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1 MIT OpenCourseWare Principles of Chemical Science, Fall 2005 Please use the following citation format: Sylvia Ceyer and Catherine Drennan, Principles of Chemical Science, Fall (Massachusetts Institute of Technology: MIT OpenCourseWare). (accessed MM DD, YYYY). License: Creative Commons Attribution-Noncommercial-Share Alike. Note: Please use the actual date you accessed this material in your citation. For more information about citing these materials or our Terms of Use, visit:

2 MIT OpenCourseWare Principles of Chemical Science, Fall 2005 Transcript Lecture 36 All right. For this half of the course, we talked about chemical equilibrium, acid-base. We talked oxidation reduction. We talked about shapes of molecules. We talked about this VSEPR theory. We talked about transition metals and kinetics, which we just ended on Monday. All of these things represent the basic principles of how enzymes work. Enzymes work by acid-base catalysis. They catalyze oxidation reduction reactions. And so if we want to understand biochemistry, you need to understand these basic principles of chemistry. That is how I got into chemistry in the first place, was this connection. And, as I mentioned last time, inhibition of enzymes is an important area. And we need to understand how the enzymes work if we want to inhibit them. Well, why do we want to inhibit them? Well, inhibition of enzymes is used in the treatment of a lot of different conditions. Headaches. This time a year a lot of us walk around carrying a bottle. Sometimes the small size isn't going to really quite do it. We might get a little larger. Here is ibuprofen. There are a number of headaches that occur around now. And headaches are treated by inhibiting an enzyme. Someone has designed an inhibitor for an enzyme, and the enzymes are called prostaglandin and synthesis or cyclooxygenases. And these materials, like aspirin and stuff, bind to the enzyme and inhibit them. And, to get good inhibitors, we need to know how the enzyme works. And so some of you have heard a lot of news recently about Vioxx. That was a drug that was designed to target the enzymes. They were very proud of themselves for trying to make the drug very specific to a particular type of cylcooxygenase, enzymes that would only inhibit the class two and not one. And it turns out that was a great idea, except that it turned out to be dangerous. We will see how that plays out. But, all of that, they are targeting enzymes. Arthritis. A lot of drugs to treat cancer are inhibiting enzymes. I mentioned HIV, that there are inhibitors against HIV protease, which is an enzyme. And so the people have designed inhibitors against that enzyme. Designing inhibitors against enzymes is a big part of the pharmaceutical industry.

3 And, to really have a good inhibitor, a good pharmaceutical product, you need to know a lot about how the enzyme works and also something about how the cell works so that you can make a good product. And this is big money. Understanding these basic principles then allows you to understand how enzymes work. I am going to take one enzyme, and I am going to tell you about the enzyme. I am going to tell you about how this enzyme works to do its reaction. And I am going to use, in telling you about the enzyme, all of these things. All of these different topics you need to know to understand this one enzyme. And, if you understand these different topics, you will, in fact, be able to understand how this enzyme works. The enzyme, it shouldn't surprise you, is vitamin B12 dependent, my favorite vitamin. And it is called methionine synthase. I have mentioned this a little bit in the course of this semester, and we are going to go back over some of these things as a review. This is going to serve as a review of the different topics that we have covered, at the same time showing how these basic principles apply to other fields in chemistry and biology. We will start at the back. We will start with kinetics. That is easy. Methionine synthase is an enzyme, and enzymes are catalysts. Catalysts speed up reactions, make them go faster. And the speed of reactions is what kinetics is. Here is the reaction that methionine synthase catalyzes. It converts an amino acid called homocysteine into another amino acid called methionine. And it takes one form of folic acid, which is another B vitamin, and converts it to another form of folic acid. And this reaction is very important in your body. It is important for the proper formation and maintenance of your spinal cord. And women who are pregnant need to take lots of folic acid to make sure that they have enough for this reaction to go so the baby is born with a properly formed brain and spinal column. Inhibition is connected with neural tube defects. Homocysteine is linked with heart disease. You want to make sure this enzyme is working very well. You will have too much homocysteine, and that is a big indicator of problems with heart disease. Also, you need this tetrahydrofolate, this one form of folic acid for DNA biosynthesis. Some people have thought about inhibiting this enzyme to prevent this reaction, and that would lead to stopping cancer cells from growing. Of course, if they do that, they might cause people then to have heart problems.

