1 00:00:00,090 --> 00:00:02,520 The following content is provided under a Creative 2 00:00:02,520 --> 00:00:04,059 Commons license. 3 00:00:04,059 --> 00:00:06,360 Your support will help MIT OpenCourseWare 4 00:00:06,360 --> 00:00:10,720 continue to offer high quality educational resources for free. 5 00:00:10,720 --> 00:00:13,350 To make a donation or view additional materials 6 00:00:13,350 --> 00:00:17,310 from hundreds of MIT courses, visit MIT OpenCourseWare 7 00:00:17,310 --> 00:00:20,880 at ocw.mit.edu. 8 00:00:20,880 --> 00:00:22,680 BOGDAN FEDELES: Hello and welcome to 5.07 9 00:00:22,680 --> 00:00:24,630 Biochemistry online. 10 00:00:24,630 --> 00:00:27,750 I'm Dr. Bogdan Fedeles. 11 00:00:27,750 --> 00:00:32,220 This video is about pyridoxal 5 phosphate, or PLP, 12 00:00:32,220 --> 00:00:36,440 an essential metabolism cofactor derived from vitamin B6. 13 00:00:36,440 --> 00:00:40,230 All animals are auxotrophic for PLP, 14 00:00:40,230 --> 00:00:42,900 meaning they need to supplement their diet with vitamin B6 15 00:00:42,900 --> 00:00:45,180 in order to survive. 16 00:00:45,180 --> 00:00:48,780 PLP is one of the most ancient cofactors, 17 00:00:48,780 --> 00:00:50,820 and surprisingly, it can catalyze 18 00:00:50,820 --> 00:00:54,390 chemical transformation, such as a transamination 19 00:00:54,390 --> 00:00:56,970 even without an enzyme. 20 00:00:56,970 --> 00:01:00,120 PLP is actually involved in a staggering number 21 00:01:00,120 --> 00:01:03,730 of biochemical transformations. 22 00:01:03,730 --> 00:01:07,080 This video summarizes the most important reactions involving 23 00:01:07,080 --> 00:01:10,620 PLP that you will see in 5.07, and will also 24 00:01:10,620 --> 00:01:13,110 show you how to write the complete curved arrow 25 00:01:13,110 --> 00:01:15,420 mechanisms for these transformations. 26 00:01:15,420 --> 00:01:20,010 Let's talk about PLP-catalyzed reactions. 27 00:01:20,010 --> 00:01:22,590 As we just mentioned, PLP is the cofactor 28 00:01:22,590 --> 00:01:25,640 derived from vitamin B6. 29 00:01:25,640 --> 00:01:30,340 This cofactor is very important for a number of reactions. 30 00:01:30,340 --> 00:01:32,340 In this course, we're going to look particularly 31 00:01:32,340 --> 00:01:35,020 at the transamination reaction. 32 00:01:35,020 --> 00:01:37,980 This is a crucial reaction for the metabolism of all amino 33 00:01:37,980 --> 00:01:42,660 the acids, and we're also going to encounter this reaction when 34 00:01:42,660 --> 00:01:45,960 we replenish the intermediates in the TCA cycle, what 35 00:01:45,960 --> 00:01:49,280 we call anaplerotic reactions. 36 00:01:49,280 --> 00:01:53,090 And we're also going to see PLP involved in reactions 37 00:01:53,090 --> 00:01:57,240 in the malate-aspartate shuttle that transfers redox 38 00:01:57,240 --> 00:01:59,010 equivalents, reducing equivalents, 39 00:01:59,010 --> 00:02:02,470 between mitochondria and cytosol. 40 00:02:02,470 --> 00:02:05,070 Let's take a look at the structure of vitamin B6, also 41 00:02:05,070 --> 00:02:08,990 known as pyridoxine. 42 00:02:08,990 --> 00:02:12,540 This is the molecule that we ingest when we get 43 00:02:12,540 --> 00:02:15,030 our daily vitamin supplement. 44 00:02:15,030 --> 00:02:18,270 Now in the body, this gets oxidized to form 45 00:02:18,270 --> 00:02:19,940 intermediate called pyridoxal. 46 00:02:22,530 --> 00:02:24,540 Notice the aldehyde group here, which 47 00:02:24,540 --> 00:02:28,530 is going to be the business end of the molecule. 48 00:02:28,530 --> 00:02:31,560 Now, the active co-factor, PLP-- 49 00:02:31,560 --> 00:02:35,820 it's actually the phosphorylated version of pyridoxal. 