Enzyme Biochemistry PDF

Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. CHAPTER 4 @g m ai l.c om Proteins II Enzymes ia nn ot ti1 3 Just as different tools perform diff...

Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. CHAPTER 4 @g m ai l.c om Proteins II Enzymes ia nn ot ti1 3 Just as different tools perform different jobs, enzymes also can be grouped by function and form. Enzymes in Context in ag In a toolbox, there are a wide variety of tools to choose from—screwdrivers, wrenches, hammers, and saws—each with a specific structure and function. If we imagine the tool section of a large hardware or home improvement center, we may encounter unusual tools, such as a giant wrench. Even though this particular tool may be unfamiliar, it is easy to imagine a function for it, based on its appearance (the structure) and how other similar tools act (the mechanism). It is also easy to appreciate the need for a wide range of tools; for example, the differences between a nail and a screw mean that both hammers and screwdrivers are necessary. Looking at the bigger picture, we can see how tools can be grouped according to their function, that is, whether they cut, fasten, bend, hold, and so on. We can categorize, make generalizations, note exceptions and specificities, and build on our knowledge of tools. m ar Just as tools can be grouped, enzymes can be broadly characterized based on the reactions they catalyze and the chemistry they conduct. Alternatively, they can be categorized based on protein conformation or structure. Armed with a knowledge of structure, we can amass other data that can help in determining a possible reaction mechanism for an enzymatically catalyzed reaction. We can then build on this knowledge to probe the types of reactions that an enzyme will catalyze and those it will not, and explain those differences. With these differences in mind, it is then possible to identify different types of inhibitors, alter biochemical processes, develop new drugs, and diagnose and treat disease. These properties of enzymes are discussed in Dynamic Figure 4.1. ai l.c om Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. DYNAMIC FIGURE 4.1 This chapter discusses the general properties, kinetics, and mechanisms of enzymes, and how they are regulated. @g m CHAPTER OUTLINE 4.1 Regarding Enzymes ia nn ot ti1 3 4.2 Enzymes Increase Reaction Rate 4.3 The Mechanism of an Enzyme Can Be Deduced from Structural, Kinetic, and Spectral Data 4.4 Examples of Enzyme Regulation COMMON THEMES in ag m ar Evolution’s outcomes are conserved. Mutations to DNA sequences may alter an enzyme’s amino acid sequence. The new enzyme may act normally (a silent mutation), have a slightly altered function, such as a change in rate or binding of a different substrate, or be completely nonfunctional. If a mutation benefits the organism, there is an increased likelihood that it will be transmitted to the organism’s progeny. As organisms have become more complex and organs and systems have evolved, enzymes have evolved functions or regulatory mechanisms specific to that organ or tissue. The mechanisms enzymes employ to catalyze reactions are themselves repeated and conserved throughout nature; this is an example of evolution at the chemical level. Analysis of mechanisms and structures of enzymes illustrate both convergent and divergent evolution. The regulatory mechanisms employed by enzymes have coevolved with catalytic mechanisms. Structure determines function. The structure of an enzyme and the position of amino acid side chains in the active site provide important biochemical information that can be used to help elucidate an enzymatic mechanism. Regulatory mechanisms (e.g., proteolytic cleavage, phosphorylation, or binding of a small regulatory molecule) lead to changes in the tertiary or quaternary structure of the enzyme, modifying substrate binding or the geometry of the active site, and ultimately altering activity. Biochemical information is transferred, exchanged, and stored. Many enzymes play important roles in transmitting and carrying out biochemical signals in the cell. The regulation of enzymes (through either covalent modification or allosteric mechanisms) regulates both the rates of biochemical reactions and flux through biochemical pathways. Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. Biomolecules are altered through pathways involving transformations of energy and matter. Most biochemical reactions are catalyzed by enzymes. The use of enzymes affords a means to regulate biochemical reactions and a way to couple energetically unfavorable reactions with favorable ones. Data about the rate at which a catalyzed reaction proceeds can be used to describe the reaction mathematically. Spectroscopic, structural, and kinetic data can be used to describe the mechanism of an enzymatically catalyzed reaction. 