4 There are definitely balances, and that is why the pharmaceutical industry often gets in trouble, because you solve one problem and then create another one. You really need to know a lot about how the system works as a whole to be able to design good inhibitors. So, this is a really important reaction. For this reaction to go in the body, you need a catalyst, you need this enzyme. What do catalysts do? This is review of what we talked about Monday. Catalysts act by lowering the activation energy barrier, lowering EA, the activation energy barrier for the forward reaction and also for the reverse reaction. They do not change the overall thermodynamics, they do not change delta G, they do not change the equilibrium constant, but they do speed up the reaction by lowering this barrier. There is a much higher barrier without the catalyst, lower barrier. It stabilizes. Another way to think about it is it stabilizes the activated state or the transition state. Catalysts are not in the overall equation, they are not consumed in the reaction, they are just added to make the reaction go faster. That is a catalyst. Enzymes are the catalyst of life. Transition metals. We are moving backwards through the semester of transition metals. This one, lots of transmission metals. It uses vitamin B12, which has cobalt, and it used zinc. Here is the methyl form of vitamin B12, also called methylcobalamin, and it has a corn ring. A corn ring contributes four nitrogen ligands to the central cobalt, so it is a tetradentate ligand. And that tetradentate ligand coordinates the cobalt. It is a chelate. And so we can review what the chelate effect is. A chelating ligand has multiple points of attachment to the particular metal. And they are unusually stable. Why are chelates unusually stable? What factor is it that causes that unusual stability? It is entropy. It is entropic effects, because usually you are replace four bound waters, in this case, with one ligand. You are putting one thing on and releasing four. And it is easier for four things to be disordered than for one thing to be disordered. That gives you a favorable entropic effect. The chelate effect has to do with entropy. Now let's consider the zinc. It also needs zinc for this to work. Zinc plus two, what would be the d-count? How many d electrons are you going to have? For that, we would go to the Periodic Table, find zinc and see which group it is in.

5 It is 12. The oxidation number is 2. We are at 12 minus 2 or a d10 state. What would you guess would be the color of this zinc site? Colorless. And that is because you are not going to be able to have any d-to-d transitions because you have ten electrons, everything is full. There are five orbitals and they all have two electrons each, so it is completely full. You would have a colorless system. Now, people did not realize methionine synthase was a zinc enzyme for a long time. Vitamin B12 has a beautiful red color, and zinc had no color. They knew it was a B12 dependent enzyme, but people were studying this enzyme for years and years and years before they realized that zinc was bound to the protein because it has no color. What about VSEPR. VSEPR allows you to predict or rationalize shapes of molecules based on a simple idea of repulsion. That lone pairs want to fill up space and move the bound atoms away. That the structure is designed such that there is minimal repulsion between bound electrons or electrons in orbitals. If we consider this then, if we look at the steric number for the whole center around the cobalt. Again, there is this tetradentate corn ring system and there is one ligand above and one ligand below. And so what does that give for our steric number? Six. We have four in the equatorial positions and two in the axial positions. What formula would this be? What is first? A then X then six. We have a central atom A and we have six things around it. And what would be the geometry of something that has six things around it? Octahedral. And here we have our octahedral system. If I hold it like this, the equatorial positions are filled with the corn wring, have a methyl group on top. And we have another ligand on the bottom. What are the angles for octahedral? 90, thank you. All right. The zinc site, I will tell you a little bit about. Three ligands were identified to zinc. And so geometry is going to have x equals three. If we don't know what the ligands are, we might not know about lone pairs. But if you have three things bound, what is the most common geometry? Trigonal planar is pretty common with three things bound, so that might be a good guess.

6 And so it might look something like this. And what would be the angles, if that was the correct geometry? Right, 120. OK. We know something about it from VSEPR. All right. Oxidation-reduction. This enzyme catalyzes an oxidation-reduction reaction. Here is the cycle of turnover for this enzyme. We have a lot of different reactions going on in this enzyme. Here is an abbreviation for the cobalamin cofactor. We just have the cobalt and its oxidation number of one here. Sometimes, instead of going around in this primary turnover, its normal sort of cycle where it is reacting with the folic acid and reacting with the homocysteine. Instead, sometimes an electron is lost. And, for it to kind of come back into the catalytic cycle, the enzyme needs to be activated. And so it becomes co-two state. It loses an electron and becomes oxidized. And then you have to re-reduce it and you have to methylate it to go back into this form. A methyl is just a carbon with three hydrogens. The methyl group comes from this compound called S-adenosyl methionine, and it gets an electron for the reduction from flavodoxin. If we look at this oxidation reduction, and we talked about this a big earlier, too, but it is a really nice review for oxidation reduction. We have a standard potential for vitamin B12 of minus volts and a standard potential for flavodoxin of minus 0.23 volts. Which of these is a better reducing agent? B12 is a better reducing agent. A large negative reduction potential means that the reduced species is very reducing. If it is a good reducing agent, it likes to reduce other things and be oxidized itself, so B12 has a low negative number. If we do the math here and calculate a delta E for the cell, and, again, the same equations can apply, if you are talking about a battery or you are talking about a biological process. We have the standard reduction potential for the reaction that is a reduction, and the standard reduction potential for the reaction that is acting as the oxidation. And, in this biological case, flavodoxin is reducing vitamin B12. Flavodoxin is getting oxidized and B12 is getting reduced. And, if we put in the numbers, we find that delta E is minus Is that going to be a spontaneous reaction? No, it won't be a spontaneous reaction. Because if this is negative, if delta E is negative then delta G is going to be positive. And if delta G is positive then it is not spontaneous.