50 00:02:35,820 --> 00:02:38,340 This requires one molecule of ATP 51 00:02:38,340 --> 00:02:42,650 and the enzyme pyridoxal kinase. 52 00:02:42,650 --> 00:02:47,530 And we get PLP, or pyridoxal 5 phosphate. 53 00:02:47,530 --> 00:02:52,360 The name PLP comes from the initials as outlined here. 54 00:02:52,360 --> 00:02:55,540 Now, this nitrogen on the pyridine ring 55 00:02:55,540 --> 00:03:00,010 tends to be protonated because it's pKa, 56 00:03:00,010 --> 00:03:06,410 it's close to physiological pH, between 6 and 7. 57 00:03:06,410 --> 00:03:08,440 Now, for the rest of this presentation 58 00:03:08,440 --> 00:03:13,120 we're going to be abbreviating this phosphate group as such, 59 00:03:13,120 --> 00:03:15,640 and throughout the course. 60 00:03:15,640 --> 00:03:19,570 Now, a related molecule is pyridoxamine 5 phosphate, 61 00:03:19,570 --> 00:03:21,160 which we'll see, it's an intermediate 62 00:03:21,160 --> 00:03:23,870 in the mechanism of PLP-catalyzed reactions. 63 00:03:28,270 --> 00:03:31,490 Also known as PMP. 64 00:03:31,490 --> 00:03:35,590 Now, notice the PMP has an amine group here 65 00:03:35,590 --> 00:03:39,670 which replaces the aldehydic group, which is the business 66 00:03:39,670 --> 00:03:41,950 end of the molecule. 67 00:03:41,950 --> 00:03:45,460 Now, in all reactions with PLP, this co-factor 68 00:03:45,460 --> 00:03:49,960 is actually covalently bound to the enzyme that uses it. 69 00:03:49,960 --> 00:03:54,790 Typically, there's a lysine in the active site of the enzyme. 70 00:03:54,790 --> 00:03:58,060 As you remember, lysine has an amine group, 71 00:03:58,060 --> 00:04:01,490 and this can form a Schiff base with the aldehyde. 72 00:04:01,490 --> 00:04:03,280 The reaction proceeds in two steps. 73 00:04:06,400 --> 00:04:09,890 First, we form a tetrahedral intermediate. 74 00:04:16,570 --> 00:04:18,100 As such. 75 00:04:18,100 --> 00:04:20,800 And then, we form the Schiff's base. 76 00:04:30,460 --> 00:04:36,360 So this will be the enzyme-bound PLP. 77 00:04:36,360 --> 00:04:39,400 And this is where all the PLP-catalyzed reactions start. 78 00:04:43,340 --> 00:04:47,830 Let's take a closer look at the transamination reaction. 79 00:04:47,830 --> 00:04:51,140 Transamination reaction occurs between an amino acid 80 00:04:51,140 --> 00:04:53,510 and an alpha keto acid. 81 00:04:53,510 --> 00:04:57,680 We have here amino acid 1, where we highlighted the amine group, 82 00:04:57,680 --> 00:05:00,440 and alpha keto acid 2. 83 00:05:00,440 --> 00:05:04,190 As you can see, there's a keto group next to the carboxyl. 84 00:05:04,190 --> 00:05:08,380 Now, in a transamination reaction catalyzed by PLP, 85 00:05:08,380 --> 00:05:13,070 the amine group moves from the amino acid 86 00:05:13,070 --> 00:05:17,570 to the keto carbon of the alpha keto acid. 87 00:05:17,570 --> 00:05:23,510 And we obtain a new alpha keto acid, and a new amino acid. 88 00:05:26,500 --> 00:05:30,870 So in effect, the PLP-catalyzed transamination reaction 89 00:05:30,870 --> 00:05:33,300 facilitates the transfer of the amine group 90 00:05:33,300 --> 00:05:35,660 from an amino acid to an alpha keto acid. 91 00:05:35,660 --> 00:05:38,850 Now, this reaction actually occurs in two steps. 92 00:05:38,850 --> 00:05:41,820 In the first step, the amino acid transfers the group 93 00:05:41,820 --> 00:05:43,140 to the co-factor itself. 94 00:05:52,410 --> 00:05:55,130 So if you remember from the previous slide, 95 00:05:55,130 --> 00:06:00,260 the PMP contains an amino group, and that will actually 96 00:06:00,260 --> 00:06:01,760 contain this amino group that was 97 00:06:01,760 --> 00:06:04,560 taken from the amino acid 1. 