13_14 Biochemistry Dr. Elliot: Enzymes Pt.

I Jan 8, 2014: 9-10am NT #36 Slide 2: Heres the outline of what were going to cover in the next couple days Slide 3: Heres what we need to know about enzymology. You lovely bunch of people all of whom are the cream of the crop, the top 190 of 13,000 applications, here you are, congratulations. Still in spite of that you are nothing more than a bag of chemicals reacting. And if you are reacting at normal room temperature and normal concentrations of salt solutions at 1 atmosphere pressure I think the temperature outside right now is what 10. How fast are you reacting outside, not very fast. Those biologically relevant biochemical reactions would be going so slow that they are not compatible with life. So we need to speed stuff up and that is what enzymes are for. Enzymes are catalysts. And here is what they are catalyzing. This is the KEGG pathway its Japanese from Kyoto University and it shows you basically where all the interactions of all the different pathways are. Well here they are and were going to be talking about carbohydrate metabolism and the Krebs cycle. So it kind of helps to know what an enzyme is and so we dont just assume you know. Slide 4: Reaction rates this should chem. 101 review its just the change in the concentration of the substrate per unit time. In this case it is a decrease in substrate and an increase in product. There are essential requirements for all reactions, biochemical or not, whether it is happening in a test tube or in a chemistry lab or in one of your cells you have to have a collision between the two molecules that are reacting. It has to be in the proper orientation and you have sufficient energy for the reaction. You can do this by putting it in a test tube and heating it up all the molecules will banging into each other and smashing it up and boom form a new bond. You dont do that in human cell. You have to have a catalyst that helps position the substrates in the right orientation, stabilizes a transition state intermediate and get a favourable energy profile. Those are terms you should have ingrained in your brain. Increasing the temperature, as far as were concerned, increasing the temperature by 10 degrees might double the reaction rate but if I start increasing your temperature by 10 degrees increments Im going to get to the point where Im going to denature you and in that case youre dead although the reactions may go on for a while. So what you need is something that increases the reaction rates without changing the equilibrium. What is a equilibrium constant? It is a constant. So what it is a constant? It is a constant it doesnt change. A constant is a constant is a constant is a constant and each one of these equilibrium constants are unique to those conditions. And if start messing with conditions like throwing out some sodium and bringing in chloride I might tweak the equilibrium a little bit. For the most part the equilibrium constants are constant and enzymes do not change the equilibrium constant they just accelerate the rate at which you

achieve equilibrium. Now here is a word about equilibrium in a biological system. It better not happen. You are a steady state of dynamic equilibrium what does that mean? You take in chemical food. It does magic and you get rid of chemical waste. The it does magic black box and all the intermediates are pretty much at the same concentration throughout all of these pathways. What changes is how much comes in at one end and what comes out at the other. You are a steady state and everything is pretty much constant. Its dynamic so it can change with environmental input. Dynamic equilibrium thats what you are. If you reached true equilibrium that would probably be several hours after you finished rigor mortis. You are still reacting and doing lots of chemistry when you are dead. So the reactions are optimized collisions, lower activation energy and they stabilize the reactive transition state. Slide 5: So here we go, two very common reaction where we break peptide bonds or we break an ester bond. Breaking the peptide bonds, you can do them by just heating the system up and adding an acid. It will hydrolyze the bond. But the peptide bonds under normal conditions are very stable and very difficult to breakdown unless you have a catalyst that can accelerate the rate of hydrolyzing a peptide bond. The same thing down here below. This ester linkage is kind of like what youre going to find with acetylcholine. Were going to talk about acetylcholine and nerve blocking agents in a little bit. This ester linkage is a little easier to break with acid hydrolysis. Its not as strong due to resonance of the peptide bond but if you put in an enzyme boom much faster catalysis. Slide 6: Were talking about magnificent rates of reaction here. OMP is orotidine monophosphate it is one of the key regulatory in pyridine biosynthesis. Pyridine biosynthesis is all the uridines and diamines and cytodines that you are going to making from RNA and DNA later on. This reaction rate is a little small. Its kind of slow. One reaction every 78 million years. Its not compatible with life but put the enzyme in there and you can get 39 reactions per second, which is a 1017 increase in reaction rate. Thats what enzymes do they increase the rate of enhancement. If I take carbon dioxide and water I will make bicarbonate eventually. It happens spontaneously in about 5 seconds but we want to be able to do this much more rapidly so we can adjust pHs in cells and in the circulatory system almost instantaneously without

