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Chapter 6 ENZYMES © 2012 Pearson Education, Inc. Enzymes: The Catalysts of Life • Enzyme catalysis: virtually all cellular processes or reactions are mediated by protein (sometimes RNA) catalysts called enzymes • The presence of the appropriate enzyme makes the difference between whether a reaction can take place and whether it will take place © 2012 Pearson Education, Inc. Activation Energy and the Metastable State • Many thermodynamically feasible reactions in a cell that could occur do not proceed at any appreciable rate • For example, the hydrolysis of ATP has G = –7.3 kcal/mol • ATP + H2O ADP + Pi • However, ATP dissolved in water remains stable for several days © 2012 Pearson Education, Inc. Before a Chemical Reaction Can Occur, the Activation Energy Barrier Must Be Overcome • Molecules that could react with one another often do not because they lack sufficient energy • Each reaction has a specific activation energy, EA • EA: the minimum amount of energy required before collisions between the reactants will give rise to products © 2012 Pearson Education, Inc. Transition state • Reactants need to reach an intermediate chemical stage called the transition state • The transition state has a higher free energy than that of the initial reactants © 2012 Pearson Education, Inc. Figure 6-1A © 2012 Pearson Education, Inc. Activation energy barrier • The rate of a reaction is always proportional to the fraction of molecules with an energy equal to or greater than EA • The only molecules that are able to react at a given time are those with enough energy to exceed the activation energy barrier, EA © 2012 Pearson Education, Inc. Figure 6-1B © 2012 Pearson Education, Inc. The Metastable State Is a Result of the Activation Barrier • For most reactions at normal cell temperature, the activation energy is so high that few molecules can exceed the EA barrier • Reactants that are thermodynamically unstable, but lack sufficient EA, are said to be in a metastable state • Life depends on high EAs that prevent most reactions in the absence of catalysts © 2012 Pearson Education, Inc. Catalysts Overcome the Activation Energy Barrier • The EA barrier must be overcome in order for needed reactions to occur • This can be achieved by either increasing the energy content of molecules or by lowering the EA requirement © 2012 Pearson Education, Inc. Increasing the energy content of a system • The input of heat can increase the kinetic energy of the average molecule, ensuring that more molecules will be able to take part in a reaction • This is not useful in cells, however, which are isothermal • Isothermal: constant in temperature © 2012 Pearson Education, Inc. Lowering activation energy • If reactants can be bound on a surface and brought close together, their interaction will be favored and the required EA will be reduced • A catalyst enhances the rate of a reaction by providing such a surface and effectively lowering EA • Catalysts themselves proceed through the reaction unaltered © 2012 Pearson Education, Inc. Figure 6-1C © 2012 Pearson Education, Inc. Figure 6-1D © 2012 Pearson Education, Inc. Enzymes as Biological Catalysts • All catalysts share three basic properties – They increase reaction rates by lowering the EA required – They form transient, reversible complexes with substrate molecules – They change the rate at which equilibrium is achieved, not the position of the equilibrium • Organic catalysts are enzymes © 2012 Pearson Education, Inc. Most Enzymes Are Proteins • Most enzymes are known to be proteins • However, recently, it has been discovered that some RNA molecules also have catalytic activity • These are called ribozymes and will be discussed later © 2012 Pearson Education, Inc. The Active Site • Every enzyme contains a characteristic cluster of amino acids that forms the active site • This results from the three dimensional folding of the protein, and is where substrates bind and catalysis takes place • The active site is usually a groove or pocket that accommodates the intended substrate(s) with high affinity © 2012 Pearson Education, Inc. Figure 6-2 © 2012 Pearson Education, Inc. Amino acids involved in the active site • Of the 20 different amino acids, only a few are involved in the active site • These are cys, his, ser, asp, glu, and