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Charles Saladino Introduction Think of emptying your bedroom and then filling it with golf balls, so as occupy every cubic inch of space – wall to wall, floor to ceiling. Without a catalyst, it would take a year to remove one cubic foot of those golf balls. Add a catalyst, and the room is emptied out in one second! Does that sound dramatic? I certainly think so; and that is why I use this analogy to emphasize the importance of the class of proteins called enzymes – biological catalysts. They speed up a chemical (metabolic) reaction without themselves being consumed in the reaction. Enzymes provide an alternate, energetically favorable reaction pathway, not the same as the uncatalyzed reaction. Slow acting enzymes only speed up reaction rates about 1000 times. Fast enzymes, however, can increase the rate of a reaction by as much as 1011 times. Just think of that number. In fact, it is absolutely safe to say that having no enzymes in the cell would be incompatible with life. Metabolic reactions would proceed just too slowly to sustain a living system, as almost all reactions in the body are enzyme-mediated. The term “enzyme” is derived from the Greek, en, “in” and zyme, “leaven.” The term was first coined in 1878 by Kuhne to designate a catalytically active substance, previously known as ferments. Although there are literally thousands of different enzymes within the living cell, they can be classified into six general categories, according to their respective functions: 1. Oxidoreductases. This class of enzymes is so named for obvious reasons. They catalyze oxidation-reduction reactions. Dehydrogensases are a prominent example of enzymes from this group. 2. Hydrolases catalyze bond cleavage by adding a single water molecule across the broken bond. Cleavage of a peptide bond is such an example. 3. Transferases catalyze the transfer of groups contining N, C, or P, for example, the addition of a carboxyl group during a carboxylation reaction. 4. Lyases catalyze the breaking of C-C or C-S, and some C-N. An example is a decarboxylation reaction, where carbon dioxide is removed from an organic acid. 5. Isomerases catalyze rearrangements of molecules in the formation of pairs of optical or geometric isomers, for example. You will be more aware of this when we discuss glycolysis. 6. Ligases catalyze the formation of bonds between C-O, C-S, C-N, for example. However, the important distinction for this class of enzymes is the energy requirement in the form of ATP. Repairing a break in the sugar/phosphate backbone of DNA is an example where a ligase is required. We must now start to consider exactly what an enzyme does, how it does it, and what the consequences of its action are. That is a lot of material to cover. Let’s start with the
simple, such as the molecule upon which the enzyme acts is called the substrate, which you probably already know. We start with the simple notion that: k1 k2 E (enzyme) + S (substrate) <======> ES <======> E + P (product) k-1 k-2 Although we show a reversible arrow after the ES complex, it is highly unlikely that once that complex is formed that the reaction would not proceed to completion. (When we look at energy curves later, you will see precisely why.) Note that the enzyme is recovered after the reaction is over. Obviously, the world of biochemical reality is far more complex than the above equation implies. I personally consider it as much conceptual as actual, because many reactions require multiple enzymes, multiple substrates, and many critical cofactors, in order for the enzyme to function at all. What value this has, however, is in understanding the kinetics - the rate of reaction over time. Why discuss this? Is this just an academic exercise in chemistry? Or, as is decidedly the case, does kinetics bear strongly upon enzyme function in metabolism and in the interpretation of enzyme inhibition? This is where we are headed in this week’s lecture. You will notice at the arrows in the above equation, that there are four rate constants. The value of the rate constant determines which direction the reaction will take (i.e. which direction the equilibrium favors). Again, once the ES complex is formed, the reaction will go to completion. We now need to talk about the famous Michaelis/Menton equation, which describes how the velocity of an enzyme-catalyzed reaction will vary as a function of the substrate concentration. Thus: Vo = Vmax [S] Km +[S] where: Vo = initial reaction velocity Vmax = maximum velocity S = substrate concentration Km= Michaelis constant
Now a picture will be worth a thousand words to describe the above equation graphically. The two graphs below are two different representations of Michaelis/Menton kinetics. In the first graph we plot the velocity of the enzyme-catalyzed reaction against the substrate concentration, assuming a small amount of enzyme present. Notice that the initial velocity rises in a straight line, which represents the velocity or rate at which the number of substrate molecules are converted to product per unit time (usually μmol/minute). This is a first order reaction with respect to substrate, due to the approximately proportional relationship of velocity to substrate concentration. Eventually, at higher substrate concentrations, the reaction reaches a maximum velocity (Vmax), no matter how much additional substrate is present. Each enzyme has its own characteristic Vmax, which really represents the saturation with substrate of all available binding sites on the enzymes present. Another very useful parameter of this plot is the Km. Clearly it is the substrate