coming up with acidosis. We have and enzyme that increases the rate of reaction by nearly a million fold. This is compatible with human physiology. Slide 7: Now this is the scary slide. This is basic chemistry and were talking about the concept of free energy. Everybody knows what free energy is right? Free energy of a reaction. If a reaction happens and it gives off energy thats good. It will be spontaneous. If the reaction has to absorb energy from the environment that may not be spontaneous unless I connect that reaction with another reaction that gives off a lot of energy. Does that make sense? Everybody recalls that? What is important here is that the standard free energy of any reaction and remember this happens in a test tube. Its rarefied and not necessarily biologically relevant. But in a test tube youre going to end up with your equilibrium constant and with what your final product and starting materials are going to be. And this relates to the free energy through this equation over here. Any equilibrium for any reaction is going to be related to the free energy of that reaction to the power of 10. Each 10-fold change in equilibrium constant changes the delta G by 1.36 kcal mol-1. Why do I care about this? Here is a very improbable reaction. So if a reaction requires energy to get to equilibrium but you prevent it from getting to equilibrium by removing the product as soon as its made you can mess with this equation. By removing the product in the next reaction, the reason for pathways, you can get an improbable reaction to go forward, by removing product in the next step. The more readily I remove the product the more negative this is going to get or the more spontaneous this is going to get. That is the purpose of this slide. You do not need to memorize any equations. Slide 8: Heres an example if you change this ratio of equilibrium constants by getting rid of the products you can see that youre going to start getting strongly negative free energy releases on removing product. Youre skewing the equilibrium constant in this steady state dynamic equilibrium and the improbable reaction is going forward. That is the essence of pathways and here it is giving off free energy to compensate. Slide 9: Now you can either remove the product in the next step of the pathway or you can couple an improbably reaction to a very highly exothermic energy releasing reaction. So the enzyme is not only altering the reaction rate but you can couple of couple of reactions and alter the overall net negative release of energy in kilocalories or kilojoules. This reaction generates two products. One of the products is then converted to a secondary product and that releases a whole lot of energy and if you couple these reactions together you end up with a net negative. So you can couple two or five reactions together and end up with a whole net negative free energy it can still spontaneously go forward.

One of the biggest ways of doing this is coupling endothermic reactions to highly exothermic reactions and ATP happens to be our energy currency and that shouldnt be a surprise to anybody in the room. If its kilocalories hydrolysis of ATP to ADP is 7.3 kcal/mol or a little over 30 kJ. You can hydrolyze 2 terminal phosphates as a pyrophosphate leaving you with AMP. This is a little higher energy release because you have loss a couple of resonance structures so the energies go up a bit. You dont need to worry about resonance structures yet. So theres more energy given off here and this can drive an even more improbable endothermic reaction by taking ATP to AMP. This also eliminates its reversibility but well go over that later on. So here we have our universal currency of hydrolysis to drive things, the most common reactions and Im not going to go over, there is a lot of them. This is just one big example. I have glucose. Glucose is in my circulatory system after my wonderful breakfast of overly sugared coffee. Sucrose gets broken down into fructose and glucose and it gets taken up by the gut lining and all this glucose is sitting around inside the circulatory system and what little goes into the muscle for contracting purposes does that. The rest of it ends up going into the liver and the liver stores it as glycogen. And when the glycogen stores in the liver are nice and fat and happy they convert the glucose eventually into fat. Thats the livers job. Excess glucose stored to make glycogen. After that you take the glucose and make fat out of it. But you have to trap the glucose inside the cell. The gluc-2 transporter we talked about. Its always there in the liver cell. So the glucose level is high in the circulatory system and so the glucose comes into the liver cell. The glucose can then turn right back around and go out of the liver cell. In fact that is one of the livers main functions, to regulate the glucose levels in the circulatory system, as youll find out when we go to the rest of metabolism. In order to trap glucose inside the cell for metabolism or storage you have to phosphorylate it. Hexokinase / Glucokinase: o Once it is Glucose + Pi Glucose-6-P G = + 3.7 kcal/mol phosphorylated it o ATP + H2O ADP + Pi G = -7.3 kcal/mol cant go back to the o transporter. Its too Glc + ATP Glc-6-P + ADP G = -3.6 kcal/mol charged, too big and heavy and its go this negative charge on its and a shell of hydration. It wont bind to or go to the transporter anymore. Youve trapped it and youve metabolically activated it. But this costs you some thing. It takes energy to put a phosphate on glucose but if I couple it with ATP. If I literally pass the phosphate from ATP to glucose then I get this as an exothermic reaction that will go forward spontaneously. Makes sense? Good. And you can further mess with the system by removing the product. If the glucose-6-phosphate goes on to another product you can decrease the amount of accumulating product so that the reaction goes forward. Were going to talk a little further alter on about hexokinase and glucokinase because they both catalyze the same reaction but they do that in different tissues. And when we talk about Km values well talk about these same reactions. Slide 10:

How enzymes actually work is that they stabilize this transition state intermediate. There is an intrinsic amount of energy in the substrate and that is usually not enough to react. What will happen is that as the enzyme binds to the substrate. It stably binds to a number of different aspects of the substrate. Ill give you an example of this in a little bit. But it is stably binding different aspects of the substrate. Its going to torque the substrate, bend the substrate and put one bond and I call that the scissile bond, so if you see it written down in the little text box below the slide, the scissile bond is the bond that is being broken or possibly being made. Its going to put that bond at a higher energy state and that higher energy state is this transition state intermediate. If you tweak that bond high enough its going to get to the point where its going to want to react and it can do so spontaneously and go forward and make a product and this product is going to release the free energy so it can actually go forward. Of course its possible that the substrate can decide I dont want to be here anymore and release from the enzyme. So what you end up doing if you want to look at this figure down here. This is a bell shaped curve of the intrinsic energy level of the substrate. Without an enzyme around, most of these substrates sit an energy level that is below the energy level needed to react. So the uncatalyzed reaction are these high-energy molecules over here. The bulk of the material doesnt have enough energy. But with the catalyst sitting there stabilizing the substrate and torqing the reactive bond into a higher energy state, now each one of those molecules has the intrinsic energy needed to react. So you have a huge number of molecules now with the intrinsic energy to react so they go forward. The more molecules that react the faster the reaction rate and as you saw earlier the reaction rates go up a million fold. Q: When you remove a product from a reaction is that a change in K and G. A: Yes, if you change the equilibrium constant the standard G changes too. Remember the equilibrium constants are constants are constants but the constants are determined usually in some test tube somewhere and what Im telling you is ignore the test tube and think about the cell. You never get to the equilibrium for that reaction in that cell because you are always moving the next product and moving forward so equilibrium constants in a test tube in a lab can be very different from the effective actual applied equilibrium constant in a living cell. Alright?

So what we do here is that enzymes stabilize transition states and they bring down the amount of energy required for the substrate to react. There are now a lot more molecules with sufficient energy to react so the velocity of the reaction goes way up. Slide 11: So factors that affect reaction rates make sense if you have more substrate, reaction rates go up. If you have more enzyme that is going to be more catalyst and the reaction rates are going to go up. If you increase the temperature a little bit the reaction rate is going to up. We are probably having a problem with that biologically denaturing some of the enzymes. Some of these catalysts are very sensitive to temperature increases. Some of these catalysts are proteins, some are RNA structures. If you change the pH of the system much you might be ionizing a group like the carboxylic acid side chain or aspartic acid or that five membered nitrogen containing ring found on histidine. All of those can pick up a proton. Histidine would suddenly have a positive charge and that may change a salt link and the negative charges on aspartic acid and glutamic acid pick up protons and become neutralized and that may break a salt link and if I start readjusting salt bridges in proteins Im going to readjust the structure and structure equals??? [Class responds: function] and if I change the structure I change the??? [Class responds: function] and that may not be good. And then we have various inhibitors for enzymes and were going to talk about those in some detail here in a little bit. Slide 12: Heres graphical representation of this. Here is the substrate increasing and the reaction rate increasing until it flattens out. This says Vmax what does that mean to you? Maximum velocity for that enzyme and that enzyme concentration and conditions. So youll notice you can get to a point where you can add an infinite amount of substrate way out here and it hits a wall is it going to get any faster than Vmax? No. What have I done? Saturated the active site it cant go any faster. The uncatalyzed reaction can. With carbonic anhydrase even if the enzyme is saturated water and carbon dioxide are still going to