lys • These can participe in binding the substrate and several serve as donors or acceptors of protons © 2012 Pearson Education, Inc. Cofactors • Some enzymes contain nonprotein cofactors needed for catalytic activity, often because they function as electron acceptors • These are called prosthetic groups and are usually metal ions or small organic molecules called coenzymes • Coenzymes are derivatives of vitamins © 2012 Pearson Education, Inc. Prosthetic groups • Prosthetic groups are located at the active site and are indispensable for enzyme activity • Each molecule of the enzyme catalase has a multimeric structure called a porphyrin ring to which a necessary iron atom is bound • The requirement for certain prosthetic groups on some enzymes explains our requirements for trace amounts of vitamins and minerals © 2012 Pearson Education, Inc. Enzyme Specificity • Due to the shape and chemistry of the active site, enzymes have a very high substrate specificity • Inorganic catalysts are very nonspecific whereas similar reactions in biological systems generally have a much higher level of specificity © 2012 Pearson Education, Inc. Figure 6-3 © 2012 Pearson Education, Inc. Group specificity • Some enzymes will accept a number of closely related substrates • Others accept any of an entire group of substrates sharing a common feature • This group specificity is most often seen in enzymes involved in degradation of polymers © 2012 Pearson Education, Inc. Enzyme Diversity and Nomenclature • Thousands of different enzymes have been identified, with enormous diversity • Names have been given to enzymes based on substrate (protease, ribonuclease, amylase), or function (trypsin, catalase) • Under the Enzyme Commission (EC), enzymes are divided into six major classes based on general function © 2012 Pearson Education, Inc. Six classes of enzymes • • • • • • Oxidoreductases Transferases Hydrolases Lysases Isomerases Ligases © 2012 Pearson Education, Inc. Table 6-1 © 2012 Pearson Education, Inc. Sensitivity to Temperature • Enzymes are characterized by their sensitivity to temperature • This is not a concern in homeotherms, birds and mammals, that maintain a constant body temperature • However, many organisms function at their environmental temperature, which can vary widely © 2012 Pearson Education, Inc. Enzyme activity and temperatures • At low temperatures, the rate of enzyme activity increases with temperature due to increased kinetic activity of enzyme and substrate molecules • However, beyond a certain point, further increases in temperature result in denaturation of the enzyme molecule and loss of enzyme activity © 2012 Pearson Education, Inc. Optimal temperature • The temperature range over which an enzyme denatures varies among enzymes and organisms • The reaction rate of human enzymes is maximum at 37oC (the optimal temperature), the normal body temperature • Most enzymes of homeotherms are inactivated by temperatures above 50–55oC © 2012 Pearson Education, Inc. Figure 6-4A © 2012 Pearson Education, Inc. Ranges of heat sensitivity • Some enzymes are unusually sensitive and will denature at temperatures as low as 40oC • Some enzymes retain activity at unusually high temperatures, such as the enzymes of archaea that live in acidic hot springs • Enzymes of cryophilic (cold-loving) organisms such as Listeria bacteria can function at low temperatures, even under refrigeration © 2012 Pearson Education, Inc. Sensitivity to pH • Most enzymes are active within a pH range of about 3–4 units • pH dependence is usually due to the presence of charged amino acids at the active site or on the substrate • pH changes affect the charge of such residues, and can disrupt ionic and hydrogen bonds © 2012 Pearson Education, Inc. Figure 6-4B © 2012 Pearson Education, Inc. Sensitivity to Other Factors • Enzymes are sensitive to factors such as molecules and ions that act as inhibitors or activators • Most enzymes are also sensitive to ionic strength of the environment • This affects hydrogen bonding and ionic interactions needed to maintain tertiary conformation © 2012 Pearson Education, Inc. Substrate Binding, Activation, and Catalysis Occur at the Active Site • Because of the precise chemical fit between the active site of the enzyme and its substrates, enzymes are highly specific © 2012 Pearson Education, Inc. Substrate