Chapter 6 Enzymes: The Catalysts of Life Lectures by Kathleen Fitzpatrick Simon Fraser University
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 + H 2 O ADP + P i However, ATP dissolved in water remains stable for several days
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, E A E A : the minimum amount of energy required before collisions between the reactants will give rise to products
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
Figure 6-1A
Activation energy barrier The rate of a reaction is always proportional to the fraction of molecules with an energy equal to or greater than E A The only molecules that are able to react at a given time are those with enough energy to exceed the activation energy barrier, E A
Figure 6-1B
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 E A barrier Reactants that are thermodynamically unstable, but lack sufficient E A, are said to be in a metastable state Life depends on high E A s that prevent most reactions in the absence of catalysts
Catalysts Overcome the Activation Energy Barrier The E A 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 E A requirement
Lowering activation energy If reactants can be bound on a surface and brought close together, their interaction will be favored and the required E A will be reduced A catalyst enhances the rate of a reaction by providing such a surface and effectively lowering E A Catalysts themselves proceed through the reaction unaltered
Figure 6-1C
Figure 6-1D
An increase in temperature increases the rate at which a spontaneous reaction occurs in a test tube because. a. an increase in temperature lowers the energy of activation (E A ) b. an increase in temperature makes all molecules more reactive c. an increase in temperature increases the proportion of molecules that have sufficient kinetic energy to react d. an increase in temperature acts like a catalyst
A catalyst increases the rate of a reaction by. a. lowering E A and thus making G more negative b. lowering E A without having any effect on G c. lowering E A and thus shifting the equilibrium in favor of a negative G d. lowering E A and thus increasing the chance that reactants will collide
Enzymes as Biological Catalysts All catalysts share three basic properties They increase reaction rates by lowering the E A 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
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
Figure 6-2
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
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
Figure 6-3
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
The active site of an enzyme is important because it. a. provides a small compartment with a higher temperature, allowing reactants to have enough energy to react b. is altered by each reaction, explaining why cells must continuously take in energy and food to synthesize new proteins c. provides a reactive surface to which products bind tightly d. provides a reactive surface that lowers E A
Enzymes exhibit more specificity than inorganic catalysts because. a. enzymes are genetically determined b. enzymes operate over a narrower range of temperatures than inorganic catalysts c. inorganic catalysts are able to catalyze a much wider range of redox reactions d. the shape and chemistry of the active site of an enzyme restricts the molecules that can bind to it
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
Table 6-1
Figure 6-4A
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
Figure 6-4B
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 affects hydrogen bonding and ionic interactions needed to maintain tertiary conformation
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
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
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
Figure 6-5
Video: Closure of hexokinase via induced fit
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
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
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
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
Figure 6-6
Figure 6-7
When an enzyme binds to a substrate and activates it, activation means that. a. the enzyme has increased the kinetic energy in the substrate, making it more likely to react b. the enzyme has lowered G for the reaction c. the induced fit has subjected the substrate to a chemical environment that lowers E A d. the enzyme has undergone a conformational change, consistent with the induced fit model
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
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
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]
Figure 6-8
To find V max for an enzyme, it s important to measure the initial velocity because. a. enzymes can catalyze both the forward and backward reaction as the ratio of products:reactants increases b. the initial velocity is the only one that comes from the induced fit caused by the enzyme c. the initial velocity is the only one directly related to G for the reaction d. degradation of the enzyme as the reaction proceeds makes the reaction progressively slower, artificially lowering V max
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
The Michaelis Menten Equation (continued) The above model, under steady state conditions gives the Michaelis Menten equation K m (the Michaelis constant) = the concentration of substrate that gives half maximum velocity
Figure 6-8
What Is the Meaning of V max and K m? We can understand the relationship between v and [S], and the meaning of V max and K m by considering three cases regarding [S]
Case 1: Very Low Substrate Concentration ([S] << K m ) If [S] << K m Then, K m + [S] = [K m ] So at very low [S], the initial velocity of the reaction is roughly proportional to [S]
Case 2: Very High Substrate Concentration ([S] >> K m ) If [S] >> K m Then, K m + [S] = [S] So at very high [S], the initial velocity of the reaction is independent of variation in [S] and V max is the velocity at saturating substrate concentrations
V max V max 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 V max is to increase enzyme concentration
Figure 6-9
Case 3: ([S] = K m ) If [S] is equal to K m [ This shows that K m is the specific substrate concentration at which the reaction proceeds at one half its maximum velocity
Why Are K m and V max Important to Cell Biologists? The lower the K m value for a given enzyme and substrate, the lower the [S] range in which the enzyme is effective V max is important, as a measure of the potential maximum rate of the reaction By knowing V max, K m, and the in vivo substrate concentration, we can estimate the likely rate of the reaction under cellular conditions
Table 6-2
The Double-Reciprocal Plot Is a Useful Means of Linearizing Kinetic Data Lineweaver and Burk inverted both sides of equation 6-7 to give This is known as the Lineweaver Burk equation
The double-reciprocal plot A plot of 1/v vs 1/[S] is called the double-reciprocal plot This linear plot takes the general form of y = mx + b, where m is the slope and b the y-intercept The slope is K m /V max, the y-intercept is 1/V max, and the x-intercept is 1/K m
Figure 6-10
Determining K m and V max: An Example Consider the following reaction, important in energy metabolism: glucose + ATP glucose-6-phosphate + ADP hexokinase To analyze this reaction, begin by determining initial velocity at several substrate concentrations For two substrates, they must be varied one at a time, with saturating levels of the other
Figure 6-12
Figure 6-13
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
Inhibitors important to enzymologists Inhibitors of greatest use to enzymologists are substrate analogs and transition state analogs
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
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
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
Figure 6-14A
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
Figure 6-14B
How would you expect a competitive inhibitor to affect enzyme function? a. By raising the K m without affecting V max, because infinite amounts of substrate would wash out the inhibitor. b. By lowering the V max without affecting K m, because the enzyme still binds well to its natural substrate. c. By lowering the V max without affecting K m, because in the presence of the inhibitor, there is essentially less enzyme, and V max is directly proportional to the enzyme concentration.
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
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
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
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
Figure 6-15
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
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
Figure 6-16A
Figure 6-16B
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
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
Which of the following must be true of enzymes that are regulated allosterically? a. The enzyme must have at least one domain or subunit that binds to the regulatory compound, and at least one catalytic domain or subunit. b. The enzyme must never catalyze the reverse reaction. c. The allosteric regulator may bind to the active site. d. The enzyme must be part of a biosynthetic pathway (such as one that synthesizes the amino acid tryptophan).
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.
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
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
Figure 6-17A
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
Figure 6-17B
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
Figure 6-18
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
Which of the following steps in the regulation of glycogen phosphorylase is incorrect? a. First, glycogen phosphorylase b and ATP bind to the active site of phosphorylase kinase. b. Next, glycogen phosphorylase catalyzes the transfer of phosphate molecules from ATP onto glycogen phosphorylase, resulting in active glycogen phosphorylase a. c. Active glycogen phosporylase a catalyzes the breaking down of glycogen. d. Glycogen phosphorylase a and water bind to the active site of phosphorylase phosphatase, which catalyzes the removal of the phosphate groups from glycogen phosphorylase a. e. The result is inactive glycogen phosphorylase b.