WEBVTT 00:00:00.090 --> 00:00:02.520 The following content is provided under a Creative 00:00:02.520 --> 00:00:04.059 Commons license. 00:00:04.059 --> 00:00:06.360 Your support will help MIT OpenCourseWare 00:00:06.360 --> 00:00:10.720 continue to offer high quality educational resources for free. 00:00:10.720 --> 00:00:13.350 To make a donation or view additional materials 00:00:13.350 --> 00:00:17.310 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.310 --> 00:00:20.880 at ocw.mit.edu. 00:00:20.880 --> 00:00:22.680 BOGDAN FEDELES: Hello and welcome to 5.07 00:00:22.680 --> 00:00:24.630 Biochemistry online. 00:00:24.630 --> 00:00:27.750 I'm Dr. Bogdan Fedeles. 00:00:27.750 --> 00:00:32.220 This video is about pyridoxal 5 phosphate, or PLP, 00:00:32.220 --> 00:00:36.440 an essential metabolism cofactor derived from vitamin B6. 00:00:36.440 --> 00:00:40.230 All animals are auxotrophic for PLP, 00:00:40.230 --> 00:00:42.900 meaning they need to supplement their diet with vitamin B6 00:00:42.900 --> 00:00:45.180 in order to survive. 00:00:45.180 --> 00:00:48.780 PLP is one of the most ancient cofactors, 00:00:48.780 --> 00:00:50.820 and surprisingly, it can catalyze 00:00:50.820 --> 00:00:54.390 chemical transformation, such as a transamination 00:00:54.390 --> 00:00:56.970 even without an enzyme. 00:00:56.970 --> 00:01:00.120 PLP is actually involved in a staggering number 00:01:00.120 --> 00:01:03.730 of biochemical transformations. 00:01:03.730 --> 00:01:07.080 This video summarizes the most important reactions involving 00:01:07.080 --> 00:01:10.620 PLP that you will see in 5.07, and will also 00:01:10.620 --> 00:01:13.110 show you how to write the complete curved arrow 00:01:13.110 --> 00:01:15.420 mechanisms for these transformations. 00:01:15.420 --> 00:01:20.010 Let's talk about PLP-catalyzed reactions. 00:01:20.010 --> 00:01:22.590 As we just mentioned, PLP is the cofactor 00:01:22.590 --> 00:01:25.640 derived from vitamin B6. 00:01:25.640 --> 00:01:30.340 This cofactor is very important for a number of reactions. 00:01:30.340 --> 00:01:32.340 In this course, we're going to look particularly 00:01:32.340 --> 00:01:35.020 at the transamination reaction. 00:01:35.020 --> 00:01:37.980 This is a crucial reaction for the metabolism of all amino 00:01:37.980 --> 00:01:42.660 the acids, and we're also going to encounter this reaction when 00:01:42.660 --> 00:01:45.960 we replenish the intermediates in the TCA cycle, what 00:01:45.960 --> 00:01:49.280 we call anaplerotic reactions. 00:01:49.280 --> 00:01:53.090 And we're also going to see PLP involved in reactions 00:01:53.090 --> 00:01:57.240 in the malate-aspartate shuttle that transfers redox 00:01:57.240 --> 00:01:59.010 equivalents, reducing equivalents, 00:01:59.010 --> 00:02:02.470 between mitochondria and cytosol. 00:02:02.470 --> 00:02:05.070 Let's take a look at the structure of vitamin B6, also 00:02:05.070 --> 00:02:08.990 known as pyridoxine. 00:02:08.990 --> 00:02:12.540 This is the molecule that we ingest when we get 00:02:12.540 --> 00:02:15.030 our daily vitamin supplement. 00:02:15.030 --> 00:02:18.270 Now in the body, this gets oxidized to form 00:02:18.270 --> 00:02:19.940 intermediate called pyridoxal. 00:02:22.530 --> 00:02:24.540 Notice the aldehyde group here, which 00:02:24.540 --> 00:02:28.530 is going to be the business end of the molecule. 00:02:28.530 --> 00:02:31.560 Now, the active co-factor, PLP-- 00:02:31.560 --> 00:02:35.820 it's actually the phosphorylated version of pyridoxal. 