98 00:06:04,560 --> 00:06:08,330 Now in the second step, the PMP will transfer its amino group 99 00:06:08,330 --> 00:06:11,990 to a different alpha keto acid to generate a new amino acid. 100 00:06:20,100 --> 00:06:22,400 Now, there are enzymes for virtually 101 00:06:22,400 --> 00:06:24,950 every single amino acid that can accomplish 102 00:06:24,950 --> 00:06:28,220 this first transformation, where by using PLP, 103 00:06:28,220 --> 00:06:31,350 to transfer the amine group and form an alpha keto 104 00:06:31,350 --> 00:06:33,574 acid and PMP. 105 00:06:33,574 --> 00:06:35,240 Now, in the second part of the reaction, 106 00:06:35,240 --> 00:06:40,040 however, the alpha keto acid 2 is typically alpha keto 107 00:06:40,040 --> 00:06:42,050 gluterate or oxaloacetate. 108 00:06:42,050 --> 00:06:46,550 So in this case, not any alpha keto acid can function. 109 00:06:49,262 --> 00:06:53,430 Alpha keto gluterate or oxaloacetate. 110 00:06:53,430 --> 00:06:55,920 Now, let's take a look at an example. 111 00:06:55,920 --> 00:07:00,990 For example, glutamate. 112 00:07:00,990 --> 00:07:02,900 It's going to be our amino acid. 113 00:07:02,900 --> 00:07:10,320 And oxaloacetate is going to be our alpha keto acid. 114 00:07:10,320 --> 00:07:13,290 And in a PLP-catalyzed transformation, 115 00:07:13,290 --> 00:07:16,760 we will obtain the alpha keto acid corresponding 116 00:07:16,760 --> 00:07:18,540 to glutamate, which is alpha keto 117 00:07:18,540 --> 00:07:22,500 glutarate, and the amino acid corresponding to oxaloacetate, 118 00:07:22,500 --> 00:07:23,990 which is aspartate. 119 00:07:31,020 --> 00:07:33,480 This enzyme that catalyzes this transformation 120 00:07:33,480 --> 00:07:35,790 is in fact ubiquitous, and it's found 121 00:07:35,790 --> 00:07:42,690 both in the liver and the muscles, and it is in fact a-- 122 00:07:42,690 --> 00:07:45,250 we can call it, depending and the product, 123 00:07:45,250 --> 00:07:52,640 we can call it aspartate, transaminase or glutamate 124 00:07:52,640 --> 00:07:55,440 oxaloacetate transaminase. 125 00:07:55,440 --> 00:07:59,960 In fact, if we find this in the bloodstream, 126 00:07:59,960 --> 00:08:02,280 this enzyme acts as a biomarker. 127 00:08:02,280 --> 00:08:04,080 And it tells us about some damage 128 00:08:04,080 --> 00:08:08,340 that might have occurred in muscle or liver, which 129 00:08:08,340 --> 00:08:11,460 forced the cells to spill out their contents. 130 00:08:11,460 --> 00:08:15,780 This biomarker is-- you'll often see as SGOT-- 131 00:08:15,780 --> 00:08:20,910 serum glutamate oxaloacetate transaminase. 132 00:08:20,910 --> 00:08:24,170 And this is just one of the biomarkers that 133 00:08:24,170 --> 00:08:27,900 are measured in blood tests that tells us about heart 134 00:08:27,900 --> 00:08:29,420 disease or liver disease. 135 00:08:32,320 --> 00:08:34,700 Let's take a look at the mechanism of the transamination 136 00:08:34,700 --> 00:08:35,795 reaction. 137 00:08:35,795 --> 00:08:37,669 And in particular, we're going to take a look 138 00:08:37,669 --> 00:08:40,280 at part one, which as we discussed before, 139 00:08:40,280 --> 00:08:46,450 the amino acid reacts with PLP to form an alpha keto 140 00:08:46,450 --> 00:08:48,340 acid and PNP. 141 00:08:56,610 --> 00:09:00,210 Here is our co-factor PLP, covalently bound 142 00:09:00,210 --> 00:09:04,220 to the lysine in the active side of the enzyme via a Schiff's 143 00:09:04,220 --> 00:09:06,940 base. 144 00:09:06,940 --> 00:09:10,395 And here is our amino acid starting material. 145 00:09:13,284 --> 00:09:17,340 So in the first step, the lysine that forms the Schiff base 146 00:09:17,340 --> 00:09:19,680 with the co-factor is going to be replaced 147 00:09:19,680 --> 00:09:23,250 by the amine functionality of the amino acid, 148 00:09:23,250 --> 00:09:27,520 and it will form a new Schiff base with the co-factor. 