react together but its going to be much slower a million fold slower rate. So sometimes you have to be concerned with the uncatalyzed reaction but for the most part no. Vmax is the border. Here is an enzyme. If I put in, here is the uncatalyzed reaction (bottom line) and here is the catalyzed reaction (middle line) and if I double the amount of enzyme (top line) I double the reaction rate. Its actually a linear process. So the rates are directly proportional to the enzyme concentration (refer to rate of reaction vs. enzyme conc. graph with the red line). And when I say enzyme concentration Im saying that an enzyme equals an active site. Some enzymes have more that one active site. Some enzymes have temperature issues as I increase the temperature, increase increase increase, in a test tube this would start to become exponential reaction rates. In a living cell, whoops, youve just denatured the cell and it might be good on the BBQ but it certainly wont be doing a good job of reacting. Here is a pH profile. Some proteins like being in an acidic environment. Can you name one? Pepsin. Pepsin likes to be an acidic environment to work. In fact it has to be in an acidic environment to work and the protons protonates some ionisable groups and when they do that it changes its confirmation and literally cuts itself first. It cuts a little peptide linkage that is sitting over the active site physically blocking it. When I acidify the proteins coming out of the cytosol of the gut lining cell in the stomach at about normal pH it hits the gastric juices at about a pH of 1 -3 or somewhere in there. It will protonate that protein and reorganize this whole blocking peptide right down into the active site and it cuts itself and then it reorganizes and starts chewing up acid denatured proteins in your diet so you can present fragmented proteins to your small intestine for the pancreatic and liver protease enzymes. A neutrophile protein are most of the ones working in the cytosol and like 5.5 9 because remember there are micro domains in your cell. The pH in the lysosome likes to run about 4.5 5. Its not 7.4. In fact if a lysosome bursts inside a cell and releases all these digestive enzymes, chances are its not going to kill that cell because all those enzymes prefer to work in acidic pHs and all those proteolytic and lipolytic enzymes sitting in the lysosome prefer a pH of 4 -5. If you release it into the cytosol where its 7.5 they are not going to work because they are outside their optimal pH. Nature designed it that way so you could save yourself the problem of auto digestion. On the other hand if you like that really nice tenderized Omaha stake it comes and it sits and it ages and it sits at the right temperature for a few days its tenderizing itself as it slowly

ruptures lysosomes in the muscle mass and its supposed to digest the muscle mass from the inside. Slide 13: So that is sort of like the kinetics and how you regulate enzyme reactions and were going to come back and look at more kinetics in a second. Now we know what enzymes do. They enhance reaction rates, they dont change equilibrium, and they bind to and are saturated by the substrate. But there are 6 major classes of enzymes. Each one of these classes of enzymes have a quite a few family members. Oxidoreductase like dehydrogenase transport electrons and protons around. Transferase are group transfers like kinases are like that glucokinase we talked about earlier that moves the phosphate from ATP to glucose. Hydrolases are any protease, or lipase or glycosylase anytime you are breaking the bond using water thats a hydrolase. Lyase remove a group leaving a double bond. There are only a few of these. But one you will run in to is fumarase in the Krebs cycle. Isomerases they isomerise and move groups around. Ligases form bonds, new bonds using ATP usually. So these are the 6 groups that all enzymes have been divided into and in fact there is a IUPAC type nomenclature, the international union for applied molecular biology and they have numbers for each every one of the enzymes. That big colourful pathway I had up front when you look it up online youll see where those lactate dehydrogenases are supposed to be but it wont be lactate dehydrogenase it will be 1.11127 and that is the official designation of lactate dehydrogenase. You will not have to know that unless you need to go to the pathway to look up some information because you are big structural biologist of some sort. Practising clinicians never usually do that but its important to know that if Im going to look up a pathway and you cant remember what the name of the enzyme was and this enzyme 6, that a ligase, 6.2319 you can go back to your textbook and look it up in there. Slide 14: Enzymes may use some friends to get accomplishments in their reactive duties. There are a couple of major players here there are coenzymes, which are organic molecules, and typically they are derived from some vitamin. And here are examples of enzymes that use coenzymes. You dont need to memorize that! You need to know that coenzymes generally are derived from vitamins. You need to know that coenzymes are molecules that participate in the reaction normally. Cofactors are metals and a bunch of enzymes used these metals like Glutathione reductase, a very very important enzyme, youll come across that later on. When you are trying to save red blood cell membrane throughout the damage youre going to want to make sure that red blood cell has a lot of reducing power so its going to need glutahionine reductase so you are going to need selenium as a required nutrient. Some of these like Carboxypeptidase the zinc actually participates in the reaction. Others like zinc for carbonic anhydrase the zinc just helps hold the protein in the right conformation so they can be reacted. They can either stabilize the structure or participate in the reaction. For the most part the coenzymes actually participate in the reaction. Now down here we have some terms the holoenzyme = apoenzyme + coenzyme/cofactor. That means that the final intact active enzyme is just the protein part, the apoenzyme, plus its organic coenzyme or inorganic cofactor. So those are terms you should know.