00:02:35.820 --> 00:02:38.340 This requires one molecule of ATP 00:02:38.340 --> 00:02:42.650 and the enzyme pyridoxal kinase. 00:02:42.650 --> 00:02:47.530 And we get PLP, or pyridoxal 5 phosphate. 00:02:47.530 --> 00:02:52.360 The name PLP comes from the initials as outlined here. 00:02:52.360 --> 00:02:55.540 Now, this nitrogen on the pyridine ring 00:02:55.540 --> 00:03:00.010 tends to be protonated because it's pKa, 00:03:00.010 --> 00:03:06.410 it's close to physiological pH, between 6 and 7. 00:03:06.410 --> 00:03:08.440 Now, for the rest of this presentation 00:03:08.440 --> 00:03:13.120 we're going to be abbreviating this phosphate group as such, 00:03:13.120 --> 00:03:15.640 and throughout the course. 00:03:15.640 --> 00:03:19.570 Now, a related molecule is pyridoxamine 5 phosphate, 00:03:19.570 --> 00:03:21.160 which we'll see, it's an intermediate 00:03:21.160 --> 00:03:23.870 in the mechanism of PLP-catalyzed reactions. 00:03:28.270 --> 00:03:31.490 Also known as PMP. 00:03:31.490 --> 00:03:35.590 Now, notice the PMP has an amine group here 00:03:35.590 --> 00:03:39.670 which replaces the aldehydic group, which is the business 00:03:39.670 --> 00:03:41.950 end of the molecule. 00:03:41.950 --> 00:03:45.460 Now, in all reactions with PLP, this co-factor 00:03:45.460 --> 00:03:49.960 is actually covalently bound to the enzyme that uses it. 00:03:49.960 --> 00:03:54.790 Typically, there's a lysine in the active site of the enzyme. 00:03:54.790 --> 00:03:58.060 As you remember, lysine has an amine group, 00:03:58.060 --> 00:04:01.490 and this can form a Schiff base with the aldehyde. 00:04:01.490 --> 00:04:03.280 The reaction proceeds in two steps. 00:04:06.400 --> 00:04:09.890 First, we form a tetrahedral intermediate. 00:04:16.570 --> 00:04:18.100 As such. 00:04:18.100 --> 00:04:20.800 And then, we form the Schiff's base. 00:04:30.460 --> 00:04:36.360 So this will be the enzyme-bound PLP. 00:04:36.360 --> 00:04:39.400 And this is where all the PLP-catalyzed reactions start. 00:04:43.340 --> 00:04:47.830 Let's take a closer look at the transamination reaction. 00:04:47.830 --> 00:04:51.140 Transamination reaction occurs between an amino acid 00:04:51.140 --> 00:04:53.510 and an alpha keto acid. 00:04:53.510 --> 00:04:57.680 We have here amino acid 1, where we highlighted the amine group, 00:04:57.680 --> 00:05:00.440 and alpha keto acid 2. 00:05:00.440 --> 00:05:04.190 As you can see, there's a keto group next to the carboxyl. 00:05:04.190 --> 00:05:08.380 Now, in a transamination reaction catalyzed by PLP, 00:05:08.380 --> 00:05:13.070 the amine group moves from the amino acid 00:05:13.070 --> 00:05:17.570 to the keto carbon of the alpha keto acid. 00:05:17.570 --> 00:05:23.510 And we obtain a new alpha keto acid, and a new amino acid. 00:05:26.500 --> 00:05:30.870 So in effect, the PLP-catalyzed transamination reaction 00:05:30.870 --> 00:05:33.300 facilitates the transfer of the amine group 00:05:33.300 --> 00:05:35.660 from an amino acid to an alpha keto acid. 00:05:35.660 --> 00:05:38.850 Now, this reaction actually occurs in two steps. 00:05:38.850 --> 00:05:41.820 In the first step, the amino acid transfers the group 00:05:41.820 --> 00:05:43.140 to the co-factor itself. 00:05:52.410 --> 00:05:55.130 So if you remember from the previous slide, 00:05:55.130 --> 00:06:00.260 the PMP contains an amino group, and that will actually 00:06:00.260 --> 00:06:01.760 contain this amino group that was 00:06:01.760 --> 00:06:04.560 taken from the amino acid 1. 