Binding • Once at the active site, the substrate molecules are bound to the enzyme surface in the right orientation to facilitate the reaction • Substrate binding usually involves hydrogen bonds, ionic bonds, or both • Substrate binding is readily reversible © 2012 Pearson Education, Inc. The induced-fit model • In the past, the enzyme was seen as rigid, with the substrate fitting into the active site like a key in a lock (lock-and-key model) • A more accurate view is the induced-fit model, in which substrate binding at the active site induces a conformational change in the shape of the enzyme © 2012 Pearson Education, Inc. Figure 6-5 © 2012 Pearson Education, Inc. Video: Closure of hexokinase via induced fit © 2012 Pearson Education, Inc. Conformational change • The induced conformational change brings needed amino acid side chains into the active site, even those that are not nearby • Sometimes these are not nearby unless the substrate is bound to the active site © 2012 Pearson Education, Inc. Substrate Activation • The role of the active site is to recognize and bind the appropriate substrate and also to activate it by providing the right environment for catalysis • This is called substrate activation, which proceeds via several possible mechanisms © 2012 Pearson Education, Inc. Three common mechanisms of substrate activation • Bond distortion, making it more susceptible to catalytic attack • Proton transfer, which increases reactivity of substrate • Electron transfer, resulting in temporary covalent bonds between enzyme, substrate © 2012 Pearson Education, Inc. The Catalytic Event • The sequence of events – 1. The random collision of a substrate molecule with the active site results in it binding there – 2. Substrate binding induces a conformational change that tightens the fit, facilitating the conversion of substrate into products © 2012 Pearson Education, Inc. Figure 6-6 © 2012 Pearson Education, Inc. The Catalytic Event (continued) • The sequence of events – 3. The products are then released from the active site – 4. The enzyme molecule returns to the original conformation with the active site available for another molecule of substrate © 2012 Pearson Education, Inc. Figure 6-7 © 2012 Pearson Education, Inc. Enzyme Kinetics • Enzyme kinetics describes the quantitative aspects of enzyme catalysis and the rate of substrate conversion into products • Reaction rates are influenced by factors such as the concentrations of substrates, products, and inhibitors © 2012 Pearson Education, Inc. Initial reaction rates • Initial reaction rates are measured over a brief time, during which the substrate concentration has not yet decreased enough to affect the rate of reaction © 2012 Pearson Education, Inc. Most Enzymes Display Michaelis– Menten Kinetics • Initial reaction velocity (v), the rate of change in product concentration per unit time, depends on the substrate concentration [S] • At low [S], doubling [S] will double v, but as [S] increases each additional increase in [S] results in a smaller increase in v • When [S] becomes very large the value of v reaches a maximum © 2012 Pearson Education, Inc. Figure 6-8 © 2012 Pearson Education, Inc. Vmax and saturation • As [S] tends toward infinity, v approaches an upper limiting value, maximum velocity (Vmax) • The value of Vmax can be increased by adding more enzyme • The inability of increasingly higher substrate concentrations to increase the reaction velocity beyond a finite upper value is called saturation © 2012 Pearson Education, Inc. The Michaelis–Menten Equation • Michaelis and Menten postulated a theory of enzyme action • Enzyme E first reacts with the substrate, to form a transient complex, ES • ES then undergoes the catalytic reaction to generate E and P © 2012 Pearson Education, Inc. The Michaelis–Menten Equation (continued) • • The above model, under steady state conditions gives the Michaelis–Menten equation • • Km (the Michaelis constant) = the concentration of substrate that gives half maximum velocity © 2012 Pearson Education, Inc. What Is the Meaning of Vmax and K m? • We can understand the relationship between v and [S], and the meaning of Vmax and Km by considering three cases regarding [S] © 2012 Pearson Education, Inc. Case 1: Very Low Substrate Concentration ([S] << Km) • If [S] << Km • Then, Km + [S] = [Km] • • So