01.02 Enzyme Kinetics

Hey there. What we’re going to talk about today are enzyme catalyzed reactions. We’re going to look at some thermodynamic aspects and also active sites of enzymes, and also kinetic analyses. There are a lot of words associated with enzyme kinetics, and it’s a good idea to have a good understanding of what those words are. So basically here's a reaction. In chemistry, we would think of a reaction as being reactant to product. So it’s a chemical change. So that means we’re going to have movement of electrons from one type of bond to another type of bond. And it’s going to convert the reactant to a product. In biochemistry, we usually say substrate instead of reactant, and we draw a slightly different type of reaction. So the way we write it out is: enzyme plus substrate--and then we use the little double arrows--it can make an enzyme substrate complex. The idea is that you’re finding an equilibrium between individual enzyme and substrate, just floating around by themselves, and a complex between the enzyme and substrate (ES). And it just means that the enzyme is basically binding to that substrate there. The point is when you go forward and make enzyme plus product, S is getting turned into P (that is substrate gets turned into product) and the enzyme gets recycled. You start off with just an individual free-floating enzyme and that’s how you end up. That’s really what a catalyst is. It is not being used up in the reaction. So when I say that it’s a macromolecule, it could be either RNA or protein. The majority of enzymes are protein, but it’s good to know that there are also some RNA enzymes that are out there. When we talk about reaction rates, it’s a good idea to understand that what we’re talking about here is...remember concentration brackets...if this is product, what we’re looking at is the change in product over the change in time. It is basically like moles per minute, that would be, you know, so the higher the [P] over time, the higher the reaction rate is. Reactions take place in an enzyme active site. And on the next slide, actually a couple of slides, we’ll take a look at what an active site looks like. But before doing that, let’s talk a little bit about how the catalysis happens from an energetic perspective. We’re going to talk about activation energy. Let’s start by looking over here at the graph. A reaction that has no enzyme has to have this much energy put into it to get the bonds to break and reform...to go from reactants over to product. In this case we have two reactants, A plus B, and they must be getting stuck together. That’s how we make AB. But if you bring an enzyme into the picture, the enzyme lowers that activation barrier. That’s what this is here: it is the activation barrier.

This is energy over here on the Y axis. It takes that amount of energy, but having the enzyme there lowers it. I’m going to make this a little messier looking because what’s really happening is that it goes down and then comes up like that. But this is where that enzyme substrate complex is. So in this case, that would be the enzyme plus the A plus the B...they all get bound up together. And because they’re forming bonds, that’s why the energy goes a little bit lower. If you remember that when you make bonds, you release energy. So making the bonds between the enzyme and the substrate lowers the energy, and then it’s got this really special little environment for the reaction to take place. That’s why the energy doesn’t have to go as high here.

So this thing is called a reaction diagram...that’s the energy as a function of what we call the 'reaction coordinate' here on the X axis, which is a little bit of a vague term, but it shows how the energy is used and released as the reaction progresses. And then as I mentioned, this is the very important enzyme substrate complex that is formed and stabilizes the substrate. Now to the reaction...let’s have a closeup view here of a real enzyme. This is hexokinase. It catalyzes, the first step of glycolysis. What it does is it takes this glucose--which I’ll abbreviate as GLC, that’s a little glucose molecule. This is a little ATP molecule. What we’re going to have happen is one phosphate is going to come off of the ATP and get added onto one of the hydroxyls--the OHs--on the glucose. That can happen at a low rate, outside of the enzyme. But when they come into the enzyme active site--into this sort of nice cleft like-place here--it creates this really special environment that then can increase the reaction rate. When you have a really high activation barrier, that’s going to be a slow rate. Because it took so much energy to get up over it. By having that enzyme there, you lower the barrier and that makes it faster. So that’s, what’s going on. When you put these two guys into this enzyme active site, now you’ll notice over here, look at how it clamped down. You’ve got those substrates that are sitting in there, but it clamped down and that’s really important. We’ll talk about that on the next slide. Some different important aspects of catalysis: Proximity. What that means is the enzyme is going to bring those two substrates close together, right? You can imagine if there’s no enzyme, these guys are just floating all around and it’s hard to get them to come close to each other. So the enzyme helps to bring them close to each other. It also orients them just right. If we’ve got this ATP here, we’ll put a PPP on it. I’m going to break that one up, beause that’s the guy that’s going to move. This is your ATP and here’s your glucose. I’m not going to draw the whole glucose, because it’ll take a little while to do that, but we’ll just do part of it. We are trying to put this phosphate over here on this oxygen. That is the reaction. So orientation means that that phosphate is going to be oriented just right, so that the electrons that are over here between the O and the H can come and attack at this bond and let the rest of this go. That’s how the P gets attached to the OH. If they weren’t oriented right, this could be flipped around and the reaction wouldn’t happen near as fast. So that’s an example of how it is that orientation can help the reaction go faster. And then the other cool thing is that clamping down is going to make it so that undesired reactions can’t happen in the active site. Notice how, if we have that H and OH, if we were to just put a little H on that, do you see that we just have H2O? So that means that these electrons could also be coming over here and doing that. And we’ve got lots and lots of water all around these molecules. So being inside the enzyme active site, water can get excluded. And so this does not happen. And that’s another thing that helps this reaction to go faster. There is an equation that can help describe how an enzyme reaction occurs. You’ve got a term called Vmax in it, and that is the fastest that the enzyme can go. And then you’ve got this thing called KM. KM is kind of a weird definition, but it’s the substrate concentration when the enzyme is running at half Vmax. It’s easier to look at like this. When you look at the graph, this is the reaction velocity, and officially it’s called the initial velocity...that’s kind of a little bit of complexity that you probably don’t need to remember specifically, but this would be initial velocities over here. This is substrate concentration down here. So this would be either the glucose or the ATP in the last example that I gave. And as you increase the amount of substrate, you’re going to increase the amount of product. So you’re going to have more reaction as you increase the substrate. However, it’s going to get to a place if this were to continue on and on and on and on and on and on. Eventually this is actually the Vmax up here. And I know that it kind of seems crazy because that’s way, way, way out here, but that I made in this equation here, I put a 10 right there to make that be the Vmax. The definition for KM is: You go to half Vmax, which is five, and you would look across here and then you would come down here, and so the KM for this guy is 0.8. (I don’t even have a unit on it, but, you know, we could say it’s pointing molar or pointed millimolar or something like that.) That’s basically how this curve works is it’s. This line is defined by this equation and you can see that you have these two numbers that would be set and they would describe the enzyme itself. And then you would have the substrate concentrations varying.

