© 2012 pearson education, inc. enzymes chapter 6

© 2012 Pearson Education, Inc.

ENZYMESChapter 6


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


Activation Energy and the Metastable State

• Many thermodynamically feasible reactions in a cell that could occur do not proceed at any appreciable rate (on their own)

• For example, the hydrolysis of ATP has G = –7.3 kcal/mol or oxidation of glucose

• ATP + H2O ADP + Pi

• 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, EA

• EA: 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 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


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 EA barrier

• Reactants that are thermodynamically unstable (seemingly stable), 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-critical why?


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


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- in lab we heat solutions

• This is not useful in cells, however, which are isothermal

• Isothermal: constant in temperature


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, not permanently changes or consumed as the reaction proceeds


Figure 6-1C


Figure 6-1D


The reaction of catalase (enzyme) in the decomposition of living tissue:

2 H2O2 → 2 H2O + O2


What is quantum tunneling???


Enzymes as Biological Catalysts

• All catalysts share three basic properties

– They increase reaction rates by lowering the EA required (is thermal activation required?)

– They form transient, reversible complexes with substrate molecules

– They change only the rate at which equilibrium is achieved, not the position of the equilibrium. Cannot change G´

• Organic catalysts are enzymes


Most Enzymes Are Proteins

• Most enzymes are known to be proteins– Fermentation studies Buchner 1897– Urease was first enzyme crystallized

• However, recently, it has been discovered that some RNA molecules also have catalytic activity

• These are called ribozymes and will be discussed later


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


Amino acids involved in the active site

• Substrate binding can depend on amino acids at various positions. Catalysis is usually very specific amino acids– Of the 20 different amino acids, only a few are

involved in the active site– These are cysteine, histine, serine, asparate,

glutamate, and lysine

• These can participate in binding the substrate and several serve as donors or acceptors of protons


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 (Mg, Zn, Fe) or small organic molecules called coenzymes

• Coenzymes are derivatives of vitamins (niacin, riboflavin, vit. B)


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


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 (trans fat)


Figure 6-3


Group specificity

• Not all enzymes are quite specific– 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 (carboxypeptidase A)


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


Six classes of enzymes

• Oxidoreductases• Transferases• Hydrolases• Lysases• Isomerases• Ligases


Table 6-1


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 (independent of environment)

• However, many organisms function at their environmental temperature, which can vary widely (insects, reptiles, worms, plants)


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


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


Figure 6-4A


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 (yeast/molds) can function at low temperatures, even under refrigeration


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 (dissolved ions) of the environment

• This affects hydrogen bonding and ionic interactions needed to maintain tertiary conformation

• Magnesium


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


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


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


Figure 6-6


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 6-7


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]

• 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


Figure 6-8


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


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

•

• Km (the Michaelis constant) = the concentration of substrate that gives half maximum velocity


What Is the Meaning of Vmax and Km?

• We can understand the relationship between v and [S], and the meaning of Vmax and Km by considering three cases regarding [S]


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]


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


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


Figure 6-9


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


Table 6-2


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

• 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


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


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


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 inter-convertible 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

© 2012 pearson education, inc. enzymes chapter 6

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