WEBVTT 00:00:00.500 --> 00:00:02.840 The following content is provided under a Creative 00:00:02.840 --> 00:00:04.380 Commons license. 00:00:04.380 --> 00:00:06.680 Your support will help MIT OpenCourseWare 00:00:06.680 --> 00:00:11.070 continue to offer high quality educational resources for free. 00:00:11.070 --> 00:00:13.670 To make a donation or view additional materials 00:00:13.670 --> 00:00:17.630 from hundreds of MIT courses, visit MIT OpenCourseWare 00:00:17.630 --> 00:00:18.800 at ocw.mit.edu. 00:00:30.782 --> 00:00:33.240 DR. BOGDAN FEDELES: Hello, and welcome to 5.07 Biochemistry 00:00:33.240 --> 00:00:34.300 online. 00:00:34.300 --> 00:00:36.100 I'm Dr. Bogdan Fedeles. 00:00:36.100 --> 00:00:38.250 Let's metabolize some problems. 00:00:38.250 --> 00:00:41.830 Today we're discussing problem 2 of problem set 6. 00:00:41.830 --> 00:00:43.990 Here we're going to explore in more detail 00:00:43.990 --> 00:00:47.170 the mechanism of phosphoglycerate mutase, which 00:00:47.170 --> 00:00:48.920 is the eighth enzyme in glycolysis. 00:00:48.920 --> 00:00:51.430 It's the enzyme that catalyzes the conversion 00:00:51.430 --> 00:00:54.820 of 3-phosphoglycerate to 2-phosphoglycerate. 00:00:54.820 --> 00:00:57.130 Generally speaking, mutases are enzymes 00:00:57.130 --> 00:00:58.900 that catalyze the shift of a functional 00:00:58.900 --> 00:01:02.540 group between two similar positions of a molecule. 00:01:02.540 --> 00:01:04.540 In the case of phosphoglycerate mutase, 00:01:04.540 --> 00:01:06.820 this enzyme catalyzes the transfer 00:01:06.820 --> 00:01:09.935 of the phosphate group from the 3 position of glycerate 00:01:09.935 --> 00:01:12.190 to the 2 position of glycerate. 00:01:12.190 --> 00:01:15.730 In 5.07, you will encounter several mutases. 00:01:15.730 --> 00:01:17.860 Similar to phosphoglycerate mutase, 00:01:17.860 --> 00:01:20.020 there is a bisphosphoglycerate mutase, 00:01:20.020 --> 00:01:22.420 which converts 1,3-bisphosphoglycerate 00:01:22.420 --> 00:01:24.480 to 2,3-bisphosphoglycerate. 00:01:24.480 --> 00:01:26.350 Now, this reaction is very important 00:01:26.350 --> 00:01:29.020 when it happens in the red blood cells. 00:01:29.020 --> 00:01:31.960 Another mutase you will encounter 00:01:31.960 --> 00:01:34.470 is in the glycogen breakdown pathway. 00:01:34.470 --> 00:01:37.390 It's called phosphoglucomutase and converts 00:01:37.390 --> 00:01:40.780 glucose 1-phosphate to glucose 6-phosphate. 00:01:40.780 --> 00:01:43.720 Now finally, the most intriguing of them 00:01:43.720 --> 00:01:46.540 all is the methylmalonyl-coa mutase, 00:01:46.540 --> 00:01:50.650 which is a fascinating enzyme that converts methylmalonyl-coA 00:01:50.650 --> 00:01:52.530 to succinyl-coA. 00:01:52.530 --> 00:01:57.250 In this reaction, it rearranges this carbon skeleton 00:01:57.250 --> 00:02:01.360 of the molecule, and it requires adenosylcobalamin, 00:02:01.360 --> 00:02:04.330 which is a co-factor derived from vitamin B12. 00:02:04.330 --> 00:02:08.139 Back to phosphoglycerate mutase, this is a fascinating enzyme 00:02:08.139 --> 00:02:12.110 because it uses a phosphorylated histidine in the active site. 00:02:12.110 --> 00:02:15.490 And this is actually an example of a phosphorous-nitrogen bond, 00:02:15.490 --> 00:02:18.820 one of the very few available in biochemistry. 