4.1 Regarding Enzymes Most of the thousands of reactions that occur in the cell require a catalyst to run at biologically significant rates. At room temperature, a bag of sugar (sucrose) is reacting with oxygen in the air, gradually forming CO2 and H2O. This reaction is ai l.c om happening far too slowly for us to see; even so, it is possible to measure this reaction chemically and understand from general chemistry that it is happening. The energetics of the reaction indicates that the reaction is exothermic; that is, heat is given off in the process. Examining other thermodynamic parameters, we find that there is a negative free energy (ΔG) for this reaction, indicating spontaneity in the forward direction. Finally, as this reaction runs, there is an increase in the entropy—the number of possible microstates or the disorder of the system. All of these thermodynamic parameters are true, yet they reveal nothing of the speed or rate of the reaction. If you were to leave a bag of sugar exposed to oxygen in your kitchen and return even several years later, you would see no appreciable change. @g m If that same bag of sugar were dissolved in water and incubated with yeast, the same net reactions would occur. Sucrose would be consumed, and CO2 and H2O would be generated, as would some heat from the making of chemical bonds. The entropy of the system would increase, and the reaction would proceed favorably. However, this reaction would be visible to the naked eye because the microbes would generate CO2 so quickly that it would bubble out of solution. The same overall reaction is happening 4.1.1 Enzymes are protein catalysts ia nn ot ti1 3 in the two cases, and the thermodynamics are unchanged. However, what has changed is that a biological system has contributed a catalyst (or, in this case, multiple catalysts) to increase the rates of reactions to the point where the yeast can use these reactions at room temperature. The catalysts are protein molecules termed enzymes. m ar in ag Enzymes are the cell’s solution to most of its catalytic problems. Enzymes are usually globular proteins, although some have irregular shapes, or form filaments. Some enzymes are monomeric, meaning they contain a single subunit, while others are multimeric; that is, they have two or more subunits (Figure 4.2). Multimeric enzymes may have both catalytic subunits and regulatory subunits, or may have multiple catalytic subunits that interact and help to regulate one another, an example of allosteric regulation. ia nn ot ti1 3 @g m ai l.c om Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. in ag Figure 4.2 General topology of enzymes. Enzymes can be A. monomeric with a single subunit or B. multimeric with several subunits. m ar The reactants in an enzymatically catalyzed reaction are referred to as substrates, and the molecules produced are referred to as products. Although this chapter focuses on reactions that have a single substrate, over 60% of reactions use more than one substrate and have more than one product. The substrate binds to the enzyme at the location where catalysis occurs, termed the active site. This site is on the surface of the enzyme but is often in a cleft, pocket, or trench. The residues that make up the surface of the active site are responsible for substrate binding and for catalysis, although some residues have more specialized functions. The active site is generally formed by residues on turns or coils, although sometimes sections of helixes or sheets may also participate in the reaction. Several models are used to describe the interaction of the substrate with the enzyme. Some substrates interact with enzymes like a key in a lock, known as the lock and key model (Figure 4.3). In this analogy, only the correct key will fit the lock; that is, the correct steric interactions are needed for the substrate to fit into the enzyme’s active site. This model explains some aspects of enzymesubstrate interactions particularly well. For example, enzymes are generally quite specific in terms of what substrates they will bind to, being able to distinguish, for example, between glutamate and aspartate or between the carbohydrates glucose and galactose (which differ only in the chirality of a single chiral center). In this regard, enzymes may indeed be like a key in a lock. However, other aspects of enzyme molecules do not fit this model. In induced fit, the interaction of the substrate with the enzyme helps to form the active site. An analogy for this is a latex or nitrile laboratory glove. The glove itself is roughly hand shaped, but in the absence of a hand, the glove is flat and floppy. The hand filling the glove is like a substrate in an active site. The induced fit interaction helps to describe many enzyme-substrate interactions, as well as some conformational changes the enzyme may undergo as a result of substrate binding. @g m ai l.c om Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. ia nn ot ti1 3 Figure 4.3 Models of substrate binding. A. In the lock and key model, the substrate fits in the active site like a key fitting into a lock. B. In induced fit, the substrate fits into the enzyme like a hand inserting into a glove. C. Enzymes are specific enough to discern the difference between very similar structures, such