:28:25,750 --> 00:28:28,100 And, I think, the most amazing thing, of course, 532 00:28:28,100 --> 00:28:31,860 is that pyridoxal phosphate without any enzyme-- 533 00:28:31,860 --> 00:28:35,180 I told you can catalyze many, many reactions you 534 00:28:35,180 --> 00:28:37,960 do all the reactions with the en-- it does it 535 00:28:37,960 --> 00:28:41,870 spontaneously, at room temperature, at pH 7. 536 00:28:41,870 --> 00:28:45,810 What the enzyme does, is only allows one of these reactions 537 00:28:45,810 --> 00:28:50,250 by having everything positioned exactly the right way 538 00:28:50,250 --> 00:28:51,750 in the active site. 539 00:28:51,750 --> 00:28:53,830 So this is one of the cofactors, that I 540 00:28:53,830 --> 00:28:55,490 thought was amazingly cool when I 541 00:28:55,490 --> 00:28:56,900 was in graduate school, that made 542 00:28:56,900 --> 00:29:00,360 me want to become a biochemist.