00:02:18.820 --> 00:02:21.100 Here is a schematic of the mechanism 00:02:21.100 --> 00:02:23.500 of phosphoglycerate mutase. 00:02:23.500 --> 00:02:27.310 Now, the reaction starts where the enzyme 00:02:27.310 --> 00:02:28.870 is already phosphorylated. 00:02:28.870 --> 00:02:30.430 We'll call it a phospho enzyme. 00:02:30.430 --> 00:02:32.260 And the histidine in the active site 00:02:32.260 --> 00:02:34.380 contains the phosphate group. 00:02:34.380 --> 00:02:38.480 Then the enzyme binds the substrate 3-phosphoglycerate. 00:02:38.480 --> 00:02:41.470 And then it's going to transfer this phosphate group onto the 2 00:02:41.470 --> 00:02:45.400 position, the 2-hydroxyl of the 3-phosphoglycerate to generate 00:02:45.400 --> 00:02:47.860 the 2,3-bisphosphoglycerate. 00:02:47.860 --> 00:02:50.800 Then the phosphate at the 3 position 00:02:50.800 --> 00:02:53.740 is transferred to the histidine to generate 00:02:53.740 --> 00:02:56.440 the product of the reaction 2-phosphoglycerate 00:02:56.440 --> 00:03:01.130 and regenerate the phosphoenzyme. 00:03:01.130 --> 00:03:04.430 Note that the phosphate group is in fact not transferred. 00:03:04.430 --> 00:03:06.890 This phosphate group here is not the same 00:03:06.890 --> 00:03:09.050 that ends up on the 2 position. 00:03:09.050 --> 00:03:12.740 But rather, this phosphate group gets transferred to the enzyme. 00:03:12.740 --> 00:03:15.080 And the phosphate group from the enzyme 00:03:15.080 --> 00:03:18.872 ends up on the second position of glycerate. 00:03:22.570 --> 00:03:25.260 As we just mentioned, the active form of the enzyme 00:03:25.260 --> 00:03:28.632 has already the phosphate bound to histidine. 00:03:28.632 --> 00:03:30.840 Now the question is, how did this phosphate get there 00:03:30.840 --> 00:03:32.160 in the first place? 00:03:32.160 --> 00:03:35.370 Presumably, the enzyme is first synthesized in a form 00:03:35.370 --> 00:03:38.850 that we call apo form, which does not have the phosphate. 00:03:38.850 --> 00:03:41.580 And the phosphate is then added as a post-translational 00:03:41.580 --> 00:03:43.170 modification. 00:03:43.170 --> 00:03:46.710 Now, our problem suggests that one source for this phosphate 00:03:46.710 --> 00:03:48.780 is phosphoenolpyruvate. 00:03:48.780 --> 00:03:50.540 And there's some experimental evidence 00:03:50.540 --> 00:03:53.580 that phosphoenolpyruvate can transfer their phosphate 00:03:53.580 --> 00:03:56.970 and phosphorylate the histidine in this enzyme. 00:03:56.970 --> 00:03:59.190 Now, we are asked to comment on how 00:03:59.190 --> 00:04:01.500 reasonable this proposal is. 00:04:01.500 --> 00:04:04.260 We're going to evaluate the proposed transformation 00:04:04.260 --> 00:04:07.770 between phosphoenolpyruvate and phosphoglycerate mutase 00:04:07.770 --> 00:04:09.400 from two points of view. 00:04:09.400 --> 00:04:11.400 First of all, is this transformation 00:04:11.400 --> 00:04:13.830 thermodynamically accessible? 00:04:13.830 --> 00:04:17.990 And second, is this structurally feasible? 