149 00:09:27,520 --> 00:09:36,460 This starts with the amine group attack on the pyridoxal carbon 150 00:09:36,460 --> 00:09:38,400 to form a tetrahedral intermediate. 151 00:09:45,480 --> 00:09:49,730 And following an additional proton transfer, 152 00:09:49,730 --> 00:09:52,920 the lysine can be kicked off to form the new Schiff base. 153 00:10:01,090 --> 00:10:05,170 So far, we have started with the Schiff's base corresponding 154 00:10:05,170 --> 00:10:10,150 to the PLP bound to the enzyme and we now 155 00:10:10,150 --> 00:10:14,650 obtain a co-factor forming a Schiff base 156 00:10:14,650 --> 00:10:17,170 with the incoming amino acid 1. 157 00:10:17,170 --> 00:10:21,640 So this portion of the mechanism is called transamination, 158 00:10:21,640 --> 00:10:24,070 because we're starting with one amine 159 00:10:24,070 --> 00:10:26,680 and we're forming a different amine. 160 00:10:26,680 --> 00:10:28,960 Now, let's take a look at the alpha proton 161 00:10:28,960 --> 00:10:30,790 attached to the alpha carbon, which 162 00:10:30,790 --> 00:10:33,770 we're going to highlight here. 163 00:10:33,770 --> 00:10:39,320 This proton is now in between two carbonyl-like groups. 164 00:10:39,320 --> 00:10:43,370 Here is the carboxyl group and here is the Schiff base group. 165 00:10:43,370 --> 00:10:46,580 So it becomes acidic enough that it 166 00:10:46,580 --> 00:10:50,030 can be removed by an active side base, for example 167 00:10:50,030 --> 00:10:52,200 the lysine in the active site. 168 00:10:52,200 --> 00:10:54,950 This will generate a carbanium alpha carbon. 169 00:11:00,351 --> 00:11:03,580 This carbanium can only form because it 170 00:11:03,580 --> 00:11:05,710 is resonance stabilized. 171 00:11:05,710 --> 00:11:08,650 And indeed, the PLP link system-- 172 00:11:08,650 --> 00:11:13,610 it's highly conjugated, and it's a good electron sink. 173 00:11:13,610 --> 00:11:15,980 For this carbanium, we can write, 174 00:11:15,980 --> 00:11:18,235 in fact, many different resonance structures. 175 00:11:18,235 --> 00:11:19,610 Let's take a look at one of them. 176 00:11:23,840 --> 00:11:26,366 This symbol denotes resonance structures. 177 00:11:38,030 --> 00:11:41,350 Notice in this structure that the positive charge 178 00:11:41,350 --> 00:11:44,200 on the pyrodine nitrogen is now gone, 179 00:11:44,200 --> 00:11:48,190 and highlights the fact that this is a good electron sink. 180 00:11:48,190 --> 00:11:52,060 And the ring now looks more like a quinone. 181 00:11:52,060 --> 00:11:54,520 That's why we call this a quinoid structure, 182 00:11:54,520 --> 00:11:56,401 or intermediate. 183 00:11:56,401 --> 00:12:00,550 This quinoid structure shows us a glimpse into how the reaction 184 00:12:00,550 --> 00:12:03,730 will proceed, because the alpha carbon now-- it's 185 00:12:03,730 --> 00:12:06,730 doubly bonded to a nitrogen, which anticipates 186 00:12:06,730 --> 00:12:11,200 how this alpha carbon will become a keto group 187 00:12:11,200 --> 00:12:16,390 and a product of the reaction will be an alpha keto acid. 188 00:12:16,390 --> 00:12:17,250 What happens? 189 00:12:17,250 --> 00:12:19,420 The quinoid structure can be re-protonated, 190 00:12:19,420 --> 00:12:20,830 but at a different place. 191 00:12:20,830 --> 00:12:25,780 For example, on the aldehydic carbon of pyridoxal. 192 00:12:28,380 --> 00:12:29,850 To highlight that these facts-- 193 00:12:29,850 --> 00:12:34,285 these structures are in fact resonance structures, 194 00:12:34,285 --> 00:12:35,785 we're going to put them in brackets. 