BIOCHEM DISCUSSION 3
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06BasicConceptsofEnzymeAction-2.pptxBiochemistry: A Short Course Fourth Edition CHAPTER 6 Basic Concepts of Enzyme Action
Tymoczko • Berg • Gatto • Stryer
© 2019 Macmillan Learning
Created by Brett Barbaro
So now that we have talked about the three-dimensional structure of proteins, we are going to talk about some of the basic concepts of enzyme action, which is what most proteins do. They are enzymes.
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Chapter 6: Outline
6.1 Enzymes Are Powerful and Highly Specific Catalysts
6.2 Many Enzymes Require Cofactors for Activity
6.3 Gibbs Free Energy Is a Useful Thermodynamic Function for Understanding Enzymes
6.4 Enzymes Facilitate the Formation of the Transition State
Created by Brett Barbaro
So just an overview of what we're going to talk about in this chapter:
6.1: The enzymes are powerful and highly specific catalysts. You will find that to be very true.
6.2: Many enzymes require cofactors for activity, and we'll go over some of those cofactors.
6.3: Energy is useful for understanding enzymes and you are probably familiar with some concepts such as Gibbs free energy from chemistry classes. We will be covering that to some extent, but I'm not going to go too deeply into the mathematics because, frankly, I haven't really used them in all my experience as far as going to graduate school and working and studying in several labs. The actual mathematics of Gibbs free energy and all of that is the best way to understand it, but is also a very difficult way to understand it. I think there are some easier, more intuitive ways of imagining how enzymes work. So that's what we are going to be talking about.
6.4: And then finally, enzymes facilitate the formation of the transition state. So we'll talk about transition states and how enzymes facilitate their formation.
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Section 6.1 Enzymes Are Powerful and Highly Specific Catalysts
Enzymes are protein catalysts that can accelerate the rate of a reaction by factors of as much as a million or more.
Even a reaction as simple as adding a molecule of water to carbon dioxide requires an enzyme, carbonic anhydrase, in red blood cells.
Created by Brett Barbaro
So, enzymes are catalysts that accelerate the rate of a reaction. And you can see at the bottom a reaction of carbon dioxide and water to form carbonic acid, which is how the carbon dioxide that is created in your blood ends up getting to your lungs to be exhaled. Now this is a very simple reaction and it happens naturally, even in the air around you. If you have a glass of water, carbon dioxide will dissolve in it and form a small amount of carbonic acid. So why do you need an enzyme?
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Table of Rate Enhancement by Selected Enzymes
=.13, or 13%
Created by Brett Barbaro
Well, if you look here at the bottom of this list you can see carbonic anhydrase and the non-enzymatic half-life is 5 seconds. That means after 5 seconds, about half of the carbon dioxide dissolved in the water will have turned into carbonic acid. Another way of looking at that is the uncatalyzed rate, which in this case is 1.3 x 10-1, which I would interpret as meaning that 13% of the carbon dioxide that is dissolved in the water converts to carbonic acid every second.
I'd like to mention at this point that the units listed at the top, the kun and s-1, are a little unclear to me. I think what they are trying to say is that the k-uncatalyzed rate, the rate constant, is expressed in units of “per second” (s to the negative 1), but there is not a really clear explanation of that anywhere in the text. So it's a little bit difficult to interpret. And I'm only telling you this because I don't want you to feel like there's something wrong with you if you don't understand all of these things immediately. Actually, a lot of the time they are very poorly expressed, and that's true sometimes in this textbook and sometimes in the literature in general. I've seen full professors struggle with trying to figure out what exactly a graph or a chart is trying to say. So don't feel bad.
But, in this case we're going to say that 13% of the carbon dioxide is turned into carbonic acid every second, just at a regular rate. If you introduce carbonic anhydrase however, that rate goes to 106, or a million (not worrying right now what a million percent of the concentration would be - the rate, at least, we can say, is about 10 million times faster than it was without the enzyme). And that's important because you want to try to clear the carbon dioxide out of your tissues as quickly as possible, and carbonic anhydrase allows you to do that.
Taking a look at some of these other enzymes listed on the table, some of these reactions would happen very slowly, all the way up to 78 million years in the case of OMP decarboxylase. But at a catalyzed rate, it goes quite a bit faster. You can clear all that stuff within a second or so. It's an enhancement of 1017 times faster, which is absolutely enormous. It's really hard to imagine how much faster that is.