as glucose and galactose. m ar in ag The active site is a tight fit. Binding of substrate usually fills the site entirely. Even water is excluded. If water molecules are present, they are usually involved in substrate binding or the reaction mechanism rather than being passive bystanders. This observation explains several reactions that would otherwise make no sense. Reactions such as transesterifications occur only because there is no water available to hydrolyze a bond, whereas other reactions add a hydroxyl group stereospecifically, through the specific positioning of a water molecule by the enzyme. The enzyme hexokinase catalyzes the transfer of a phosphate group from ATP to a hydroxyl group in glucose. To accomplish this, the enzyme binds both substrates and closes down around them, excluding water or any other molecule from the active site before catalysis occurs (Figure 4.4). Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. Figure 4.4 Topology of the active site. Shown is the enzyme glucokinase and a close-up view of its active site. Once the substrate is bound, the enzyme closes around it like the two halves of a clam shell, excluding all molecules except the two substrates. This is an example of induced fit. 4.1.2 Enzymatically catalyzed reactions can be categorized Tens of thousands of different reactions can occur in an organism. However, these reactions can be divided into six different classes, each having different subclasses, based on the type of substrate and the mechanism of action of the enzyme involved (Table 4.1). The classes are: ai l.c om 1. Oxidoreductases—catalyze reactions involving the gain or loss of electrons. 2. Transferases—transfer one group to another. 3. Hydrolases—cleave a bond with water. 4. Lyases—break double bonds using some other means than oxidation or hydrolysis. 5. Isomerases—catalyze a rearrangement of the molecule. 6. Ligases—join two molecules. Table 4.1 Classes of Enzymes Production Class General reaction Important subclasses Examples Dehydrogenases @g m Oxidases, peroxidases 1. Oxidoreductases Reductases Monooxygenases Cytochrome oxidase Lactate dehydrogenase ia nn ot ti1 3 Dioxygenases C-1 Transferases 2. Transferases Glycosyltransferases Acetate kinase Aminotransferases Alanine deaminase Phosphotransferases m 4. Lyases (“synthases”) ar 3. Hydrolases in ag Esterases Glycosidases Lipase Peptidases Sucrase Amidases C-C-Lyases C-O-Lyases Oxalate decarboxylase C-N-Lyases Isocitrate lyase C-S-Lyases Epimerases 5. Isomerases cis trans Isomerases Intramolecular transferases Glucose-phosphate isomerase Alanine racemase C-C-Ligases 6. Ligases (“synthetases”) C-O-Ligases Acetyl-CoA synthetase C-N-Ligases DNA ligase C-S-Ligases These groups were decided upon by the Enzyme Commission (EC) of the International Union of Biochemistry and Molecular Biology (IUBMB), and are called EC numbers. Every enzyme has a unique EC number, based on its category and further subclassifications. Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. For example, adenylate cyclase, the enzyme that catalyzes the formation of the signaling molecule cyclic AMP from ATP, is a lyase (Class 4) but is then further subclassified into a phosphorus-oxygen lyase that acts on ATP. One way our understanding of enzyme chemistry has assisted in the clinic through the measurement of enzyme activity in the blood is discussed in Medical Biochemistry: Enzymes in diagnosis. Medical Biochemistry Enzymes in diagnosis ai l.c om Physicians often request blood work on a patient to aid in a diagnosis. A comprehensive metabolic panel (referred to as a CHEM-20) is a commonly requested battery of blood tests. Many parameters will be already familiar: pH and the concentrations of different ions and metabolites, such as glucose and urea. These values provide the clinician with a general view of the function of various organs and systems. Other values, such as the activities of several enzymes typically found in the liver or muscle, can probe deeper. Because these enzymes are not typically found in blood, their appearance indicates organ damage. Such enzymes include lactate dehydrogenase, alanine transaminase, aspartate aminotransferase, and creatine phosphokinase. Lactate dehydrogenase (LDH) is usually localized to specific tissues, including the heart, the liver, the muscle, and/or the kidney. If LDH is released from those tissues, it will accumulate in the blood rather than being excreted by the kidney. LDH is therefore used as a marker for general tissue damage. @g m Alanine transaminase (ALT) and aspartate aminotransferase (AST) are localized to the liver; hence, as with LDH, elevated levels of these enzymes in blood indicate liver damage. Similarly, the liver enzyme gamma-glutamyl transpeptidase (gamma-GT) is a marker for liver damage. Among its multiple functions, gamma-GT catalyzes the transfer of the gamma-linked glutamate from glutathione to an acceptor molecule. ia nn ot ti1 3 One final molecule that is often included on chemistry panels is creatine phosphokinase (CPK). This enzyme is specifically found in muscle tissue and the liver, where it functions in creatine metabolism. Elevated levels of CPK indicate muscle damage or, more frequently, heart attack. 