concentration at which one half Vmax is reached. This very important substrate concentration value assesses the affinity of the enzyme for the substrate. Now, a large Km value means a low affinity of the enzyme for the substrate. That makes sense, if you consider that you would need more substrate to get to half maximum velocity than you would with a low Km, where the affinity of enzyme for substrate is greater, and thus requiring less substrate to achieve half Vmax. Make sense? Please tell me if you are having any problems with this very important concept. The second graph is called the Lineweaver/Burke plot, otherwise known as the double reciprocal plot, 1/V vs. 1/S. Obviously, the Km is calculated from where the line intersects the substrate axis, whereas the Vmax is calculated from where the velocity axis is intersected. The real advantage of this plot vs. the first one is that calculating a precise Vmax value is more accurate with a Linweaver/Burke plot, because in the first graph, it is difficult to say exactly at what substrate concentration the Vmax is achieved. Only good estimates can be achieved, but not as accurately as with the double reciprocal plot.
Now getting away from the theoretical for a moment, by way of practical example, lets look at the Km values (μmolar) of a few enzymes. Lysozyme has a value of 6 μM, whereas that of carbonic anhydrase is 8000 μM. Why such a difference? The action of lysozyme on a bacterial cell wall need not be instantaneous in molecular time. The reaction can occur more slowly with a Km of 6 μM and a maximum turnover of only 0.5 molecules of bacterial cell wall substrate (Hexa-N-acetylglucosamine) per second. In stark contrast, the Km of carbonic anhydrase is 8000 μM, with a maximum turnover rate of 600,000 molecules of carbonic acid being converted to water and carbon dioxide per second! This is because the carbon dioxide/oxygen exchange is very rapid, and the
enzyme is critical to that exchange, as we have previously discussed. Therefore, the Vmax reflects the need (or lack thereof) for the speed with which the enzyme must act. It also acts as an indirect proof that an ES complex exists, because uncatalyzed reactions do not show the saturation effect (a Vmax). Active Site of the Enzyme In the proteins lecture, I stressed the need for specificity of action in the case of many classes of proteins. The enzyme is a prime example of the need for specificity between enzyme and substrate. Before going to the heart of enzyme specificity, let’s illustrate this principle by way of example. When we study carbohydrates, you will learn, if you don’t already know, that galactose and glucose are sterioisomers of each other, and that they are otherwise identical, except for the direction one hydroxyl group is pointing in glucose vs. galactose. This small difference in the angle of a small group is picked up by the enzyme glucose kinase, which phosphorylates glucose, but will not phosphorylate galactose. This is a prime example of how specific an enzyme can be. What accounts for this specificity? The answer primarily is the active site of the enzyme. The active site of an enzyme is a region that binds the substrate and a coenzyme and possibly other cofactors. Although the substrate can bind to other regions of the enzyme in a less specific manner, the active site is where true specificity resides. Let’s look at some properties of all active sites. 1. First, the active site is a three-dimensional crevice or cleft formed by groups that join different parts of the primary sequence of the protein. In other words, the active site might contain amino acids # 12, 44, 67, 140, and 230, all brought together through secondary and tertiary levels of folding. In other words, residues far away from one another in the primary sequence might interact more significantly than adjacent ones. 2. As state in the first point, active sites are cervices or clefts to which the substrate bonds. This area is generally hydrophobic, unless water is a reactant. This non-polar area enhances catalysis and substrate binding. 3. The active site occupies but a small part of the entire volume of the enzyme. Here is an email assignment question: Why is there such a big enzyme with such a small active site? There are at least two good answers. 4. A substrate will bind an enzyme by many weak interactions. These are non-covalent bonds, such as hydrogen, hydrophobic interactions, and van der Waals forces (these being so weak, that only when the substrate and enzyme are very close do they matter, and there must be a large number of them before they become significant). 5. The enzyme and the substrate must be a complementary fit. However, this does not start off as a lock and key, but rather becomes complementary as the enzyme and substrate approach each other and possibly after the substrate binds. This is called the induced fit model, which replaces the antiquated lock and key theory. This required high degree of specificity is satisfied, in part, by the directional character of hydrogen bonds between enzyme and substrate. In summary of this point, the high degree of specificity of bonding between enzyme and substrate at the active site is dependent upon a preciselydefined arrangement of atoms in the active site.