195 00:12:44,340 --> 00:12:46,020 So let's take a look at what happened 196 00:12:46,020 --> 00:12:49,180 in this past couple of steps. 197 00:12:49,180 --> 00:12:53,390 So we had an alpha proton that was fairly acidic, 198 00:12:53,390 --> 00:12:57,720 it was able to be removed by the active site lysine. 199 00:12:57,720 --> 00:13:00,960 And then this proton came back to a different position. 200 00:13:00,960 --> 00:13:04,340 So all that's happened in just a couple of steps 201 00:13:04,340 --> 00:13:07,600 was a proton transfer. 202 00:13:07,600 --> 00:13:09,980 Now, looking at this intermediate, 203 00:13:09,980 --> 00:13:14,110 we can see that in fact the Schiff's base or the imine 204 00:13:14,110 --> 00:13:19,810 of the PMP form of the co-factor and the alpha keto acid 205 00:13:19,810 --> 00:13:22,090 corresponding to amino acid 1. 206 00:13:22,090 --> 00:13:27,680 So via a hydrolysis reaction, these two can come apart. 207 00:13:27,680 --> 00:13:29,910 So in the first step, an activated water molecule 208 00:13:29,910 --> 00:13:37,631 attacks alpha carbon, forming a tetrahedral intermediate. 209 00:13:46,480 --> 00:13:48,950 And one more proton transfer and we're 210 00:13:48,950 --> 00:13:51,480 going to kick off the pyridoxamine form 211 00:13:51,480 --> 00:13:53,110 of the co-factor. 212 00:14:03,320 --> 00:14:09,060 And notice we obtain PMP and the alpha keto acid 213 00:14:09,060 --> 00:14:13,390 corresponding to the amino acid 1. 214 00:14:13,390 --> 00:14:16,800 This last step is, in fact, just a hydrolysis reaction 215 00:14:16,800 --> 00:14:17,660 of a Schiff base. 216 00:14:21,600 --> 00:14:24,470 Now, let's take a look at the second part of the PLP 217 00:14:24,470 --> 00:14:27,340 transamination reaction. 218 00:14:27,340 --> 00:14:30,830 The part one left us off with formation of PMP. 219 00:14:30,830 --> 00:14:37,280 So in this second part, PMP will react with a new alpha keto 220 00:14:37,280 --> 00:14:45,670 acid to regenerate PLP and a new amino acid. 221 00:14:45,670 --> 00:14:48,020 In fact, this part of the mechanism-- 222 00:14:48,020 --> 00:14:52,420 it's the exact reverse of part one. 223 00:14:52,420 --> 00:14:57,290 Here is PMP and our alpha keto acid. 224 00:14:59,830 --> 00:15:02,590 In the first step, we're going to form-- 225 00:15:02,590 --> 00:15:04,570 as we've gotten used so far-- 226 00:15:04,570 --> 00:15:08,616 to a new imine between the keto group of alpha keto acid 227 00:15:08,616 --> 00:15:09,740 and the amine group of PLP. 228 00:15:15,390 --> 00:15:19,056 As usual, first we're going to get a tetrahedral intermediate. 229 00:15:24,900 --> 00:15:29,300 And one more proton transfer, and we can kick off 230 00:15:29,300 --> 00:15:31,670 the OH group to form the imine. 231 00:15:42,500 --> 00:15:44,960 Now, this portion of the reaction 232 00:15:44,960 --> 00:15:47,090 is, in fact, imine formation, which 233 00:15:47,090 --> 00:15:52,880 is the reverse of the hydrolysis step that we saw in part one. 234 00:15:52,880 --> 00:16:01,100 Now, as you remember, there is an active site lysine which 235 00:16:01,100 --> 00:16:03,800 can act as a general base. 236 00:16:03,800 --> 00:16:13,730 And it's going to de-protonate one of these two protons 237 00:16:13,730 --> 00:16:15,930 on the pyridoxal ring. 238 00:16:28,820 --> 00:16:30,830 The reason that we can form this carbanion 239 00:16:30,830 --> 00:16:33,290 here is because this negative charge 240 00:16:33,290 --> 00:16:38,430 is delocalized throughout the entire ring system. 241 00:16:38,430 --> 00:16:41,381 And let's show one important resonance structure. 242 00:16:51,980 --> 00:16:54,590 Which is none other than the quinoid structure 243 00:16:54,590 --> 00:16:57,790 we saw before. 