So, enzymes really allow these important reactions to take place on a reasonable time scale. Because otherwise you wouldn't be able to see this reaction take place appreciably, and cells need these reactions.
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Proteolytic Enzymes Illustrate the Range of Enzyme Specificity
Reactants in enzyme-catalyzed reactions are called substrates.
Proteolytic enzymes catalyze the hydrolysis of peptide bonds.
DID YOU KNOW?
Hydrolysis reactions, the breaking of a chemical bond by the addition of a water molecule, are prominent in biochemistry.
Created by Brett Barbaro
Enzymatic reactants (the stuff that enzymes act on) are called substrates. And one of the most common reactants is water, because it's everywhere so it tends to be a substrate of enzymatic reactions a great deal of the time.
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Diagram of Enzyme Specificity
(A) Trypsin cleaves on the carboxyl side of arginine and lysine residues.
(B) Thrombin cleaves Arg–Gly bonds in particular sequences only.
but
Created by Brett Barbaro
Figure 6.1 Enzyme specificity. (A) Trypsin cleaves on the carboxyl side of arginine and lysine residues, whereas (B) thrombin cleaves Arg–Gly bonds in particular sequences only.
Not only are enzymes fast, but they are also very specific. In this case, we're comparing trypsin and thrombin. Trypsin will cleave a peptide bond only on the carboxyl side of arginine and lysine residues. That's all it does. It doesn't cleave any other peptide bonds appreciably or have any other major effects in the cell, as far as we know. Thrombin is specific for arginine only. So as trypsin will cleave next to lysine or arginine, thrombin only cleaves next to arginine. And if you'll recall, lysine and arginine are both positively charged at physiological pHs, so one might imagine that both of these are able to recognize that positive charge. But the thrombin is able to discriminate between lysine and arginine! That's a pretty good example of how specific these enzymes can be.
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There Are Six Major Classes of Enzymes
Oxidoreductases catalyze oxidation–reduction reactions.
Transferases move functional groups between molecules.
Hydrolyases cleave bonds with the addition of water.
Lyases remove atoms to form double bonds or add atoms to double bonds.
Isomerases move functional groups within a molecule.
Ligases join two molecules at the expense of ATP.
Created by Brett Barbaro
There are, this textbook at least quantifies, six major classes of enzymes. You are free to look at them and try to figure out what you want from them. I personally haven't found that to be very useful for myself. All of the enzymes do something slightly different.
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Section 6.2 Many Enzymes Require Cofactors for Activity
Cofactors are small molecules that some enzymes require for activity. The two main classes of cofactors are coenzymes—organic molecules derived from vitamins—and metals.
Tightly bound coenzymes are called prosthetic groups.
Created by Brett Barbaro
Now, enzymes are proteins, and they therefore have only twenty functional groups to work with - but they can recruit other molecules if they need to do something a little bit more specific. And those other molecules are called cofactors. And there are two main classes of cofactors. There's organic molecules, which are often derived from vitamins, and then there's metals, like iron or copper.
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Table of Enzyme Cofactors
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Created by Brett Barbaro
Here's a brief list of some of the enzyme cofactors. I've put red stars by the ones that we are going to be discussing in more detail later. These would probably be the most important ones. I'm a little surprised they didn't put iron on there since we already were talking about that. But just be aware this is not a complete list.
Cofactors are something to be aware of, but for now we're going to move on. And we will be talking about specific cofactors later in the course.
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Section 6.3 Gibbs Free Energy is a Useful Thermodynamic Function for Understanding Enzymes
Learning objective 1: Describe the relations between the enzyme catalysis of a reaction, the thermodynamics of the reaction, and the formation of the transition state.
Free energy (G) is a measure of energy capable of doing work. The change in free energy when a reaction occurs is symbolized by ΔG.
Enzymes do not alter the ΔG of a reaction.
Created by Brett Barbaro
But I'm not going to get into the mathematics of it. Frankly, I don't understand them that well myself. But some basics are: free energy (which we'll call G, for Gibbs free energy) is the ability of something to do work. And when there's a change in the Gibbs free energy we represent that by ΔG (delta G). So basically what you need to know is that if the change in free energy is negative, then the reaction will happen. If the change is positive, then it won't happen. This has nothing to do with enzymes, though - enzymes actually don't alter the ΔG, they just change the rate of the reaction.