4.1.3 How do enzymes work? Enzymes are catalysts. All catalysts work by decreasing the activation energy (Ea or ΔG‡) of a reaction. As mentioned above, enzymes do not change the thermodynamic parameters of a reaction. Those are equations of state, and they do not depend on how reactants are transformed into products, merely that they are transformed. Likewise, catalysts do not affect equilibrium. If an equilibrium for a reaction lies heavily to the left or to the right, it will still be that way in the presence of an enzyme; the only difference is that the reaction will achieve that equilibrium more quickly in the presence of a catalyst than without. m ar in ag Catalysts increase reaction rate by lowering the energy of the transition state (Figure 4.5). General chemistry indicates that the number of molecules that can cross the energy barrier from reactants, or substrate, to products is limited by the height of the barrier, known as the activation energy, through the Arrhenius equation: @g m ai l.c om Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. ia nn ot ti1 3 Figure 4.5 Enzymes lower activation energy. Shown is a reaction coordinate with energy on the y-axis and the progress of the reaction on the x-axis. All reactions have an energetic barrier they need to cross (the activation energy, Ea). Enzymes work by lowering this barrier. This makes both the forward and reverse directions more favorable. In this instance, the higher the barrier (Ea, the activation energy), the slower the reaction. Catalysts work by lowering this barrier and thus increasing the reaction rate. A quick calculation shows how significant this can be. A 36 kJ/mol change in the activation energy results in an astounding 106-fold change in the rate constant (k) at the same temperature. As an analogy, think about living in one town in the mountains and wishing to go to a second town, located in a nearby valley. We would be limited by the height of the mountain we need to climb to go to the second town; a tunnel through the mountain would facilitate our travel. ar in ag Enzymes lower activation energy in both a surprisingly simple and terribly complex way. Enzymes bind substrates using a combination of weak forces and steric interactions that align functional groups, polarize bonds, promote electron flow, constrain molecules, and otherwise place molecules in a favorable geometry for reactions to occur. That is, enzymes literally form a pocket that brings groups together or pulls them apart to form a structure similar to the transition state. Once at the peak of this energetic barrier some fraction of molecules will decompose back into substrates, while the remainder will convert into products. m WORKED PROBLEM 4.1 | Describing an enzymatically catalyzed reaction Lysozyme is an enzyme that breaks bonds in the carbohydrate chains found in bacterial cell walls. Describe this reaction using the vocabulary found in this section. Strategy Examine the image and the reaction shown. Describe in your own words what is going on. Next, determine whether there are specific terms or names for what you have described, and put what you have written into scientific terms. Solution Lysozyme is a monomeric enzyme that catalyzes the hydrolytic cleavage of carbohydrate chains in the bacterial cell wall; thus it belongs to EC classification 3, hydrolases. The carbohydrate chains are the substrate, and they bind to the active site of the enzyme using induced fit. This binding stabilizes the transition state, lowering the activation energy—the barrier to catalysis. The broken fragments of the carbohydrate chain are the products of the reaction. Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. Follow-up question Choose any three enzymatically catalyzed reactions you can think of (either from this text or from other sources). Categorize these reactions based on the six types of reactions found in section 4.1.2. 4.1 Summary ai l.c om Enzymes are protein catalysts that increase rates of reaction by lowering activation energy. Enzymes are often globular proteins that may be monomeric or multimeric. Substrates (the reactants) are converted to products in the active site of the enzyme, a surface cleft that specifically binds the substrate. The active site binds substrate like a key in a lock or a hand in a glove. The binding is specific and tight, with solvent often being excluded. Enzymes can be categorized based on the type of reaction they catalyze. Enzymes do not alter equilibrium, but they do allow systems to achieve equilibrium more quickly than would be the case in the absence of a catalyst. 4.1 Concept Check 4.2 Enzymes Increase Reaction Rate @g m Describe what generally happens in an enzymatically catalyzed reaction. Describe the interactions of substrate and enzyme using the proper biochemical terminology. Describe the two models (lock and key and induced fit) that are used to describe these interactions. Use a simple thermodynamic argument to describe why enzymes increase the rate of a reaction. 