00:06:04.560 --> 00:06:08.330 Now in the second step, the PMP will transfer its amino group 00:06:08.330 --> 00:06:11.990 to a different alpha keto acid to generate a new amino acid. 00:06:20.100 --> 00:06:22.400 Now, there are enzymes for virtually 00:06:22.400 --> 00:06:24.950 every single amino acid that can accomplish 00:06:24.950 --> 00:06:28.220 this first transformation, where by using PLP, 00:06:28.220 --> 00:06:31.350 to transfer the amine group and form an alpha keto 00:06:31.350 --> 00:06:33.574 acid and PMP. 00:06:33.574 --> 00:06:35.240 Now, in the second part of the reaction, 00:06:35.240 --> 00:06:40.040 however, the alpha keto acid 2 is typically alpha keto 00:06:40.040 --> 00:06:42.050 gluterate or oxaloacetate. 00:06:42.050 --> 00:06:46.550 So in this case, not any alpha keto acid can function. 00:06:49.262 --> 00:06:53.430 Alpha keto gluterate or oxaloacetate. 00:06:53.430 --> 00:06:55.920 Now, let's take a look at an example. 00:06:55.920 --> 00:07:00.990 For example, glutamate. 00:07:00.990 --> 00:07:02.900 It's going to be our amino acid. 00:07:02.900 --> 00:07:10.320 And oxaloacetate is going to be our alpha keto acid. 00:07:10.320 --> 00:07:13.290 And in a PLP-catalyzed transformation, 00:07:13.290 --> 00:07:16.760 we will obtain the alpha keto acid corresponding 00:07:16.760 --> 00:07:18.540 to glutamate, which is alpha keto 00:07:18.540 --> 00:07:22.500 glutarate, and the amino acid corresponding to oxaloacetate, 00:07:22.500 --> 00:07:23.990 which is aspartate. 00:07:31.020 --> 00:07:33.480 This enzyme that catalyzes this transformation 00:07:33.480 --> 00:07:35.790 is in fact ubiquitous, and it's found 00:07:35.790 --> 00:07:42.690 both in the liver and the muscles, and it is in fact a-- 00:07:42.690 --> 00:07:45.250 we can call it, depending and the product, 00:07:45.250 --> 00:07:52.640 we can call it aspartate, transaminase or glutamate 00:07:52.640 --> 00:07:55.440 oxaloacetate transaminase. 00:07:55.440 --> 00:07:59.960 In fact, if we find this in the bloodstream, 00:07:59.960 --> 00:08:02.280 this enzyme acts as a biomarker. 00:08:02.280 --> 00:08:04.080 And it tells us about some damage 00:08:04.080 --> 00:08:08.340 that might have occurred in muscle or liver, which 00:08:08.340 --> 00:08:11.460 forced the cells to spill out their contents. 00:08:11.460 --> 00:08:15.780 This biomarker is-- you'll often see as SGOT-- 00:08:15.780 --> 00:08:20.910 serum glutamate oxaloacetate transaminase. 00:08:20.910 --> 00:08:24.170 And this is just one of the biomarkers that 00:08:24.170 --> 00:08:27.900 are measured in blood tests that tells us about heart 00:08:27.900 --> 00:08:29.420 disease or liver disease. 00:08:32.320 --> 00:08:34.700 Let's take a look at the mechanism of the transamination 00:08:34.700 --> 00:08:35.795 reaction. 00:08:35.795 --> 00:08:37.669 And in particular, we're going to take a look 00:08:37.669 --> 00:08:40.280 at part one, which as we discussed before, 00:08:40.280 --> 00:08:46.450 the amino acid reacts with PLP to form an alpha keto 00:08:46.450 --> 00:08:48.340 acid and PNP. 00:08:56.610 --> 00:09:00.210 Here is our co-factor PLP, covalently bound 00:09:00.210 --> 00:09:04.220 to the lysine in the active side of the enzyme via a Schiff's 00:09:04.220 --> 00:09:06.940 base. 00:09:06.940 --> 00:09:10.395 And here is our amino acid starting material. 