Transcript Enzymes - Clayton State University

Chapter 6
ENZYMES
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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
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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
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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
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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
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Figure 6-1A
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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
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Figure 6-1B
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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
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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
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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
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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
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Figure 6-1C
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Figure 6-1D
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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
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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
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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
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Figure 6-2
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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
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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
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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
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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
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Figure 6-3
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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
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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
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Six classes of enzymes
•
•
•
•
•
•
Oxidoreductases
Transferases
Hydrolases
Lysases
Isomerases
Ligases
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Table 6-1
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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
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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
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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
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Figure 6-4A
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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
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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
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Figure 6-4B
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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
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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
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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
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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
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Figure 6-5
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Video: Closure of hexokinase
via induced fit
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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
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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
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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
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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
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Figure 6-6
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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
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Figure 6-7
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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
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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
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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
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Figure 6-8
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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
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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
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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
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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]
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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]
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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
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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
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Figure 6-9
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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
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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
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Table 6-2
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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
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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
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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
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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
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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
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Figure 6-14A
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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
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Figure 6-14B
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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
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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
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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
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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
•
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Figure 6-15
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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
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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
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Figure 6-16A
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Figure 6-16B
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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
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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
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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.
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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
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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
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Figure 6-17A
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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
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Figure 6-17B
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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
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Figure 6-18
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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
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