00:04:17.990 --> 00:04:20.690 We know that phosphoenolpyruvate contains 00:04:20.690 --> 00:04:23.150 a high-energy phosphate bond that 00:04:23.150 --> 00:04:26.540 can release a lot of energy upon hydrolysis. 00:04:26.540 --> 00:04:30.050 Now, if we look in our book, this 00:04:30.050 --> 00:04:32.400 is the Voet & Voet, Third Edition. 00:04:32.400 --> 00:04:35.240 If we look here, phosphoenolpyruvate, 00:04:35.240 --> 00:04:39.260 it says, releases about 62 kilojoules per mole 00:04:39.260 --> 00:04:41.060 upon hydrolysis. 00:04:41.060 --> 00:04:44.510 Now this is significantly more than what 00:04:44.510 --> 00:04:47.210 energy is released by the hydrolysis of ATP 00:04:47.210 --> 00:04:52.150 going to ADP, which is only about 31 kilojoules per mole. 00:04:52.150 --> 00:04:54.260 Now, this should not be surprising to you 00:04:54.260 --> 00:04:57.530 because PEP, phosphoenolpyruvate, 00:04:57.530 --> 00:04:59.790 is used in the last step of glycolysis, 00:04:59.790 --> 00:05:03.620 the pyruvate kinase, to phosphorylate ADP and generate 00:05:03.620 --> 00:05:04.710 ATP. 00:05:04.710 --> 00:05:07.640 So the fact it has a higher energy of hydrolysis, 00:05:07.640 --> 00:05:09.830 it just makes that transformation 00:05:09.830 --> 00:05:11.900 thermodynamically accessible. 00:05:11.900 --> 00:05:14.600 Let's now take a look at the arrow pushing mechanism 00:05:14.600 --> 00:05:17.860 of how phosphoenolpyruvate can phosphorylate 00:05:17.860 --> 00:05:20.180 phosphoglycerate mutase. 00:05:20.180 --> 00:05:22.880 Here is the phosphoenolpyruvate molecule, 00:05:22.880 --> 00:05:26.600 and here is the histidine in the active site of the enzyme. 00:05:26.600 --> 00:05:31.330 Now, as you know, histidine has a pKa of about 6, 00:05:31.330 --> 00:05:34.150 so an important fraction of the histidine 00:05:34.150 --> 00:05:36.670 will be protonated at physiological pH. 00:05:36.670 --> 00:05:38.560 However, for this reaction to work 00:05:38.560 --> 00:05:40.600 we need the histidine to act as a nucleophile 00:05:40.600 --> 00:05:41.950 to attack the phosphate. 00:05:41.950 --> 00:05:45.830 Therefore, we're going to consider it deprotonated. 00:05:45.830 --> 00:05:48.100 Now, the reaction starts by assuming 00:05:48.100 --> 00:05:50.470 there's a base in the active site that's 00:05:50.470 --> 00:05:53.100 going to deprotonate the histidine, 00:05:53.100 --> 00:05:56.170 and then it's going to attack the phosphate. 00:05:56.170 --> 00:05:57.640 And finally, the phosphate group is 00:05:57.640 --> 00:06:02.110 going to leave with the assistance of a general acid. 00:06:02.110 --> 00:06:06.180 So these are the products that we obtain. 00:06:06.180 --> 00:06:09.240 This is the phosphoenzyme with the histidine that now has 00:06:09.240 --> 00:06:11.070 the phosphate group attached. 00:06:11.070 --> 00:06:15.810 And this is the enol that is released 00:06:15.810 --> 00:06:17.880 from the phosphoenolpyruvate, which is 00:06:17.880 --> 00:06:20.200 the enol form of the pyruvate. 00:06:20.200 --> 00:06:24.900 Now, if we evaluate the starting material and the product 00:06:24.900 --> 00:06:27.150 in terms of their ability to stabilize 00:06:27.150 --> 00:06:30.030 negative charge, such as the charges on the phosphate, 00:06:30.030 --> 00:06:31.170 by resonance-- 00:06:31.170 --> 00:06:34.440 We notice that there is no significant difference. 