00:09:13.284 --> 00:09:17.340 So in the first step, the lysine that forms the Schiff base 00:09:17.340 --> 00:09:19.680 with the co-factor is going to be replaced 00:09:19.680 --> 00:09:23.250 by the amine functionality of the amino acid, 00:09:23.250 --> 00:09:27.520 and it will form a new Schiff base with the co-factor. 00:09:27.520 --> 00:09:36.460 This starts with the amine group attack on the pyridoxal carbon 00:09:36.460 --> 00:09:38.400 to form a tetrahedral intermediate. 00:09:45.480 --> 00:09:49.730 And following an additional proton transfer, 00:09:49.730 --> 00:09:52.920 the lysine can be kicked off to form the new Schiff base. 00:10:01.090 --> 00:10:05.170 So far, we have started with the Schiff's base corresponding 00:10:05.170 --> 00:10:10.150 to the PLP bound to the enzyme and we now 00:10:10.150 --> 00:10:14.650 obtain a co-factor forming a Schiff base 00:10:14.650 --> 00:10:17.170 with the incoming amino acid 1. 00:10:17.170 --> 00:10:21.640 So this portion of the mechanism is called transamination, 00:10:21.640 --> 00:10:24.070 because we're starting with one amine 00:10:24.070 --> 00:10:26.680 and we're forming a different amine. 00:10:26.680 --> 00:10:28.960 Now, let's take a look at the alpha proton 00:10:28.960 --> 00:10:30.790 attached to the alpha carbon, which 00:10:30.790 --> 00:10:33.770 we're going to highlight here. 00:10:33.770 --> 00:10:39.320 This proton is now in between two carbonyl-like groups. 00:10:39.320 --> 00:10:43.370 Here is the carboxyl group and here is the Schiff base group. 00:10:43.370 --> 00:10:46.580 So it becomes acidic enough that it 00:10:46.580 --> 00:10:50.030 can be removed by an active side base, for example 00:10:50.030 --> 00:10:52.200 the lysine in the active site. 00:10:52.200 --> 00:10:54.950 This will generate a carbanium alpha carbon. 00:11:00.351 --> 00:11:03.580 This carbanium can only form because it 00:11:03.580 --> 00:11:05.710 is resonance stabilized. 00:11:05.710 --> 00:11:08.650 And indeed, the PLP link system-- 00:11:08.650 --> 00:11:13.610 it's highly conjugated, and it's a good electron sink. 00:11:13.610 --> 00:11:15.980 For this carbanium, we can write, 00:11:15.980 --> 00:11:18.235 in fact, many different resonance structures. 00:11:18.235 --> 00:11:19.610 Let's take a look at one of them. 00:11:23.840 --> 00:11:26.366 This symbol denotes resonance structures. 00:11:38.030 --> 00:11:41.350 Notice in this structure that the positive charge 00:11:41.350 --> 00:11:44.200 on the pyrodine nitrogen is now gone, 00:11:44.200 --> 00:11:48.190 and highlights the fact that this is a good electron sink. 00:11:48.190 --> 00:11:52.060 And the ring now looks more like a quinone. 00:11:52.060 --> 00:11:54.520 That's why we call this a quinoid structure, 00:11:54.520 --> 00:11:56.401 or intermediate. 00:11:56.401 --> 00:12:00.550 This quinoid structure shows us a glimpse into how the reaction 00:12:00.550 --> 00:12:03.730 will proceed, because the alpha carbon now-- it's 00:12:03.730 --> 00:12:06.730 doubly bonded to a nitrogen, which anticipates 00:12:06.730 --> 00:12:11.200 how this alpha carbon will become a keto group 00:12:11.200 --> 00:12:16.390 and a product of the reaction will be an alpha keto acid. 00:12:16.390 --> 00:12:17.250 What happens? 00:12:17.250 --> 00:12:19.420 The quinoid structure can be re-protonated, 00:12:19.420 --> 00:12:20.830 but at a different place. 00:12:20.830 --> 00:12:25.780 For example, on the aldehydic carbon of pyridoxal. 00:12:28.380 --> 00:12:29.850 To highlight that these facts-- 00:12:29.850 --> 00:12:34.285 these structures are in fact resonance structures, 00:12:34.285 --> 00:12:35.785 we're going to put them in brackets. 