We have been discussing the active site, as though there were always just one per enzyme. In fact, that is not true. Many enzymes have multiple active sites. Then how could we know that? There are several technological methods that can be used. However, if we look at the kinetics of the enzyme, the answer can be forthcoming. You notice that the Michaelis/Mention kinetics curve is hyperbolic, that is, the graph starts off with a straight line portion, followed by an approach to and then the achievement of saturation. This curve defines the kinetics of enzymes with one active site. However, if the curve represents an enzyme with multiple subunits and active sites and/or the enzyme is controlled allosterically, the following sigmoidal curve appears:
If you remember our discussion about allosterism, one hallmark would be the changing the shape or activity of a protein (like an enzyme) by binding an allosteric effector to it, at a place in the structure other than the active site. This type of enzyme manifests the sigmoidal kinetics curve. If this enzyme were to be inhibited, it would likely follow a hyperbolic shape again, but below that of the sigmoidal curve. Enzyme Inhibition While we are still on the topic of enzyme kinetics, let’s look at two types of enzyme inhibition, competitive and non-competitive. To understand them, you will have needed to grasp the concept of the Km. Below is the Michaelis/Menton kinetics curve for normal kinetics and for competitive inhibition. A competitive inhibitor is one that ties up the active site, making it unavailable to the substrate. Only a flood of substrate will reverse this inhibition. Notice how in the inhibition curve, the Km is much greater, but Vmax is eventually achieved. Do you understand why the Km is larger? The answer is because the affinity of the enzyme for the substrate is compromised, due to the inhibitor occupying the active site of the enzymes. Obviously, this is a dose effect, in that the more inhibitor, the greater the number of enzymes with an unavailable active site will be present, and the greater amount of substrate will be needed to reverse the competitive inhibition.
The next curve shows a non-competitive inhibition. Here the inhibitor binds the enzyme, but not at the active site. Perhaps by allosterism or some other structural change, the enzyme is not able to form the ES complex as well. You will notice that the Km is affected very little, whereas Vmax can never be achieved. By comparing these two curves, or two Lineweaver/Burk plots, one can differentiate competitive from noncompetitive inhibition. Adding more substrate here still does not allow Vmax to be reached.
Allosterism and Enzyme Regulation Many enzymes have multiple active sites and multiple subunits. They do not follow the usual Michaelis/Menton hyperbolic curve. Rather, there shape is signmoidal (refer to the text-see below). Their regulation then is going to be allosteric. This critically important concept states that an enzyme (or other types of protein) can be activated or deactivated by 3-dimensional conformation changes in the structure. This is usually brought about by an allosteric effector(s) that bind to key domain sites. Such regulators can be a variety of substances, including metals, phosphates, cAMP, etc. If the enzymes in activated by such binding, then the ligand doing the binding is sais to be a positive allosteric effector. If the ligand deactivates the enzyme, then it is a negative allosteric effector. In either case, the enzyme’s activity is changed due to a conformational change brought about by the binding of the respective effector. (We will see some good examples of this when we
study glycogen formation and breakdown by the highly-regulated glycogen synthase and glycogen phosphorylase enzymes, respectively.) Such regulations are very common in the living cell. Remember, what we are dealing with here is not more or less enzyme, but rather an enzyme that is active or inactive, due to the 3-dimensionality of the structure that the enzyme acquires. For more clarification on allosteric regulation of enzymes, read Section 10.10 in Devlin and learn that material well for the next exam. In addition to the ways of controlling metabolism through regulation of enzymes, two more items can be added. First, enzymes can be up-regulated or down-regulated. This simply means synthesizing more or less enzyme, respectively. The second issue is feedback regulation, otherwise known as end product inhibition. What this means is the end product of a metabolic pathway regulates the rate of its own synthesis by inhibiting a key enzyme in the metabolic pathway that produces that end product. For example, in helping to control blood glucose levels, glucose-6-P inhibits the hexokinase enzyme that allows phosphorylation of the glucose in the first place. This occurs in the muscles and other tissues. On the other hand, glucokinase phosphorylates glucose in the liver, and the kinase is not subject to feedback regulation. Because of this, and because