00:06:34.440 --> 00:06:37.980 Here we have two negative charges and one set 00:06:37.980 --> 00:06:40.440 of phosphorus oxygen bonded. 00:06:40.440 --> 00:06:42.910 The charge can delocalize on this oxygen. 00:06:42.910 --> 00:06:47.830 We also have this carboxylate group, which we also have here. 00:06:47.830 --> 00:06:52.110 So there's not a lot of resonant stabilization between starting 00:06:52.110 --> 00:06:54.010 materials and products so far. 00:06:54.010 --> 00:06:57.870 Therefore, this reaction is thermodynamically 00:06:57.870 --> 00:07:00.550 close to neutral. 00:07:00.550 --> 00:07:03.960 However, notice the enol form of pyruvate. 00:07:03.960 --> 00:07:07.230 Now this is in fact a very unstable product. 00:07:07.230 --> 00:07:11.460 And it likes to tautomerize, basically 00:07:11.460 --> 00:07:13.590 isomerize in acid base conditions 00:07:13.590 --> 00:07:16.260 to the keto form of a pyruvate. 00:07:16.260 --> 00:07:19.300 The mechanism would be, as such, the base 00:07:19.300 --> 00:07:21.130 can deprotonate the enol. 00:07:21.130 --> 00:07:26.960 And then, the general acid can protonate CH2 group 00:07:26.960 --> 00:07:29.120 to generate the keto form of the pyruvate. 00:07:29.120 --> 00:07:31.880 It turns out the delta G for this transformation 00:07:31.880 --> 00:07:33.580 is very negative. 00:07:33.580 --> 00:07:40.810 Delta G here is approximately minus 40 kilojoules per mole. 00:07:40.810 --> 00:07:45.490 So that means this reaction is strongly going to the right, 00:07:45.490 --> 00:07:47.770 and strongly favors the keto form. 00:07:47.770 --> 00:07:51.910 So that means per ensemble the transformation going from PEP 00:07:51.910 --> 00:07:53.950 in our histidine in the active site 00:07:53.950 --> 00:07:57.850 is going to be strongly driven to the right because 00:07:57.850 --> 00:08:00.240 of this keto equilibrium. 00:08:00.240 --> 00:08:02.830 Therefore, the entire process shown here 00:08:02.830 --> 00:08:04.930 is expected to be thermodynamically very 00:08:04.930 --> 00:08:06.190 favorable. 00:08:06.190 --> 00:08:09.130 Now let's take a look at some structural considerations. 00:08:09.130 --> 00:08:11.560 In order for PEP to phosporylate the enzyme, 00:08:11.560 --> 00:08:14.380 it has to be able to reach the histidine that's 00:08:14.380 --> 00:08:16.070 deep in the active site. 00:08:16.070 --> 00:08:18.190 Notice that the 2-phosphoglycerate 00:08:18.190 --> 00:08:22.040 is one of the products or substrates of the enzyme. 00:08:22.040 --> 00:08:25.480 And therefore, it fits very nicely in the active site. 00:08:25.480 --> 00:08:29.650 Now phosphoenolpyruvate looks a lot like 2-phosphoglycerate. 00:08:32.380 --> 00:08:36.840 Going to sketch it here, going to have the phosphate there, 00:08:36.840 --> 00:08:40.200 and then there's the double bond in this position. 00:08:40.200 --> 00:08:44.159 So because phosphoenolpyruvate looks 00:08:44.159 --> 00:08:47.820 a lot like 2-phosphoglycerate it should have no problem fitting 00:08:47.820 --> 00:08:49.830 inside the active site of the enzyme 00:08:49.830 --> 00:08:53.120 and reaching the active site histidine. 