244 00:16:57,790 --> 00:17:00,450 Just as before, the protonated lysine 245 00:17:00,450 --> 00:17:03,110 can now donate proton on a different position. 246 00:17:03,110 --> 00:17:06,920 For example, the alpha carbon of the alpha keto acid. 247 00:17:25,540 --> 00:17:28,480 As you can see here, now the proton 248 00:17:28,480 --> 00:17:30,970 is on the alpha position. 249 00:17:30,970 --> 00:17:34,480 And now where this starts to look more 250 00:17:34,480 --> 00:17:38,410 like the Schiff's base formed by an amino acid 251 00:17:38,410 --> 00:17:42,940 with the PLP version of the co-factor. 252 00:17:42,940 --> 00:17:47,080 So from here on onwards, we're just 253 00:17:47,080 --> 00:17:50,028 going to substitute the PLP-- 254 00:17:50,028 --> 00:17:52,170 the amino acid bound to the PLP-- 255 00:17:52,170 --> 00:17:56,320 with the active site lysine in the transimination reaction 256 00:17:56,320 --> 00:17:58,650 that we saw before. 257 00:17:58,650 --> 00:18:14,950 So first, the lysine can attack this carbon, 258 00:18:14,950 --> 00:18:17,506 forming a tetrahedral intermediate. 259 00:18:21,240 --> 00:18:25,980 And then, following some proton transfer, 260 00:18:25,980 --> 00:18:30,240 we can kick off the amino acid and generate the Schiff's base 261 00:18:30,240 --> 00:18:33,840 corresponding to the PLP co-factor bound to the enzyme. 262 00:18:44,720 --> 00:18:49,620 So here we have PLP, enzyme bound, 263 00:18:49,620 --> 00:18:54,680 and the new amino acid 2. 264 00:18:54,680 --> 00:18:58,370 We mentioned PLP is a very versatile co-factor, 265 00:18:58,370 --> 00:19:00,350 so let's take a look what other reactions 266 00:19:00,350 --> 00:19:04,360 besides transamination can PLP catalyze. 267 00:19:04,360 --> 00:19:07,600 One interesting reaction, used especially by bacteria, 268 00:19:07,600 --> 00:19:09,980 is a racemization. 269 00:19:09,980 --> 00:19:19,820 This involves taking an L amino acid, for example L alanine, 270 00:19:19,820 --> 00:19:24,900 and converting it via a PLP-catalyzed reaction to D 271 00:19:24,900 --> 00:19:25,832 alanine. 272 00:19:30,960 --> 00:19:32,880 This is an important reaction for bacteria, 273 00:19:32,880 --> 00:19:35,040 because they incorporate the alanine into the cell 274 00:19:35,040 --> 00:19:38,130 walls, which make it very hard to recognize 275 00:19:38,130 --> 00:19:41,250 by the immune system, and makes it very hard 276 00:19:41,250 --> 00:19:46,860 to digest by the host proteases. 277 00:19:46,860 --> 00:19:49,230 Let's take a look at a key intermediate 278 00:19:49,230 --> 00:19:51,630 in the PLP-catalyzed reaction. 279 00:19:51,630 --> 00:19:54,000 As before, L alanine is going to react 280 00:19:54,000 --> 00:19:56,507 with the PLP bound to the enzyme, 281 00:19:56,507 --> 00:19:57,840 and it's going to form an amine. 282 00:20:06,570 --> 00:20:08,800 Here we're highlighting the stereochemistry 283 00:20:08,800 --> 00:20:20,460 of the alpha hydrogen. And here is our active site lysine. 284 00:20:20,460 --> 00:20:22,480 As we've seen before, this alpha hydrogen 285 00:20:22,480 --> 00:20:25,580 is acidic enough that it can be removed by the lysine. 286 00:20:28,960 --> 00:20:31,903 And it's going to form a carbanion at this position. 287 00:20:42,270 --> 00:20:45,960 Now, this carbanion, as we've seen before, 288 00:20:45,960 --> 00:20:49,650 is able to form because the charge is, in fact, delocalized 289 00:20:49,650 --> 00:20:54,780 through the entire system of the pyridoxal ring. 290 00:20:54,780 --> 00:20:57,800 So for this structure, we can write 291 00:20:57,800 --> 00:21:00,450 a number of resonance structures, which we're not 292 00:21:00,450 --> 00:21:02,890 going to mention here. 293 00:21:02,890 --> 00:21:07,440 Now, this carbanion can be re-protonated. 