Slide 15: This gets us talking about enzyme active sites. Enzymes and substrates have a degree of specificity. There are some enzymes that have absolute specificity like glucokinase that only binds to glucose. This is a liver enzyme. It likes to bind to glucose and ATP it traps the glucose in the cell. Hexokinase uses any hexose sugar glucose being one of them. It phosphorylates it, traps it in its tissue. Hexokinases are usually made in cell muscle. So hexokinase is a muscles isozyme. Different gene, different protein, different structure and sequence but the same reaction. But it has different types of parameters. Hexokinase is not near as specific. It binds to any hexose sugar. It phosphorylates it and traps it inside the cell. Same thing here Chymotrypsin has a very specific substrate. It binds to proteins right next to the hydrophobic amino acids only and cuts that peptide bond. Where as cathepsin proteases youll find in lysosomes see just about any peptide bond and hydrolyzes it. So very specific versus somewhat general. And then we have something similar with nucleotides in DNA. EcoR1 is a restriction enzyme and well get around to talking about that in exam block 4. This is very specific it looks for a hexomeric sequence and just cuts that. Whereas DNase1 looks for any DNA and just randomly chops up DNA. They both chop DNA but one is highly specific and one isnt. So specificity is there for all enzymes but sometimes it can be a little more generalized. Where is this activity taking place? The enzyme active site. This giant protein. In this big protein you are the amino acids for this entire protein. You and you and you (points to people sitting near the center isle towards the front of the room) are the active site. You put your foot out I trip over it you make sure to beat down to the floor; you beat me to death and bam it makes everybody happy. Thats the active site. Does that make some sort of comedic sense? Good. So the active site is a three dimensional pocket or groove. It is generally sitting in a somewhat globular protein. Generally long elongated structures like collagen dont have active sites because they are structural proteins. These tend to be somewhat globular with specific binding pockets are you are going to have amino acids within that pocket where the side chains are either going to bind to or stabilize the binding of the substrate or they maybe the reactive side chains that induce the catalysis of the strained bond. Coenzymes are typically derived from vitamins. Some examples NAD, FAD, biotin and thiamine they all bind to reversibly generally, except biotin that becomes covalently bound, NAD is reversibly bound adjacent to the active site and participates in the reaction. FAD tends to be covalently bound to the enzyme as such is then called a prosthetic group. If its covalently bonded to the enzyme then its a prosthetic group and if its free to bind and release its generally considered a coenzyme. Cofactors these again stabilize structures and/or participate in the reaction. Once you have all these participants in the right spot then you can start talking about the chemistry