00:08:53.120 --> 00:08:56.010 Therefore, the chemical reaction proposed in this problem 00:08:56.010 --> 00:08:57.330 is quite reasonable. 00:08:57.330 --> 00:08:59.460 First of all, the thermodynamics are 00:08:59.460 --> 00:09:02.640 excellent because the hydrolysis of phosphoenolpyruvate 00:09:02.640 --> 00:09:03.990 gives a lot of energy. 00:09:03.990 --> 00:09:07.650 And then the sterics are also favorable because PEP 00:09:07.650 --> 00:09:10.080 resembles 2-phosphoglycerate, one 00:09:10.080 --> 00:09:11.420 of the products of the enzyme. 00:09:14.410 --> 00:09:18.030 Part 2 of this problem asks us to evaluate the consequences 00:09:18.030 --> 00:09:21.310 on the major function of glycolysis of this reaction 00:09:21.310 --> 00:09:24.370 that we just discussed-- of phosphoenolpyruvate 00:09:24.370 --> 00:09:27.030 with phosphoglycerate mutase. 00:09:27.030 --> 00:09:29.160 Here is the second half of glycolysis, 00:09:29.160 --> 00:09:31.980 going from glyceraldehyde phosphate, or GAP, all the way 00:09:31.980 --> 00:09:33.520 to pyruvate. 00:09:33.520 --> 00:09:35.530 As you know, the main function of glycolysis 00:09:35.530 --> 00:09:37.850 is to generate ATP. 00:09:37.850 --> 00:09:39.640 And for each molecule of glucose, 00:09:39.640 --> 00:09:42.595 we have a net generation of two molecules of ATP. 00:09:42.595 --> 00:09:45.400 Now, ATP is produced in two places. 00:09:45.400 --> 00:09:49.080 First, at the phosphoglycerate kinase when 00:09:49.080 --> 00:09:52.590 1,3-bisphosphoglycerate can phosphorylate ADP to generate 00:09:52.590 --> 00:09:53.580 ATP. 00:09:53.580 --> 00:09:55.920 And then the pyruvate kinase where 00:09:55.920 --> 00:09:57.720 phosphoenolpyruvate phosphorylates 00:09:57.720 --> 00:10:00.150 ADP to generate ATP. 00:10:00.150 --> 00:10:03.060 Now since we start glycolysis by investing some ATP, 00:10:03.060 --> 00:10:07.590 we need two molecules of ATP to phosphorylate glucose. 00:10:07.590 --> 00:10:10.170 We recover those two molecules of ATP 00:10:10.170 --> 00:10:12.540 at the phosphoglycerate kinase step. 00:10:12.540 --> 00:10:15.750 So all the net production of ATP that we get in glycolysis 00:10:15.750 --> 00:10:20.466 comes from the pyruvate kinase reaction shown here. 00:10:20.466 --> 00:10:23.880 Now, if phosphoenolpyruvate is used 00:10:23.880 --> 00:10:27.190 to phosphorylate phosphoglycerate mutase, 00:10:27.190 --> 00:10:30.210 basically, it's going to react to give 00:10:30.210 --> 00:10:32.700 the phosphate group here. 00:10:32.700 --> 00:10:38.080 It's going to generate pyruvate but without generating ATP, 00:10:38.080 --> 00:10:38.580 right? 00:10:38.580 --> 00:10:41.910 So the phosphate group goes to this phosphoglycerate mutase, 00:10:41.910 --> 00:10:45.010 and it generates pyruvate, but we 00:10:45.010 --> 00:10:47.150 get no net production of ATP. 00:10:47.150 --> 00:10:49.540 Now if PEP is used to phosphorylate 00:10:49.540 --> 00:10:51.760 phosphoglycerate mutase, it's not 00:10:51.760 --> 00:10:55.500 going to be available for the pyruvate kinase step. 00:10:55.500 --> 00:10:59.230 But we do generate pyruvate, so the whole transformation 00:10:59.230 --> 00:11:04.230 reaches pyruvate, but without producing a net amount of ATP. 00:11:04.230 --> 00:11:07.230 Of course, this should not be a significant problem, 00:11:07.230 --> 00:11:10.020 as in glycolysis we only require the enzymes 00:11:10.020 --> 00:11:11.520 in catalytic amounts. 