294 00:21:07,440 --> 00:21:10,400 And here we had a-- 295 00:21:10,400 --> 00:21:13,050 the hydrogen was pointing up on the top 296 00:21:13,050 --> 00:21:17,610 of the plane of the page, but we can re-protonate it 297 00:21:17,610 --> 00:21:22,030 from the bottom, and that will change the stereochemistry 298 00:21:22,030 --> 00:21:24,300 of this carbon. 299 00:21:24,300 --> 00:21:29,760 See if that re-protonation happens from the bottom, 300 00:21:29,760 --> 00:21:33,658 we will obtain the Schiff base corresponding to the D alanine. 301 00:21:41,640 --> 00:21:45,195 So by being able to generate this carbanion 302 00:21:45,195 --> 00:21:52,710 at the alpha position, the PLP reaction and co-factor 303 00:21:52,710 --> 00:21:54,840 allows the inversion of the configuration 304 00:21:54,840 --> 00:21:59,110 and the alpha carbon, converting L alanine to D alanine. 305 00:21:59,110 --> 00:22:02,420 Now, another interesting reaction that requires PLP 306 00:22:02,420 --> 00:22:04,020 is de-carboxylation. 307 00:22:09,090 --> 00:22:13,026 Here we're looking at an amino acid-- for example, glutamate. 308 00:22:15,942 --> 00:22:22,950 In a PLP-catalyzed reaction, you can lose this CO2 group 309 00:22:22,950 --> 00:22:36,230 and form this molecule, which is called gamma aminobutyric acid, 310 00:22:36,230 --> 00:22:38,870 or GABA. 311 00:22:38,870 --> 00:22:41,615 This is, in fact, a very important neurotransmitter 312 00:22:41,615 --> 00:22:44,300 and inhibitor, a neurotransmitter 313 00:22:44,300 --> 00:22:49,150 that is required in the brain. 314 00:22:49,150 --> 00:22:51,260 Let's take a look how this de-carboxylation 315 00:22:51,260 --> 00:22:52,850 is catalyzed by PLP. 316 00:22:52,850 --> 00:22:59,390 As always, have we seen so far, the glutamate 317 00:22:59,390 --> 00:23:02,840 will react with PLP bound in the active site of the enzyme, 318 00:23:02,840 --> 00:23:05,330 to form a Schiff base. 319 00:23:05,330 --> 00:23:06,430 Here is the Schiff base. 320 00:23:14,750 --> 00:23:15,880 Like that. 321 00:23:15,880 --> 00:23:20,030 Now instead of de-protonating at the alpha position, 322 00:23:20,030 --> 00:23:23,180 the CO2 is activated to leave. 323 00:23:23,180 --> 00:23:25,730 Because it will leave behind the carbanion. 324 00:23:28,749 --> 00:23:29,290 Such as that. 325 00:23:38,500 --> 00:23:40,300 And as we've seen before, a carbanion 326 00:23:40,300 --> 00:23:42,280 formed at this position can de-localize 327 00:23:42,280 --> 00:23:45,430 throughout the entire pyridoxal ring, 328 00:23:45,430 --> 00:23:50,470 and therefore it stabilize and it can exist long enough. 329 00:23:50,470 --> 00:23:52,824 And we're not going to draw, but there-- 330 00:23:52,824 --> 00:23:55,240 you can imagine, there are a number of different resonance 331 00:23:55,240 --> 00:23:56,230 structures. 332 00:23:56,230 --> 00:24:02,230 Now, this carbanion-- it gets protonated quickly 333 00:24:02,230 --> 00:24:15,640 by a general acid, and will generate this structure, which 334 00:24:15,640 --> 00:24:19,390 is just a Schiff base corresponding to gamma 335 00:24:19,390 --> 00:24:23,170 aminobutyric acid with PLP. 336 00:24:23,170 --> 00:24:27,310 And now, from here, a transimination 337 00:24:27,310 --> 00:24:30,460 where the active site lysine will remove the PLP 338 00:24:30,460 --> 00:24:35,390 and free up the GABA products of the reaction. 339 00:24:38,660 --> 00:24:41,830 In this video we talked about PLP-catalyzed reactions. 340 00:24:41,830 --> 00:24:45,840 PLP is the co-factor that comes from vitamin B6. 