Slide 16: Now what do I want you to take away from this. I got an enzyme that is represented by E it binds a substrate represented by S and its got brackets around here indicating thats a concentration. Say a fixed enzyme and a variable concentration substrate gives you something called the enzyme-substrate complex. Remember the transient free energy reaction it goes up to the top and then down. That is where we are. The enzymesubstrate complex is at the top of that dome. And if you stabilize it you can reduce the energy needed for the reaction and increase the reaction rate. If its not stabilized it may not react quite so well. But when it starts reacting youre going to get a decrease in the substrate concentration and an increase in product concentration. But what happens very early on is that you saturate the enzyme substrate and its either going to break down productively to form product or its going to breakdown non-productively to reform the substrate. And that concentration of enzymesubstrate complex and the concentration of the free enzyme is called the steady state. In a living system you are always at a steady state. And in the test tube youre doing all our experiments here (refer to the graph to the right). Scientists really get off on this but clinicians go why bother and they have a good point. Why do I need to worry about what happened in the test tube the test tube is not filing an insurance claim. But unfortunately this is the convention of how things work with Vmax and Km. What were interested in and when were looking at enzymes and how they work is that when were looking at an initial rate and were not yet in steady state and Ive got free enzyme and lots of substrate whats going on? What are the kinetics under those conditions and then we use the values that we determine from those conditions and pre-steady state calculations to tell us things about enzymes like what is their relative affinity for a substrate or a drug and then we can compare using those same kinds of numbers from one enzyme to another looking at the numbers to see what type of affinity does the drug bind to two or three different targets. Slide 17: Now hopefully I can point that out to you more directly here. So were looking enzyme-substrate complex this is really structure-function stuff. And what we have here are the two opposite ends of the spectrum. You have the lock and key and something called induced-fit. Lock and key is just that. Youve got a lock its fixed and it does not change structure and if you have exactly the right key it will go in and you turn the key the door opens. If you put the wrong key in its going to stay locked with the wrong substrate in its not going to react. On the other hand with have the induced-fit model and this is kind of like what is going on with glucokinase and hexokinase. Its like Pac-man. Here is the enzyme sitting there with its mouth wide open here it is and it gets in there and the enzyme begins to fold around the substrate setting up more productive interactions with the substrate, more and more specificity, the enzyme changes structure binding to the structure until it creates the active

conformation. Now to be honest all the enzymes lie within this kind of spectrum. Some enzymes are more lock and key style with just a little bit of tweaking and structure and some enzymes are more induced fit with a little more limited lock and key and some sit right in the middle. A little lock and key and little induced fit and they get in there all bound and happy. Here is an example of enzyme were going to talk about it some detail its called lysozyme. I used to do reaction diagrams and all sorts of enzymes but I decided to cut them down to two. Lysozyme is a primary defence against bacterial infection on all your membrane along the GI tract. This enzyme sees a gram positive bacteria and binds to the proteoglycan layer and cuts the glycan cut cut cut cut cuts until you have sacrificed this big covalently linked shell around the bacteria and the bacteria succumbs to shock and autolysis. So what youre doing here is using this enzyme to chop up the surface of the bacterial cell wall and here it is it is a nice stable globular structure coming out the cell and sitting out there in the mucosal membrane and its stable and its made out of alpha helixes here a couple of beta sheets and they form two primary domains one on one side of the flap and the other on the other side of the flab and you see this coloured amino side chain residues that are extending from the alpha helixes or beta sheets, these are lying in the cleft and they set up productive interactions with the substrate and as this bacterial peptidoglycan wall sits down in the cleft on this the enzyme wraps around it with more and more productive interactions and it strains one of the glycosidic bunds in the peptide backbone. That strained bond is at a really high energy and the transition state intermediate is at high energy and it can either react or not and we can show you how that works. Slide 18: So here we have this substrate and its bound to this cleft. And this shows you and well come back to this, the shows you NAG and NAM all put together with alpha and beta linkages and as it binds to this active site apparently the amino acid side chains here in this blue and down here in this blue hydrogen bonding interactions with the substrates repositioning this thing in the active site very precisely but there is a little speed bump in the enzyme that causes the substrate to get bent and when you torque this glycosidic bond from what was a stable boat conformation to a half chair conformation which is very unstable so Ive destabilized that sugar ring which makes it much more reactive and magic

happens as youll see.

Slide 19: So catalytic reaction mechanism are common to enzymes. Proximity and orientation effects are lock and key; strain and distortion are induced fit. There is acid-base catalysis and thats the example I showed you with lysozyme. Covalent catalysis, we are going to show you an example of that with a serine protease and Ill tell you why I picked serine proteases in a little bit. Metal ion catalysis, I dropped this example from the presentation just for the sake of time. Carboxypeptidase uses the same to polarize water to make it react with a hydroxyl group, which makes it take the C-terminal residue of the protein. There is electronic catalysis and intermediate states involved in all enzymes. Slide 20: There is a number of generalized concepts that go on in enzyme reactions dont memorize the details just know that there are a number of different mechanisms and were going to show you two of them. So were going to start with lysozyme and thats 20 slides in Im going to take a break now.