00:12:44.340 --> 00:12:46.020 So let's take a look at what happened 00:12:46.020 --> 00:12:49.180 in this past couple of steps. 00:12:49.180 --> 00:12:53.390 So we had an alpha proton that was fairly acidic, 00:12:53.390 --> 00:12:57.720 it was able to be removed by the active site lysine. 00:12:57.720 --> 00:13:00.960 And then this proton came back to a different position. 00:13:00.960 --> 00:13:04.340 So all that's happened in just a couple of steps 00:13:04.340 --> 00:13:07.600 was a proton transfer. 00:13:07.600 --> 00:13:09.980 Now, looking at this intermediate, 00:13:09.980 --> 00:13:14.110 we can see that in fact the Schiff's base or the imine 00:13:14.110 --> 00:13:19.810 of the PMP form of the co-factor and the alpha keto acid 00:13:19.810 --> 00:13:22.090 corresponding to amino acid 1. 00:13:22.090 --> 00:13:27.680 So via a hydrolysis reaction, these two can come apart. 00:13:27.680 --> 00:13:29.910 So in the first step, an activated water molecule 00:13:29.910 --> 00:13:37.631 attacks alpha carbon, forming a tetrahedral intermediate. 00:13:46.480 --> 00:13:48.950 And one more proton transfer and we're 00:13:48.950 --> 00:13:51.480 going to kick off the pyridoxamine form 00:13:51.480 --> 00:13:53.110 of the co-factor. 00:14:03.320 --> 00:14:09.060 And notice we obtain PMP and the alpha keto acid 00:14:09.060 --> 00:14:13.390 corresponding to the amino acid 1. 00:14:13.390 --> 00:14:16.800 This last step is, in fact, just a hydrolysis reaction 00:14:16.800 --> 00:14:17.660 of a Schiff base. 00:14:21.600 --> 00:14:24.470 Now, let's take a look at the second part of the PLP 00:14:24.470 --> 00:14:27.340 transamination reaction. 00:14:27.340 --> 00:14:30.830 The part one left us off with formation of PMP. 00:14:30.830 --> 00:14:37.280 So in this second part, PMP will react with a new alpha keto 00:14:37.280 --> 00:14:45.670 acid to regenerate PLP and a new amino acid. 00:14:45.670 --> 00:14:48.020 In fact, this part of the mechanism-- 00:14:48.020 --> 00:14:52.420 it's the exact reverse of part one. 00:14:52.420 --> 00:14:57.290 Here is PMP and our alpha keto acid. 00:14:59.830 --> 00:15:02.590 In the first step, we're going to form-- 00:15:02.590 --> 00:15:04.570 as we've gotten used so far-- 00:15:04.570 --> 00:15:08.616 to a new imine between the keto group of alpha keto acid 00:15:08.616 --> 00:15:09.740 and the amine group of PLP. 00:15:15.390 --> 00:15:19.056 As usual, first we're going to get a tetrahedral intermediate. 00:15:24.900 --> 00:15:29.300 And one more proton transfer, and we can kick off 00:15:29.300 --> 00:15:31.670 the OH group to form the imine. 00:15:42.500 --> 00:15:44.960 Now, this portion of the reaction 00:15:44.960 --> 00:15:47.090 is, in fact, imine formation, which 00:15:47.090 --> 00:15:52.880 is the reverse of the hydrolysis step that we saw in part one. 00:15:52.880 --> 00:16:01.100 Now, as you remember, there is an active site lysine which 00:16:01.100 --> 00:16:03.800 can act as a general base. 00:16:03.800 --> 00:16:13.730 And it's going to de-protonate one of these two protons 00:16:13.730 --> 00:16:15.930 on the pyridoxal ring. 00:16:28.820 --> 00:16:30.830 The reason that we can form this carbanion 00:16:30.830 --> 00:16:33.290 here is because this negative charge 00:16:33.290 --> 00:16:38.430 is delocalized throughout the entire ring system. 00:16:38.430 --> 00:16:41.381 And let's show one important resonance structure. 