glucokinase has a high Km vs. that of hexokinase with a low Km, the liver is the major regulator of plasma glucose levels. There is a critical principle for you to absorb here. Please never forget this in all your biochemistry-related courses. That is you can control an entire metabolic pathway, if you control a key enzyme in that pathway. This is extremely thermodynamically efficient. Remember our Introductory Lecture. As we go along in biochemistry, we shall see that many enzymes are simultaneously controlled by several mechanisms, in order to ensure back-up or redundancy, as NASA calls it. Before leaving the topic of enzyme inhibition, let’s look at a few practical examples. Now we all know that lead is a poison. Well, lead forms covalent (very tight bonding – strong inhibition) bonds to the sulfhydryl groups of cysteines’ side chains in proteins, including enzymes. This heavy metal shows the kinetics of non-competitive inhibition. For example, the enzyme ferrochelatase, which catalyzes the insertion of Fe++ into a heme precursor, is quite sensitive to lead inhibition. Also, some insecticides irreversibly bond to the enzyme acetylcholinesterase (thus neurotoxic effects). That they bond irreversibly defines them as suicide inhibitors. Finally, more than half the pharmaceuticals used in the U.S. are enzyme inhibitors. Both penicillin and amoxicillin inhibit enzymes involved in the synthesis of the bacterial cell wall. Angiotensin converting enzyme (ACE) inhibitors lower blood pressure by blocking this enzyme, which catalyzes the conversion of angiotensin I to angiotensin II, a powerful vasoconstrictor. Enalapril and captopril are examples of such drugs that induce vasodilation by this enzyme-inhibiting mechanism.
Enzyme Cofactors Although some enzymes require no other molecules except their own amino acid
sequence, many form associations with what we will call non-protein cofactors. Without these cofactors, such enzymes would not be active. There are several types of cofactors. The first group of cofactors is the inorganic elements. A given enzyme might require one or more metals, such as Fe++ for the cytochrome oxidases (we will discuss these later in the course), Mg++ for hexokinase and pyruvate kinase, Se, for glutathione peroxidase, and Zn++ for carbonic anhydrase. Some of these might be present in the active site, being involved with the catalysis, whereas others might play a structural role, for example. Regardless, the need for trace metals in the diet is thus evident. Also of critical importance is the group of cofactors called the coenzymes. They are usually within the active site and usually participate in the actual catalytic reaction itself. When tightly bound (covalently), any cofactor is referred to as a prosthetic group. Some enzymes require both metals and coenzymes, in order to function. A complete, catalytically-active enzyme together with its bound coenzyme and/or metal ions is referred to as a holoenzyme. The protein moiety of such an enzyme is defined as the apoenzyme. The topic of coenzymes is a large one, but a few principles should stay with you. First, coenzymes are vitamin derivatives, as we shall see in a few examples to follow. Second, it is at the coenzyme where catalytic reactions take place. Your text on page 391 shows the structure and reactive center of one of the very important coenzymes, nicotinamide adenine dinucleotide (NAD+). This coenzyme is associated with oxidation/reduction enzymes, such as the dehydrogenases, with which you will become familiar later in the course. For now, however, an oxidation/reduction reaction involves transferring electrons and hydrogen, or in this case, a hydride ion. So picture this. You have the entire complex enzyme structure with a coenzyme present in the active site, for the sole purpose of transferring a hydride ion and electrons from one substrate to another. The same situation occurs with the coenzyme, flavin adenine dinucleotide (FAD+), another coenzyme associated with the some of the dehydrogenase enzymes. See page 392 of your text for its structure. So why are cofactors necessary? We could say that the functional groups in proteins are limited to whatever amino acid side chains are present. By joining with cofactors, enzymes acquire more chemically reactive groups not available in the amino acid side chains. The NAD+ molecules described above serve as a fine example of this point, especially if you will examine the structure of NAD+. (Note: NAD+ represents the oxidized form, whereas NADH is the reduced form of the coenzyme.) What an Enzyme Does to Facilitate Catalysis The heading of this subtopic is the next question to be answered. What does this enzyme literally do to speed up a reaction? There are two main answers, one an energy consideration, the other more chemical. Let’s tackle the latter point first by using the digestive enzyme chymotrypsin as an example. We know this enzyme is designed to facilitate the breakage of certain peptide bonds during digestion by a hydrolysis reaction.