7 And we did this math before, but just as a review you can calculate delta G if you know the delta E. It is just minus the number of electrons involved times Faraday constant times your delta E, your change in the cell potential. That would be minus one, it is a one electron reduction, times Faraday constant times the cell potential we calculated. And we get positive 28.6 kilojoules per mole. That is not spontaneous. But, as I mentioned, the cells come up with a way to do this. It has to do this reaction or none of us would be alive. This is an important reaction for us. The way that it does that is it couples it with another reaction. So, at the same time the reduction is happening, you are cleaving that a S-adenosyl methionine. It is giving up the methyl group, which is important for the reaction. But also this is very favorable, minus 37.6 kilojoules per mole. That allows the reaction to go. That sort of pumps in the energy that is needed for this unfavorable oxidation-reduction reaction to occur. Cells that require energy to catalyze non-spontaneous reactions are called what? Right, electrolytic. And if it is a spontaneous reaction it is what? Galvanic, right. Here, in this one example, we talk about the connection between thermodynamics and oxidation-reduction, and we talk about oxidizing agents and reducing agents. All right. The enzyme does an oxidation-reduction. It is a catalyst, it speeds up the reaction, it has two transition metals in it and it uses acid-base catalysis as well. Let's talk about acid-base. There is an acid-base reaction. There is catalysis by a Lewis acid in this part of the reaction, the reaction with homocysteine, the conversion to methionine. Let's talk about this. The B12 is going to give up this methyl group, two homocysteine to make methionine. It turns out that only the deproteinated homocysteine is reactive. Here is homocysteine. It is an amino acid. This is proteinated. The sulfur down here has a proton. This is deproteinated homocysteine. The sulfur here has no proton. It has a negative charge. And only this homocysteine here that is deproteinated with the sulfur with the negative charge can be methylated. The reaction catalyzed transfers this methyl group, this CH3 group and puts it on the sulfur, and that makes the amino acid methionine. But if there is a proton there, the methyl transfer does not happen. It has to be deproteinated.

8 At physiological ph, we need to ask the question, how much homocysteine is deproteinated? Remember, our body is buffered. It is all around the same ph. We want to know at this ph, how much of the homocysteine is in the deproteinated form and how much of the homocysteine is in the proteinated form? What do we need to know, what value do we need to know to be able to answer, at a particular ph, how much HA you have and how much A minus you have? What do we need to know? We need to know the pka. Question. What is the pka? Here it is ten. That is a big number. If we know the physiological ph, we know the pka, what equation can we use to find the ratio of HA to A minus? Henderson-Hasselbach. We can do the math. Physiological ph is around 7.4. The pka is ten. What is the ratio of proteinated to deproteinated, the ratio of this proteinated homocysteine to the deproteinated? It turns out to be 400 proteinated for every one deproteinated. Well, that is bad news for us b/c it needs to be deproteinated to be reactive. At physiological ph then, it is proteinated and nonreactive. Here the enzyme has to come in. Enzymes catalyze reactions. The enzyme needs to get involved and make this reaction happen. Again, this is unfavorable without the enzyme. Let's think a little bit about ph and pka for a minute. Let's review some acid-base titration issues. This is not in your handout, I don't think, it has been there before, but let's just take a look at it. You should remember what these titration curves look like. Remember at the half equivalence point ph equals pka. And that happens b/c at the half equivalence point half of the acid has been converted to the conjugate base. These are equal quantities. And so they will cancel out in the Henderson-Hasselbach and this will be true. You have equal amounts. If you are at a ph that is below the half equivalence point, you will be more proteinated. If you are at a ph that is above the half equivalence point, you will be more deproteinated. We have not maybe discussed this in these terms, but that is how you can think about this problem. Now let's go back to methionine synthase, and this is in your handout. At the half equivalence point in a titration, or when the ph equals the pka, half of your material is proteinated, half of your material is deproteinated. If your ph is below that pka, you are more proteinated.