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Graph Model of How Enzymes Accelerate the Reaction Rate
Expansion of the graph:
Created by Brett Barbaro
Figure 6.2 Enzymes accelerate the reaction rate. The same equilibrium point is reached, but much more quickly in the presence of an enzyme.
You can see this very clearly in this diagram. With an enzyme, your product will increase very rapidly. And that's the purple line that you see in the graph. With no enzyme, it will increase very slowly, which is the black line you see at the bottom of the graph. But, at the end, you'll still end up with the same amount of product. It just takes a lot longer with no enzyme.
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Section 6.4 Enzymes Facilitate the Formation of the Transition State
Learning objective 2: Explain the relation between the transition state and the active site of an enzyme and list the characteristics of active sites.
A chemical reaction proceeds through a transition state, a molecular form that is no longer substrate but not yet product.
S ⇌ X‡ → P
The transition state is designated by the double dagger.
The energy required to form the transition state from the substrate is called the activation energy, symbolized by ΔG‡.
Enzymes facilitate the formation of the transition state.
Created by Brett Barbaro
A chemical reaction is when two substances come together and change their connectivity so that they end up as two different substances. And in order for that to happen...there has to be an intermediate state when there's a transition between one set of substances and the other. And that's called the transition state. We use a double dagger (‡) to represent that. There is an energy that's required to form the transition state and we symbolize that with a ΔG‡. So enzymes function by facilitating the formation of these transition states.
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http://figures.boundless.com/11846/full/rxn-coordinate-diagram-5.png
Gibbs Free Energy – Transition State
Created by Brett Barbaro
I think this is a very clear illustration of that. Notice along the bottom we have a reaction coordinate. This is not time, because the reaction can actually proceed either to the left or to the right. So it's just a measure of where the reaction is in the process. And the Gibbs free energy, on the left, will tell you how much energy these different states have.
So, if you're in a state with reactants, and this is a reaction of hydroxide ion coming to form with, I think, CH3Br, then together these two reactants have a certain amount of energy. Now, the hydroxide will crash into the CH3Br, and if it crashes into it with enough force, then it can form this transition state where the energy of that motion, of the hydroxyl ion coming in, actually turns into chemical bonding. Now this transition state is unstable, so it will break down either way, either to the left or to the right. And if it breaks down to the right, then you end up with product, which is methanol and a bromine ion. If it breaks down to the left, then it goes back to your original reactants.
But once the reaction has proceeded to the products, and you have your methanol, it takes a great deal more energy to get back up to that transition state. Your products are therefore less likely to reach that transition state and turn back into the reactants. It can happen. And it does happen. But the rate at which the products form the reactants in this case would be much slower than the reactants forming the products. And this is all talking in aggregate terms of the overall concentrations of these reactants and products in solution.
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Thermal motions cause bending, but not enough to allow the reaction.
The enzyme bends the substrate into the right position.
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How Enzymes Work
Created by Brett Barbaro
So here's what an enzyme does, basically:
1. Looking at the top line, you see on the left your reactants, in the center there's the transition state, and on the right you have your products. Now, in order for the transition state to form, you have to bend that white molecule quite a bit.
2. On the second line you can see that the molecule does bend, just as the result of thermal motion, but that's not enough to make the reaction occur.
3. Now on the third line we can see that the white molecule has some charge distribution - there's a negative on the top and the bottom and a positive in the middle - and that this charge distribution is complementary to the enzyme active site, so that the white molecule will stick to that active site and bend at the correct angle.
4. And now, on the last line, now that the white molecule is bent at that proper angle, the black atom can come in and very easily form the transition state. And then the newly formed molecule, which probably doesn't fit the enzyme active site as well, can detach and make room for another reaction to occur.
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Graph Model of How Enzymes Decrease the Activation Energy
Created by Brett Barbaro
Figure 6.3 Enzymes decrease the activation energy. Enzymes accelerate reactions by decreasing ΔG‡, the free energy of activation.
So here's another diagram that's very important, and this will tell you what the effect of the enzyme is. So you start on the left with your substrates, your reactants, and the reaction coordinate along the bottom will tell you where the reaction is. Now, normally it would take quite a bit of energy to get to that transition state, and that hump is somewhat difficult to overcome. What an enzyme does is it reduces the energy required to reach that transition state so the reaction can proceed more quickly. The overall energy change between the substrate and the products is not different - it's merely a change in the activation energy that is required to reach the transition state.