The enzyme cleaves polypeptide chains by breaking the peptide bond on the carbonyl side of the amino acid residues that include an aromatic ring (such as in phenylalanine). Briefly, a portion of the polypeptide enters the hydrophobic pocket of the active site with its hydrophobic side chain (aromatic ring), with the peptide bond to be broken opposite a serine and histidine residue. Then a hydrogen transfers from the OH of serine to the histidine. This allows formation of a strained (unstable) intermediate where the serine side chain bonds to the peptide bond carbon. The peptide bond is broken, and the segment with the new amine group leaves the active site. Later a water molecule enters the active site, and its H restores the H to the serine OH, while the OH of the water bonds to the other piece of the protein to give a new COOH terminal group, so that this piece can leave the active site. I know this example is a little hard to picture without an illustration; but it is very common and easily found on-line, if you wish. However, I am not really interested in your learning the exact mechanism of peptide breakage as I am in your getting the feel of what an enzyme is doing and how it is doing it. Now we can make some general statements about how an enzyme really works. So the enzyme 1) brings the substrate and catalytic sites together. This is called the proximity effect. It accomplishes this in such a way as to 2) bring the groups that must connect close to each other by holding the substrate(s) at the exact distance and in the exact orientation necessary for the reaction. This is called the orientation effect. Enzymes also 3) provide acid, basic, and other types of groups required for catalysis. This is called the catalytic effect. Having now explained a little about the chemical effects, we can now go to the final explanation as to how enzymes work: the energy effect. This is where the energy barrier for the reaction is lowered by the enzyme by inducing a strain in bonds within the substrate molecule, as mentioned above. Where the above-mentioned unstable strain in the bond is just as it is about to break is called the transition state. Of course the same principles apply to any reaction, where an enzyme, for example, might help bring two reactants into position. It’s the same principle. Let’s look at an energy diagram and observe the transition state, as well as other properties of the catalytic reaction.
This graph is an energy diagram showing the energy change from a substrate (A) to a product (B). The transition state is at the top of the energy diagram where the free energy is the highest – too high to be stable. Thus, the reaction proceeds to completion. Think of the transition period as being approximately the ES complex. The top ΔG value is the energy of activation or energy barrier, which must be overcome for the reaction to occur. Notice that the energy barrier is the energy difference between the initial state and the transition state. Notice how the enzyme lowers this energy barrier, compared to an uncatalyzed reaction. This energy barrier (energy of activation) represents the proximity and orientation effects and other factors occurring during the reaction. The ΔG at the bottom of the graph is the free energy yielded from this reaction. Notice that the yield is the difference in free energy between the reactants and products. Notice also that by lowering the energy barrier, and thus speeding up the reaction, we are not altering the value of the energy yield (or requirement. This is very important, because that means we are not changing the equilibrium – direction – of the reaction with the catalyst. The enzyme (catalyst) only speeds up a reaction but does not change the energy yield or energy requirement, i.e. the direction of the reaction. Additional Variables Related to Enzymes As in the case of most proteins, there is an optimal temperature and an optimal pH at which enzymes function. A bell-shaped curve would describe the activity or velocity of an enzyme at a given temperature, with the optimal temperature (the top of the curve) being that of the body. The curve is plotted as velocity on the Y-axis vs. the temperature