00:16:51.980 --> 00:16:54.590 Which is none other than the quinoid structure 00:16:54.590 --> 00:16:57.790 we saw before. 00:16:57.790 --> 00:17:00.450 Just as before, the protonated lysine 00:17:00.450 --> 00:17:03.110 can now donate proton on a different position. 00:17:03.110 --> 00:17:06.920 For example, the alpha carbon of the alpha keto acid. 00:17:25.540 --> 00:17:28.480 As you can see here, now the proton 00:17:28.480 --> 00:17:30.970 is on the alpha position. 00:17:30.970 --> 00:17:34.480 And now where this starts to look more 00:17:34.480 --> 00:17:38.410 like the Schiff's base formed by an amino acid 00:17:38.410 --> 00:17:42.940 with the PLP version of the co-factor. 00:17:42.940 --> 00:17:47.080 So from here on onwards, we're just 00:17:47.080 --> 00:17:50.028 going to substitute the PLP-- 00:17:50.028 --> 00:17:52.170 the amino acid bound to the PLP-- 00:17:52.170 --> 00:17:56.320 with the active site lysine in the transimination reaction 00:17:56.320 --> 00:17:58.650 that we saw before. 00:17:58.650 --> 00:18:14.950 So first, the lysine can attack this carbon, 00:18:14.950 --> 00:18:17.506 forming a tetrahedral intermediate. 00:18:21.240 --> 00:18:25.980 And then, following some proton transfer, 00:18:25.980 --> 00:18:30.240 we can kick off the amino acid and generate the Schiff's base 00:18:30.240 --> 00:18:33.840 corresponding to the PLP co-factor bound to the enzyme. 00:18:44.720 --> 00:18:49.620 So here we have PLP, enzyme bound, 00:18:49.620 --> 00:18:54.680 and the new amino acid 2. 00:18:54.680 --> 00:18:58.370 We mentioned PLP is a very versatile co-factor, 00:18:58.370 --> 00:19:00.350 so let's take a look what other reactions 00:19:00.350 --> 00:19:04.360 besides transamination can PLP catalyze. 00:19:04.360 --> 00:19:07.600 One interesting reaction, used especially by bacteria, 00:19:07.600 --> 00:19:09.980 is a racemization. 00:19:09.980 --> 00:19:19.820 This involves taking an L amino acid, for example L alanine, 00:19:19.820 --> 00:19:24.900 and converting it via a PLP-catalyzed reaction to D 00:19:24.900 --> 00:19:25.832 alanine. 00:19:30.960 --> 00:19:32.880 This is an important reaction for bacteria, 00:19:32.880 --> 00:19:35.040 because they incorporate the alanine into the cell 00:19:35.040 --> 00:19:38.130 walls, which make it very hard to recognize 00:19:38.130 --> 00:19:41.250 by the immune system, and makes it very hard 00:19:41.250 --> 00:19:46.860 to digest by the host proteases. 00:19:46.860 --> 00:19:49.230 Let's take a look at a key intermediate 00:19:49.230 --> 00:19:51.630 in the PLP-catalyzed reaction. 00:19:51.630 --> 00:19:54.000 As before, L alanine is going to react 00:19:54.000 --> 00:19:56.507 with the PLP bound to the enzyme, 00:19:56.507 --> 00:19:57.840 and it's going to form an amine. 00:20:06.570 --> 00:20:08.800 Here we're highlighting the stereochemistry 00:20:08.800 --> 00:20:20.460 of the alpha hydrogen. And here is our active site lysine. 00:20:20.460 --> 00:20:22.480 As we've seen before, this alpha hydrogen 00:20:22.480 --> 00:20:25.580 is acidic enough that it can be removed by the lysine. 00:20:28.960 --> 00:20:31.903 And it's going to form a carbanion at this position. 00:20:42.270 --> 00:20:45.960 Now, this carbanion, as we've seen before, 00:20:45.960 --> 00:20:49.650 is able to form because the charge is, in fact, delocalized 00:20:49.650 --> 00:20:54.780 through the entire system of the pyridoxal ring. 00:20:54.780 --> 00:20:57.800 So for this structure, we can write 00:20:57.800 --> 00:21:00.450 a number of resonance structures, which we're not 00:21:00.450 --> 00:21:02.890 going to mention here. 00:21:02.890 --> 00:21:07.440 Now, this carbanion can be re-protonated. 