00:11:11.520 --> 00:11:16.980 So initially, we're not going to be generating net amount of ATP 00:11:16.980 --> 00:11:19.530 until we phosphorylate the entire pool 00:11:19.530 --> 00:11:21.240 of phosphoglycerate mutase. 00:11:21.240 --> 00:11:23.505 After that, now we have phosphoglycerate mutase, 00:11:23.505 --> 00:11:27.210 so PEP is once again available for the pyruvate kinase 00:11:27.210 --> 00:11:29.760 reaction to generate ATP. 00:11:29.760 --> 00:11:33.210 I hope you noticed that there is a more subtle question here. 00:11:33.210 --> 00:11:36.310 If we're going to use PEP to phosphorylate 00:11:36.310 --> 00:11:39.390 phosphoglycerate mutase, how are we 00:11:39.390 --> 00:11:42.490 going to get to produce PEP in the first place 00:11:42.490 --> 00:11:45.750 since we need phosphoglycerate mutase to go 00:11:45.750 --> 00:11:48.570 from 3-phosphoglycerate to 2-phosphoglycerate, 00:11:48.570 --> 00:11:50.490 which then produces PEP. 00:11:50.490 --> 00:11:53.040 Again, it's kind of like one of these chicken and the egg 00:11:53.040 --> 00:11:54.690 problems. 00:11:54.690 --> 00:11:57.300 As you'll find out, many pathways 00:11:57.300 --> 00:12:01.050 feed into or intersect with glycolysis. 00:12:01.050 --> 00:12:03.180 And therefore, phosphoenolpyruvate 00:12:03.180 --> 00:12:06.390 could in principle be made in other ways. 00:12:06.390 --> 00:12:08.550 For example, in gluconeogenesis you'll 00:12:08.550 --> 00:12:14.790 see that pyruvate can lead to oxaloacetate, which then 00:12:14.790 --> 00:12:19.000 can lead to phosphoenolpyruvate using this enzyme called 00:12:19.000 --> 00:12:22.560 PEP carboxykinase, or PEPCK. 00:12:22.560 --> 00:12:26.070 So there are ways to produce phosphoenolpyruvate, which 00:12:26.070 --> 00:12:28.920 can then, say, phosphorylate phosphoglycerate mutase, which 00:12:28.920 --> 00:12:34.290 then allows the glycolysis to flow through in the normal way. 00:12:34.290 --> 00:12:36.120 Well, that's it for this problem. 00:12:36.120 --> 00:12:38.430 I hope you found pretty intriguing how 00:12:38.430 --> 00:12:40.690 phosphoglycerate mutase works. 00:12:40.690 --> 00:12:43.020 Now remember, this is one of the very few enzymes 00:12:43.020 --> 00:12:46.950 in biochemistry that utilizes the phosphorylated histidine. 00:12:46.950 --> 00:12:48.720 And this is one of the few examples 00:12:48.720 --> 00:12:52.470 of a phosphorus-nitrogen bond we have in biochemistry. 00:12:52.470 --> 00:12:54.990 Also remember, the phosphoenolpyruvate 00:12:54.990 --> 00:12:58.980 is the highest high-energy phosphate compound 00:12:58.980 --> 00:13:00.730 we have in the body. 00:13:00.730 --> 00:13:03.180 But all that hydrolysis energy really 00:13:03.180 --> 00:13:05.610 comes from the keto enol tautomerization 00:13:05.610 --> 00:13:10.730 equilibrium of the pyruvate that gets released upon hydrolysis.