at very low [S], the initial velocity of the reaction is roughly proportional to [S] © 2012 Pearson Education, Inc. Case 2: Very High Substrate Concentration ([S] >> Km) • If [S] >> Km • Then, Km + [S] = [S] • • So at very high [S], the initial velocity of the reaction is independent of variation in [S] and Vmax is the velocity at saturating substrate concentrations © 2012 Pearson Education, Inc. Vmax • Vmax is an upper limit determined by – The time required for the actual catalytic reaction – How many enzyme molecules are present • The only way to increase Vmax is to increase enzyme concentration © 2012 Pearson Education, Inc. Figure 6-9 © 2012 Pearson Education, Inc. Why Are Km and Vmax Important to Cell Biologists? • The lower the Km value for a given enzyme and substrate, the lower the [S] range in which the enzyme is effective • Vmax is important, as a measure of the potential maximum rate of the reaction • By knowing Vmax, Km, and the in vivo substrate concentration, we can estimate the likely rate of the reaction under cellular conditions © 2012 Pearson Education, Inc. Turnover number • Vmax can be used to determine turnover number, kcat • kcat is the rate at which substrate molecules are converted to product by a single enzyme at maximum velocity • • Turnover numbers vary greatly among enzymes © 2012 Pearson Education, Inc. Table 6-2 © 2012 Pearson Education, Inc. Enzyme Inhibitors Act Either Irreversibly or Reversibly • Enzymes are influenced (mostly inhibited) by products, alternative substrates, substrate analogs, drugs, toxins, and allosteric effectors • The inhibition of enzyme activity plays a vital role as a control mechanism in cells • Drugs and poisons frequently exert their effects by inhibition of specific enzymes © 2012 Pearson Education, Inc. Inhibitors important to enzymologists • Inhibitors of greatest use to enzymologists are substrate analogs and transition state analogs • These are compounds that resemble real substrates or transition states closely enough to occupy the active state but not closely enough to complete the reaction • Substrate analogs are important tools in fighting infectious diseases © 2012 Pearson Education, Inc. Reversible and irreversible inhibition • Irreversible inhibitors, which bind the enzyme covalently, cause permanent loss of catalytic activity and are generally toxic to cells – For example, heavy metal ions, nerve gas poisons, some insecticides • Reversible inhibitors bind enzymes noncovalently and can dissociate from the enzyme © 2012 Pearson Education, Inc. Reversible inhibition (continued) • The fraction of enzyme available for use in a cell depends on the concentration of the inhibitor and how easily the enzyme and inhibitor can dissociate • The two forms of reversible inhibitors are competitive inhibitors and noncompetitive inhibitors © 2012 Pearson Education, Inc. Competitive inhibition • Competitive inhibitors bind the active site of an enzyme and so compete with substrate for the active site • Enzyme activity is inhibited directly because active sites are bound to inhibitors, preventing the substrate from binding © 2012 Pearson Education, Inc. Figure 6-14A © 2012 Pearson Education, Inc. Noncompetitive inhibition • Noncompetitive inhibitors bind the enzyme molecule outside of the active site • They inhibit activity indirectly by causing a conformation change in the enzyme that – Inhibits substrate binding at the active site, or – Reduces catalytic activity at the active site © 2012 Pearson Education, Inc. Figure 6-14B © 2012 Pearson Education, Inc. Enzyme Regulation • Enzyme rates must be continuously adjusted to keep them tuned to the needs of the cell • Regulation that depends on interactions of substrates and products with an enzyme is called substrate-level regulation • Increases in substrate levels result in increased reaction rates, whereas increased product levels lead to lower rates © 2012 Pearson Education, Inc. Allosteric regulation and covalent modification • Cells can turn enzymes on and off as needed by two mechanisms: allosteric regulation and covalent modification • Usually enzymes regulated this way catalyze the first step of a multi-step sequence • By regulating the first step of a process, cells are able to regulate the entire process © 2012 Pearson Education, Inc. Allosteric Enzymes Are Regulated by Molecules