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Energy and Boltzmann
http://www.chemguide.co.uk/physical/basicrates/catalyst.html
Created by Brett Barbaro
Here's another way of looking at it. If the curve there, under the Boltzmann curve, represents all of the particles in the system, there are only a few particles, a portion of those, that have enough energy to reach the transition state to react. Now, initially those are represented by the green area on the right, and you can see that's a relatively small portion of the overall number of particles. Now once you introduce an enzyme, then you've reduced the energy required to reach that activation state. And as a result, you've increased the number of particles that have enough energy to react. More particles that are able to react ends up with a faster rate of reaction.
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The Formation of an Enzyme–Substrate Complex Is the First Step in Enzymatic Catalysis
Enzymes bring substrates together to form an enzyme–substrate complex on a particular region of the enzyme called the active site.
The interaction of the enzyme and substrates at the active site promotes the formation of the transition state.
Created by Brett Barbaro
So the place where these two products, or, rather, reactants come together on the enzyme is called the active site. And it's the interaction of the enzyme active site with the substrates that promotes the formation of the transition state.
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The Active Sites of Enzymes Have Some Common Features
The active site is a three-dimensional cleft or crevice created by amino acids from different parts of the primary structure.
The active site constitutes a small portion of the enzyme volume.
Active sites create unique microenvironments.
The interaction of the enzyme and substrate at the active site involves multiple weak interactions.
Enzyme specificity depends on the molecular architecture at the active site.
Created by Brett Barbaro
Figure 6.4 Active sites may include distant residues. (A) Ribbon diagram of the enzyme lysozyme with several components of the active site shown in color. (B) A schematic representation of the primary structure of lysozyme shows that the active site is composed of residues that come from different parts of the polypeptide chain. [Drawn from 6LYZ.pdb.]
There's some common characteristics of enzymatic active sites.
They are usually a three dimensional cleft or crevice, a little space that is created by amino acids from different parts of the protein. And it's important that these different amino acids come from different parts of the protein because they usually need to attack the substrates from several different angles, and that's very difficult to do with amino acids that are side by side.
So these large protein structures often exist simply to support a small microenvironment on the protein which is specific for making this reaction occur.
The interaction between the substrates and the enzyme is not covalent – they are very weak interactions, and that's important because the substrates need to be able to detach from the enzyme after the reaction is complete.
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The Binding Energy Between Enzyme and Substrate Is Important for Catalysis
Binding energy is the free energy released upon interaction of the enzyme and substrate.
Binding energy is greatest when the enzyme interacts with the transition state, thus facilitating the formation of the transition state.
This also helps to eject products from the active site, because products don’t bind the active site as well as reactants do.
Created by Brett Barbaro
The interaction between the enzyme active site and the substrate releases energy. It's a lower energy configuration than having the substrate fly around. And that energy is called the binding energy. And it's this binding energy that contributes to the reduction of the transition state formation. In fact you might say that the binding energy is not so much released as it is put into the substrate to create the transition state.
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Transition-State Analogs Are Potent Inhibitors of Enzymes
MANY DRUGS WORK THIS WAY
Created by Brett Barbaro
Figure 6.7 Inhibition by transition-state analogs. (A) The isomerization of l-proline to d-proline by proline racemase, a bacterial enzyme, proceeds through a planar transition state in which the -carbon atom is trigonal rather than tetrahedral. (B) Pyrrole 2-carboxylate, a transition-state analog because of its trigonal geometry, is a potent inhibitor of proline racemase.
So a really good way to block the reaction from occurring is to use what's called a transition state analog. We have a reaction here on A, which is L-Proline going to D-Proline - and in order to do that, it passes through a planar transition state. There is a molecule, pyrrole 2-carboxylic acid we see here on the right, and that is very similar to the transition state. It is also planar, and it still has the carboxylic acid group and the nitrogen in the correct place. So this molecule will bind very readily to the enzyme, and thus block the enzyme's interaction with proline.
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How Transition State Analogs Work
Created by Brett Barbaro
This is just another illustration of that same principle:
1. Along the top, you see the normal progression of enzymatic catalysis of a reaction. But, in the center, you see a transition state analog, which has the same charge distribution, but it has something there blocking any interaction with that central atom.
2. This transition state analog will bind just as well, or even better, to the enzyme than the original substrate.
3. And while it's bound there, it will prevent the substrates from interacting with the active site, as you see on the third line.
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