9 If you are a little bit below, you still have some deproteinated. If you are very far below, it is all proteinated. Going the other direction, if you are at ph above the pka, you have mostly deproteinated, maybe a little proteinated. And when you get far away, it is all deproteinated. Now we can put in what we know about homocysteine. The pka is 10 and we are at ph 7.4, so we are quite a bit below the pka. And we calculated that at that ph with this pka, we would have 400 proteinated to every one deproteinated. I am not going to write out 400 of these to be proteinated for every one deproteinated, but we are mostly proteinated. All right. That is the problem, b/c we don't want to be proteinated. We want to be deproteinated for the enzyme to work. What does the enzyme do? Well, part of how the enzyme catalyzes the reaction is that it lowers the pka of the homocysteine. Instead of 10, which is what the pka is with free homocysteine, the pka now, when homocysteine is bound to the enzyme is 6. That is a pretty substantial drop. The pka is substantially lowered. Now let's go and look at what that does for us in terms of the reaction that is catalyzed. All right. What is happening here is the zinc. Here comes the zinc. The zinc is acting as a Lewis acid and it is coordinating, binding that homocysteine. And that interaction between the zinc and the sulfur of the homocysteine lowers the pka of the homocysteine from 10 to 6. That is why this protein has to have the zinc to act as the Lewis acid. We can go back to Henderson-Hasselbach and plug in our new values. We are still at physiological ph 7.4. But now the pka is 6. And so our ratio then, of proteinated to deproteinated, instead of 400 to one is now one to 25. There is more deproteinated homocysteine when it is bound to the enzyme at this physiological ph b/c the pka is lower. And if we look at that in terms of this again. Here is the pka. The proteinated is equal to deproteinated, and our pka now is 6 for the bound homocysteine. And now the ph, the physiological ph is above the pka. It is at 7.4. And, above the pka, there is more deproteinated, 25 deproteinated to one proteinated. Now we are in good shape. You need the deproteinated for it to be reactive. Most of it is deproteinated. The enzyme can go.

10 The enzyme uses this Lewis acid catalysis to drive this reaction forward in the direction that you want. It is all about adjusting. Enzymes can work by adjusting pkas. All right. Chemical equilibrium, the first thing we started with in the second half of the semester. What is true about methionine synthase is that it exists in multiple confirmations. This enzyme can have multiple states. And these confirmations are in equilibrium with each other. Lots of things can be in equilibrium, including enzymes. And equilibrium is a dynamic process. What is happening? Well, let's look at why this enzyme would need multiple states. Well, it needs to react with homocysteine, it needs to react with the folic acid and it needs to react with the S-adenosyl methionine and flavodoxin to do this reaction over here. If we look at the structure of the enzyme, the vitamin B12 is in green, the methyl group that is going to be transferred is in red and all this yellow and white is more protein. Homocysteine, when it is deproteinated, needs to come in here and accept that methyl group. Folic acid needs to come in here and donate the methyl group. And also S-adenosyl methionine, in the activation reaction, needs to methylate at the same time the electron is added to the system. The enzyme needs to position all of these guys up here. And, if you look at the structure, there is no room for anything to fit into the structure. Nothing can kind of go over. You can just barely see the methyl group. But these guys are kind of big and are not going to fit in here. B/c in this one confirmation of the enzyme they do not fit. The answer has to be that the enzyme moves, it changes shape during the reaction. And this is something that had been known for enzymes, that they did this for quite some time, but my father told all our neighbors that his daughter had discovered that enzymes change shape. [LAUGHTER] But, since none of our neighbors knew anything about biochemistry, even what an enzyme was, I think that that was just fine. All right. So this needs to change shape. This is a picture of the structure. Here is the B12. The red is the methyl group again. This whole region needs to move to make room for those reactions to happen. There has got to be a number of different states of this protein. Here is a little cartoon. In orange is where the B12 binds to the protein. The B12 is in red. This region above we call the methyl cap.

11 It sits over the methyl group and protects it. There needs to be a domain to bind folic acid, another domain binds homocysteine and another domain over here binds the S-adenosyl methionine and is involved in that oxidation-reduction reaction and the remethylation. What has to be true for this enzyme is that you need at least four different confirmations. You need a confirmation where the folic acid sits on top of a B12. It needs to give the methyl group over. You need a confirmation where the homocysteine in yellow domain sits on top of the B12 and accepts the methyl group. Zinc needs to be around there, too. You need a confirmation that is resting where the region that we saw in the initial crystal structure is sitting over this cap. It is capping the B12. And you need a confirmation where this activating domain is above the B12 to do the oxidation-reduction reaction there. So we need at least these four confirmations. And what is true is that the enzyme is just kind of switching confirmation as it is going through, and that these confirmations are in equilibrium with each other and are just shifting back and forth to these different states. And, when you add something like folic acid, it helps shift the confirmation to this form. When you add homocysteine, it helps shift the confirmation to this form. This is just chemical equilibrium going on. There is a lot of things that can be in equilibrium. And enzyme confirmations can be in equilibrium, just like some of the reactions that we studied in the beginning of the course. Enzymes are dynamic. Equilibrium is a dynamic process. Chemistry is dynamic. Things are happening. I am not talking about chemistry in the solid state in this course. I am talking about chemistry in solution b/c that is chemistry of life. That is the chemistry that is happening in your body that allows you to breath and life. And so, finally, I am going to show you methionine synthase in action in its dynamic process.