Other than Reactants and Products • Allosteric regulation is the single most important control mechanism whereby the rates of enzymatic reactions are adjusted to meet the cell’s needs © 2012 Pearson Education, Inc. Feedback Inhibition • It is not in the best interests of a cell for enzymatic reactions to proceed at the maximum rate • In feedback (or end-product) inhibition, the final product of an enzyme pathway negatively regulates an earlier step in the pathway • © 2012 Pearson Education, Inc. Figure 6-15 © 2012 Pearson Education, Inc. Allosteric Regulation • Allosteric enzymes have two conformations, one in which it has affinity for the substrate(s) and one in which it does not • Allosteric regulation makes use of this property by regulating the conformation of the enzyme • An allosteric effector regulates enzyme activity by binding and stabilizing one of the conformations © 2012 Pearson Education, Inc. Allosteric regulation (continued) • An allosteric effector binds a site called an allosteric (or regulatory) site, distinct from the active site • The allosteric effector may be an activator or inhibitor, depending on its effect on the enzyme • Inhibitors shift the equilibrium between the two enzyme states to the low affinity form; activators favor the high affinity form © 2012 Pearson Education, Inc. Figure 6-16A © 2012 Pearson Education, Inc. Figure 6-16B © 2012 Pearson Education, Inc. Allosteric enzymes • Most allosteric enzymes are large, multisubunit proteins with an active or allosteric site on each subunit • Active and allosteric sites are on different subunits, the catalytic and regulatory subunits, respectively • Binding of allosteric effectors alters the shape of both catalytic and regulatory subunits © 2012 Pearson Education, Inc. Allosteric Enzymes Exhibit Cooperative Interactions Between Subunits • Many allosteric enzymes exhibit cooperativity • As multiple catalytic sites bind substrate molecules, the enzyme changes conformation, which alters affinity for the substrate • In positive cooperativity the conformation change increases affinity for substrate; in negative cooperativity, affinity for substrate is decreased © 2012 Pearson Education, Inc. Enzymes Can Also Be Regulated by the Addition or Removal of Chemical Groups • Many enzymes are subject to covalent modification • Activity is regulated by addition or removal of groups, such as phosphate, methyl, acetyl groups, etc. © 2012 Pearson Education, Inc. Phosphorylation and Dephosphorylation • The reversible addition of phosphate groups is a common covalent modification • Phosphorylation occurs most commonly by transfer of a phosphate group from ATP to the hydroxyl group of Ser, Thr, or Tyr residues in a protein • Protein kinases catalyze the phosphorylation of other proteins © 2012 Pearson Education, Inc. Dephosphorylation • Dephosphorylation, the removal of phosphate groups from proteins, is catalyzed by protein phosphatases • Depending on the enzyme, phosphorylation may be associated with activation or inhibition of the enzyme • Fisher and Krebs won the Nobel prize for their work on glycogen phosphorylase © 2012 Pearson Education, Inc. Figure 6-17A © 2012 Pearson Education, Inc. Regulation of glycogen phosphorylase • Glycogen phosphorylase exists as two interconvertible forms – An active, phosphorylated form (glycogen phosphorylase-a) – An inactive, non-phosphorylated form (glycogen phosphorylase-b) • The enzymes responsible – Phosphorylase kinase phosphorylates the enzyme – Phosphorylase phosphatase removes the phosphate © 2012 Pearson Education, Inc. Figure 6-17B © 2012 Pearson Education, Inc. Proteolytic Cleavage • The activation of a protein by a one-time, irreversible removal of part of the polypeptide chain is called proteolytic cleavage • Proteolytic enzymes of the pancreas, trypsin, chymotrypsin, and carboxypeptidase, are examples of enzymes synthesized in inactive form (as zymogens) and activated by cleavage as needed © 2012 Pearson Education, Inc. Figure 6-18 © 2012 Pearson Education, Inc. RNA Molecules as Enzymes: Ribozymes • Some RNA molecules have been found to have catalytic activity; these are called ribozymes • Self-splicing rRNA from Tetrahymena thermophila and ribonuclease P are examples • It is thought by some that RNA catalysts predate protein catalysts, and even DNA © 2012 Pearson Education, Inc.