4.2.1 A review of chemical rates ia nn ot ti1 3 Rates of enzymatically catalyzed reactions can be derived from rates of chemical reactions and are powerful tools for describing reactions. The rate of a chemical reaction is defined as the change in concentration of a reactant or product over time. Using square brackets to denote molar concentrations and the notation of calculus gives: (4.1) ar (4.2) in ag If this is the simplest reaction, where a single molecule of reactant is being changed into product, then the loss of reactant will equal the gain in product: (4.3) m In the simplest chemical reaction, such as an isomerization, a single molecule of A reacts to form a molecule of B. If we determine experimentally that this is a first-order reaction overall, we can express this mathematically using an equation termed a rate law: (4.4) In this expression, the rate is equal to a rate constant (k) multiplied by the concentration of A. This constant is unique for any individual reaction. The reaction rate also varies with the concentration of reactant A. In this example, the rate varies linearly with A. If the concentration of A were to double, the rate would double; if the concentration were to go up five times, the rate would increase five times. This indicates that the rate is first order with regard to A and first order overall; that is, it varies linearly with A and nothing else. It also indicates that, on a physical level, the reaction does not require two molecules of A to collide; rather, it proceeds when a single molecule of A rearranges into B. A reaction in which two molecules of A need to collide to generate a molecule of B is second order with regard to A: (4.5) (4.6) Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. Here, the rate of the reaction varies with the square of the concentration of A. Thus, if the concentration of A were to double, the rate would increase by four times. If the concentration were tripled, the reaction would go nine times faster. If the reaction rate did not vary at all with regard to the concentration of A, we would say that the reaction is zeroth order with regard to A (4.7) None of these rate laws relates concentration and time. To do that, the integrated rate law for each equation is required. For a firstorder reaction, the equation is first rearranged and then integrated: (4.8) ai l.c om (4.9) This gives: (4.10) which can be rearranged to give: @g m (4.11) or (4.12) ia nn ot ti1 3 The integrated form of the second-order rate law can be derived in a similar fashion and gives: (4.13) The integrated form of the zeroth-order rate law is (4.14) By integrating the rate law, it is possible to express product concentration as a function of time. in ag The rate constant (k) relates the concentration to the rate. It has two main components. First is the frequency factor, A, which describes the percentage of collisions that result in a favorable reaction, that is, the number of times that molecules must collide before they collide in the proper orientation to cause chemistry to occur. The second term is an exponential term that relates the activation energy, Ea; the temperature (measured in Kelvin), T; and the gas law constant, R. Here, e is being raised to the negative activation energy (Ea) power. Hence, the higher the Ea, the lower the value of the exponential, the lower the value of k, and the lower (4.15) m ar the rate. However, as the temperature increases, it can modify the exponential term to increase k and thus increase the reaction rate. This is a variation of a Boltzman–Maxwell distribution and describes the fraction of molecules with enough energy to overcome the activation energy barrier (Figure 4.6). Combining the frequency factor A and the exponential term gives the Arrhenius equation you may recall from general chemistry: @g m ai l.c om Printed by: [email protected]. Printing is for personal use only. No part of this book may be reproduced or transmitted without publisher's prior permission. Violators will be prosecuted. Figure 4.6 Boltzmann distribution. A Boltzmann distribution shows the distribution of particles with different energies. ia nn ot ti1 3 When a chemical reaction is catalyzed, the catalyst takes part in the reaction but is not consumed. Rather, it provides an alternative path for a reaction to occur, and this alternative mechanism has a lower activation energy. 4.2.2 The Michaelis-Menten equation relates enzymatic rates to measurable parameters Before examining an enzymatically catalyzed reaction, it is helpful to define the terms used. In such a reaction, the starting material is termed the substrate (S), and it is converted by the enzyme (E) to the product (P). The rate of an enzymatically catalyzed reaction is referred to as a velocity (v). m ar in ag In some of the first experiments to determine enzymatic rate, Adrian Brown, a professor of malting and brewing, determined that invertase (an enzyme that breaks down sucrose) is