00:21:07.440 --> 00:21:10.400 And here we had a-- 00:21:10.400 --> 00:21:13.050 the hydrogen was pointing up on the top 00:21:13.050 --> 00:21:17.610 of the plane of the page, but we can re-protonate it 00:21:17.610 --> 00:21:22.030 from the bottom, and that will change the stereochemistry 00:21:22.030 --> 00:21:24.300 of this carbon. 00:21:24.300 --> 00:21:29.760 See if that re-protonation happens from the bottom, 00:21:29.760 --> 00:21:33.658 we will obtain the Schiff base corresponding to the D alanine. 00:21:41.640 --> 00:21:45.195 So by being able to generate this carbanion 00:21:45.195 --> 00:21:52.710 at the alpha position, the PLP reaction and co-factor 00:21:52.710 --> 00:21:54.840 allows the inversion of the configuration 00:21:54.840 --> 00:21:59.110 and the alpha carbon, converting L alanine to D alanine. 00:21:59.110 --> 00:22:02.420 Now, another interesting reaction that requires PLP 00:22:02.420 --> 00:22:04.020 is de-carboxylation. 00:22:09.090 --> 00:22:13.026 Here we're looking at an amino acid-- for example, glutamate. 00:22:15.942 --> 00:22:22.950 In a PLP-catalyzed reaction, you can lose this CO2 group 00:22:22.950 --> 00:22:36.230 and form this molecule, which is called gamma aminobutyric acid, 00:22:36.230 --> 00:22:38.870 or GABA. 00:22:38.870 --> 00:22:41.615 This is, in fact, a very important neurotransmitter 00:22:41.615 --> 00:22:44.300 and inhibitor, a neurotransmitter 00:22:44.300 --> 00:22:49.150 that is required in the brain. 00:22:49.150 --> 00:22:51.260 Let's take a look how this de-carboxylation 00:22:51.260 --> 00:22:52.850 is catalyzed by PLP. 00:22:52.850 --> 00:22:59.390 As always, have we seen so far, the glutamate 00:22:59.390 --> 00:23:02.840 will react with PLP bound in the active site of the enzyme, 00:23:02.840 --> 00:23:05.330 to form a Schiff base. 00:23:05.330 --> 00:23:06.430 Here is the Schiff base. 00:23:14.750 --> 00:23:15.880 Like that. 00:23:15.880 --> 00:23:20.030 Now instead of de-protonating at the alpha position, 00:23:20.030 --> 00:23:23.180 the CO2 is activated to leave. 00:23:23.180 --> 00:23:25.730 Because it will leave behind the carbanion. 00:23:28.749 --> 00:23:29.290 Such as that. 00:23:38.500 --> 00:23:40.300 And as we've seen before, a carbanion 00:23:40.300 --> 00:23:42.280 formed at this position can de-localize 00:23:42.280 --> 00:23:45.430 throughout the entire pyridoxal ring, 00:23:45.430 --> 00:23:50.470 and therefore it stabilize and it can exist long enough. 00:23:50.470 --> 00:23:52.824 And we're not going to draw, but there-- 00:23:52.824 --> 00:23:55.240 you can imagine, there are a number of different resonance 00:23:55.240 --> 00:23:56.230 structures. 00:23:56.230 --> 00:24:02.230 Now, this carbanion-- it gets protonated quickly 00:24:02.230 --> 00:24:15.640 by a general acid, and will generate this structure, which 00:24:15.640 --> 00:24:19.390 is just a Schiff base corresponding to gamma 00:24:19.390 --> 00:24:23.170 aminobutyric acid with PLP. 00:24:23.170 --> 00:24:27.310 And now, from here, a transimination 00:24:27.310 --> 00:24:30.460 where the active site lysine will remove the PLP 00:24:30.460 --> 00:24:35.390 and free up the GABA products of the reaction. 00:24:38.660 --> 00:24:41.830 In this video we talked about PLP-catalyzed reactions. 00:24:41.830 --> 00:24:45.840 PLP is the co-factor that comes from vitamin B6. 