Now, one of the things about KM that’s useful is that the more tightly bound the substrate is, then it’s going to take even less substrate to get to that place. Let’s say that we have a really tight binding, so then the reaction can take place quickly. Let’s say we’ve got something that’s more like a curve that comes up like that. That means at this half Vmax point, you can drop it down and it would be a much lower substrate concentration at this point. And so that means that it didn’t take very much substrate to get to that half point. And so that means the enzyme and the substrate bound to each other tightly. It’s slightly more complex than that, but that’s the basic idea. You can sort of think of KM is telling you how tightly the enzyme binds to the substrate.

Now how does this relates to health and medicine? Drugs are often enzyme inhibitors. The idea is that often drugs are something that maybe looks like ATP, and could come into this active site, and it would work to block this real ATP from coming in. And so enzyme inhibitors often look a lot like an enzyme's substrate because they already have an affinity for the enzyme, but they get in there and then they block it. And there’s lots of examples of that kind of thing. And these are called competitive inhibitors because they are binding at the active site...they’re coming in and competing for the substrates in the active site. There are other types of inhibitors, noncompetitive, and uncompetitive, kind of some little complex aspects to them mathematically. But it’s like maybe they bind over here, and affect how the, a cleft here works. Maybe it doesn’t clamp down like it should. So these are a little bit more complex, but those are other examples of types of drugs that could be made. Let’s do the competitive example here, looking at the graph--because this is sort of the simple one to look at. It changes the KM, but not the Vmax. So this here in blue is minus the inhibitor. And then the orange one is plus the inhibitor. If you were to keep going out a way out, you would eventually get up to this 10-mark here where the Vmax is same for both of these. So you’re not effecting Vmax. But if we just do that little drawing across here for the half Vmax part, this is where the KM is for no inhibitor and here’s where the KM is for plus inhibitor. So you can see that we have increased the amount of substrate that it takes to get to that half Vmax point. And that should make sense because you’ve got this enzyme active site and you’ve put an inhibitor in here, you’re going to have to get that much more substrate around to get in there. You’re going to have to swamp out that inhibitor a little bit to get it to come out and put the substrate in there. That’s how come the substrate concentration has increased with the addition of the inhibitor. Let’s go over some of the key points. Enzymes are macromolecules that increase reaction rates and enzymes do this by creating an enzyme substrate complex that lowers the reaction activation energy. Remember higher versus lower...this is going to be faster if you can have lower activation energy. Enzymes create this special environment, so they have that sort of claw like shape. And here’s that special environment that helps with proximity of the substrates, orients them just right and excludes things that might also react and like water. It gets those things out of there. And we use enzyme kinetics then to describe the KM and the Vmax of an enzyme. It’s sort of like saying, "what color is it?" Or "how fast is it?" et cetera. We use KM and Vmax to say that. And then importantly, we know that many drugs are enzyme inhibitors--I mean, even just in the case of a headache--things that cause pain in us can be inhibited. Aspirin or ibuprofen, those are enzyme inhibitors that actually go function in the enzyme active site and they prevent the normal pathway from occurring. And then that’s why you feel less pain when you take aspirin or ibuprofen. All right. That’s it for enzyme kinetics. We love you guys! Go out and be your best self today. And as always happy nursing!