341 00:24:45,840 --> 00:24:50,180 Here's vitamin B6, what we call pyridoxine, 342 00:24:50,180 --> 00:24:55,365 which is the molecule that we find in our vitamin pills. 343 00:24:55,365 --> 00:24:57,540 Now, in the body, pyridoxine formed 344 00:24:57,540 --> 00:25:02,330 pyridoxal, which is activated to form pyridoxal 5 phosphate, 345 00:25:02,330 --> 00:25:03,800 or PLP. 346 00:25:03,800 --> 00:25:05,780 And typically when it reacts, PLP 347 00:25:05,780 --> 00:25:10,520 is found as a Schiff's base bound in the active site 348 00:25:10,520 --> 00:25:12,960 via a lysine. 349 00:25:12,960 --> 00:25:15,980 PLP is very important for transamination reactions, 350 00:25:15,980 --> 00:25:20,990 which are essential for the metabolism of all amino acids. 351 00:25:20,990 --> 00:25:24,230 We have seen the transamination reaction where an amino acid 352 00:25:24,230 --> 00:25:30,050 1 reacts with alpha keto acid 2 and the PLP catalyzed reaction 353 00:25:30,050 --> 00:25:33,620 forms an alpha keto acid 1 and amino acid 2, 354 00:25:33,620 --> 00:25:35,180 essentially transferring the group-- 355 00:25:35,180 --> 00:25:38,660 the amino group from the amino acid to the alpha keto acid. 356 00:25:38,660 --> 00:25:40,700 The reaction occurs in two steps, 357 00:25:40,700 --> 00:25:44,730 where first the amino acid is transferred to PLP to form PMP. 358 00:25:44,730 --> 00:25:47,365 Then PMP then transfers this group-- 359 00:25:47,365 --> 00:25:49,595 the amino group-- back to a alpha keto acid 360 00:25:49,595 --> 00:25:51,230 to generate a new amino acid. 361 00:25:54,180 --> 00:25:56,070 As we saw, the mechanism of transamination 362 00:25:56,070 --> 00:25:57,640 involves multiple steps. 363 00:25:57,640 --> 00:26:04,450 The first step is a transimination reaction 364 00:26:04,450 --> 00:26:08,080 where the Schiff's base that's formed between the active site 365 00:26:08,080 --> 00:26:12,170 lysine and the PLP becomes a Schiff's 366 00:26:12,170 --> 00:26:15,760 base between the incoming amino acid and PLP. 367 00:26:15,760 --> 00:26:18,370 Next, we have a proton transfer, which 368 00:26:18,370 --> 00:26:21,280 is allowed by the ability of the PLP ring 369 00:26:21,280 --> 00:26:25,420 to stabilize a negative charge, and move 370 00:26:25,420 --> 00:26:27,040 the proton from the alpha position 371 00:26:27,040 --> 00:26:34,460 to somewhere on the PLP ring via a quinoid structure. 372 00:26:34,460 --> 00:26:37,430 And finally, that the resulting Schiff base 373 00:26:37,430 --> 00:26:42,580 is hydrolyzed to generate PMP and an alpha keto acid. 374 00:26:42,580 --> 00:26:44,470 In the second part of the reaction, 375 00:26:44,470 --> 00:26:48,640 PMP now reacts with alpha keto acid to form a new Schiff base, 376 00:26:48,640 --> 00:26:52,860 and then the proton transfer happens in reverse, via, again, 377 00:26:52,860 --> 00:26:56,350 a quinoid structure, to generate the Schiff base 378 00:26:56,350 --> 00:27:00,730 corresponding to the PLP and the new amino acid 2. 379 00:27:00,730 --> 00:27:02,715 Which, via a transamination reaction 380 00:27:02,715 --> 00:27:07,810 will generate amino acid 2 and the PLP enzyme bound. 381 00:27:07,810 --> 00:27:10,420 Finally, we mentioned that PLP is very versatile, 382 00:27:10,420 --> 00:27:12,160 and it can catalyze other reactions, 383 00:27:12,160 --> 00:27:14,890 such as racemization, for example, 384 00:27:14,890 --> 00:27:17,920 switching the configuration of the alpha carbon from L 385 00:27:17,920 --> 00:27:22,510 alanine to D alanine, or de-carboxylation, generating 386 00:27:22,510 --> 00:27:24,520 alpha aminobutyric acid, or GABA, 387 00:27:24,520 --> 00:27:27,790 an important neurotransmitter from glutamate.