00:24:45.840 --> 00:24:50.180 Here's vitamin B6, what we call pyridoxine, 00:24:50.180 --> 00:24:55.365 which is the molecule that we find in our vitamin pills. 00:24:55.365 --> 00:24:57.540 Now, in the body, pyridoxine formed 00:24:57.540 --> 00:25:02.330 pyridoxal, which is activated to form pyridoxal 5 phosphate, 00:25:02.330 --> 00:25:03.800 or PLP. 00:25:03.800 --> 00:25:05.780 And typically when it reacts, PLP 00:25:05.780 --> 00:25:10.520 is found as a Schiff's base bound in the active site 00:25:10.520 --> 00:25:12.960 via a lysine. 00:25:12.960 --> 00:25:15.980 PLP is very important for transamination reactions, 00:25:15.980 --> 00:25:20.990 which are essential for the metabolism of all amino acids. 00:25:20.990 --> 00:25:24.230 We have seen the transamination reaction where an amino acid 00:25:24.230 --> 00:25:30.050 1 reacts with alpha keto acid 2 and the PLP catalyzed reaction 00:25:30.050 --> 00:25:33.620 forms an alpha keto acid 1 and amino acid 2, 00:25:33.620 --> 00:25:35.180 essentially transferring the group-- 00:25:35.180 --> 00:25:38.660 the amino group from the amino acid to the alpha keto acid. 00:25:38.660 --> 00:25:40.700 The reaction occurs in two steps, 00:25:40.700 --> 00:25:44.730 where first the amino acid is transferred to PLP to form PMP. 00:25:44.730 --> 00:25:47.365 Then PMP then transfers this group-- 00:25:47.365 --> 00:25:49.595 the amino group-- back to a alpha keto acid 00:25:49.595 --> 00:25:51.230 to generate a new amino acid. 00:25:54.180 --> 00:25:56.070 As we saw, the mechanism of transamination 00:25:56.070 --> 00:25:57.640 involves multiple steps. 00:25:57.640 --> 00:26:04.450 The first step is a transimination reaction 00:26:04.450 --> 00:26:08.080 where the Schiff's base that's formed between the active site 00:26:08.080 --> 00:26:12.170 lysine and the PLP becomes a Schiff's 00:26:12.170 --> 00:26:15.760 base between the incoming amino acid and PLP. 00:26:15.760 --> 00:26:18.370 Next, we have a proton transfer, which 00:26:18.370 --> 00:26:21.280 is allowed by the ability of the PLP ring 00:26:21.280 --> 00:26:25.420 to stabilize a negative charge, and move 00:26:25.420 --> 00:26:27.040 the proton from the alpha position 00:26:27.040 --> 00:26:34.460 to somewhere on the PLP ring via a quinoid structure. 00:26:34.460 --> 00:26:37.430 And finally, that the resulting Schiff base 00:26:37.430 --> 00:26:42.580 is hydrolyzed to generate PMP and an alpha keto acid. 00:26:42.580 --> 00:26:44.470 In the second part of the reaction, 00:26:44.470 --> 00:26:48.640 PMP now reacts with alpha keto acid to form a new Schiff base, 00:26:48.640 --> 00:26:52.860 and then the proton transfer happens in reverse, via, again, 00:26:52.860 --> 00:26:56.350 a quinoid structure, to generate the Schiff base 00:26:56.350 --> 00:27:00.730 corresponding to the PLP and the new amino acid 2. 00:27:00.730 --> 00:27:02.715 Which, via a transamination reaction 00:27:02.715 --> 00:27:07.810 will generate amino acid 2 and the PLP enzyme bound. 00:27:07.810 --> 00:27:10.420 Finally, we mentioned that PLP is very versatile, 00:27:10.420 --> 00:27:12.160 and it can catalyze other reactions, 00:27:12.160 --> 00:27:14.890 such as racemization, for example, 00:27:14.890 --> 00:27:17.920 switching the configuration of the alpha carbon from L 00:27:17.920 --> 00:27:22.510 alanine to D alanine, or de-carboxylation, generating 00:27:22.510 --> 00:27:24.520 alpha aminobutyric acid, or GABA, 00:27:24.520 --> 00:27:27.790 an important neurotransmitter from glutamate.