on the X-axis. Below the optimal temperature, the enzyme would function more slowly and then progressively faster as the temperature rises. However, above optimal temperature, there becomes an increasingly greater risk of denaturation of the protein, wherein the hydrogen bonds break and the proper geometric structure is lost. When you fry an egg in a pan, you notice the change in the egg white. That albumin is being denatured. Once the structure of the enzyme is compromised, then so is the function, especially in the case of an enzyme. Likewise, each enzyme has an optimal pH at which it functions. However, unlike a temperature curve – which would be similar for all enzymes – each enzyme has its own pH curve, which is bell-shaped, but which shows a varying optimal pH, depending upon the enzyme. For example, trypsin, alkaline phosphatase, and pepsin all have bell-shaped curves for enzyme velocity plotted against pH. However, the optimal pH for these three enzymes is very different. Pepsin is a proteolytic enzyme designed to function in the extreme acidity of the stomach. Thus, its optimal velocity is at about pH 1-2. Trypsin has a maximal velocity at a pH closer to that of the approximately neutral pH environment of the small intestine in which it functions. The bell-shaped curve for the velocity alkaline phosphatase shows a maximal velocity (again, high point on the curve) in the approximately mid-alkaline range. So, whereas the environment of the stomach would denature trypsin and alkaline phosphatase, pepsin is so configured to function at maximal velocity at the extremely acidic pH of the stomach. The mechanism of the effect of pH on enzyme velocity is not difficult to understand. Think back about what you learned concerning pKa and ionization. Depending upon the pKa of the respective reactive groups within the active site, pH changes will cause ionization changes, which in turn will affect the bonding environment between components within the active site and between the active site and a potential substrate. This can greatly affect the efficiency (and thus velocity) with which an enzyme functions. Zymogens Briefly, this topic refers to the fact the many proteins are synthesized as precursors or in an inactive form. Collectively, they are called zymogens – in inactive secretory products (often in granule form) of a hormone- or enzyme-producing cell. These proteins are activated later when in their proper environment. For example, many digestive enzymes secreted by the exocrine pancreas are synthesized in an inactive state. One could only imagine the pancreatitis that would follow, should the pancreatic enzymes be active when they are synthesized within that delicate organ. However, they are synthesized in the inactive form, and they are activated only when they reach the duodenum. This activation in some cases is pH driven, whereas in other cases, it involves removal of some key peptides by another already-active enzyme. In other words, depending upon the enzyme, many of these catalysts, as well as other proteins (remember insulin), are synthesized without activity, until later, when a pH-induced or enzyme-induced structural change initiates activation.
Isoenzymes (also called Isozymes) These can be defined as enzymes found in different tissues catalyzing the same reaction. However, their physical properties vary, due to genetically programmed changes in their amino acid sequence. Such amino acid changes are usually minor, but they are usually discernable by electrophoresis, which you remember recognizes charge variations (due to amino acid side chain differences). An example of an isoenzyme is hexokinase (phosphorylating glucose in the muscle, for example) vs. glucokinase (phosphorylating glucose in the liver. Different organs often have different proportions of the isoenzymes. When a given tissue is damaged, the normal plasma levels of a given isoenzyme unique to that given organ will usually rise. This is obviously of great diagnostic importance. For example, heart muscle contains a creatine kinase (CK) isoenzyme (CK-MB). During and after a myocardial infarction, many substances leak out of damaged or dead cardiac muscle, including the CK-MB isoenzymes. Thus, the proportion of plasma CK-MB rises in comparison to the plasma levels of skeletal muscle (CK-MM) and brain (CK-BB) isoenzymes. I am sure this topic will be discussed in greater detail in Clinical Biochemistry, 560D. Concluding Remarks I have given a good bit of detail in this lecture. If you have any problems understanding any aspect of it, please let me know quickly. In what I have presented, I am not trying to make biochemists out of you. Believe me, the work would be much more intense and would require a great deal of math. However, one of several of my goals in this course, as I have previously stated (but wish to reiterate), is for you to understand what is going on in the biomolecular world and why and how it is happening. Within the limited scope of this single course, I want you to have an appreciation of some of the complexities and inter-relationships that exist in biochemistry, and specifically here, enzymes. I never cease to be in awe that the cell carries on all the activities it does - and with relatively remarkable success. I need you to memorize, yes; but I also desire that you understand and appreciate what biochemistry is really all about, that is, critical thinking and understanding inter-relationships within the proper context. I hope I am beginning to succeed.