enzymes: the catalysts of life - warner pacific collegeclasspages.warnerpacific.edu/bdupriest/bio...

© 2012 Pearson Education, Inc.

Lectures by

Kathleen Fitzpatrick Simon Fraser University

Enzymes:

The Catalysts

of Life

Chapter 6


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


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,

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


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


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


Figure 6-1C


Figure 6-1D

© 2012 Pearson Education, Inc. © 2012 Pearson Education, Inc.

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 (EA)

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 EA and thus making G more negative

b. lowering EA without having any effect on G

c. lowering EA and thus shifting the equilibrium in favor of a negative G

d. lowering EA 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

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


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 EA


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

EnzymeInducedFit.html


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 EA

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 Vmax 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 Vmax


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


Figure 6-8


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


Case 3: ([S] = Km)

• If [S] is equal to Km

• [

• This shows that Km is the specific substrate

concentration at which the reaction proceeds

at one half its maximum velocity


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


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 Km/Vmax, the y-intercept is 1/Vmax, and

the x-intercept is –1/Km


Figure 6-10


Determining Km and Vmax: An Example

• Consider the following reaction, important in energy

metabolism:

– glucose + ATP → glucose-6-phosphate + ADP

• 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

hexokinase


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 Km without affecting Vmax, because infinite amounts of substrate would wash out the inhibitor.

b. By lowering the Vmax without affecting Km, because the enzyme still binds well to its natural substrate.

c. By lowering the Vmax without affecting Km, because in the presence of the inhibitor, there is essentially less enzyme, and Vmax

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 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


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.

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