Enzymes

2009
Biochemistry
Pre professional year
Dr. Waleed Tamimi
 Outline
1. Enzymes are powerful and highly specific catalysts.

2. Free energy is a useful Thermodynamic function for

understanding Enzymes.

3. Enzymes accelerate Reactions by facilitating the

formation of the transition State.

4. The Mechaelis-Menten formula accounts for the kinetics

properties of many enzymes.

5. Enzymes can be inhibited by specific molecules.

6. Vitamins are often precursors to Coenzymes.

 Definition & Function
• Characteristics of enzymes are their
catalysis and specificity.
• Catalysis takes place at a particular site
on the enzyme called the active site.
• Nearly all known enzymes are
proteins.
Functions of Enzymes Are Powerful
and Highly Specific Catalysts
• Are Powerful and Highly Specific Catalysts
• Enzymes accelerate reactions by factors of as much as a million
or more.
• Biological reactions do not take place in absence of enzymes.
• Example is the hydration of carbon dioxide is catalyzed by an
enzyme—carbonic anhydrase.
• The transfer of CO2 from the tissues into the blood and then to
the alveolar air would be less complete in the absence of this
enzyme.
 Proteolytic Enzymes
• Proteolytic Enzymes catalyze
proteolysis, the hydrolysis of a peptide
bond.
• The specificity of an enzyme is due to
the precise interaction of the substrate
with the enzyme.
 Examples of Proteolytic Enzymes
• Subtilisin is found in bacteria, it cleaves any
peptide bond.
• Trypsin, a digestive enzyme, catalyzes the
splitting of peptide bonds only on the carboxyl
side of lysine and arginine residues.
• Thrombin, an enzyme that participates in blood
clotting, catalyzes the hydrolysis of Arg-Gly
bonds.
• DNA polymerase I, adds nucleotides to a DNA
strand.
Figure 8.1. Enzyme Specificity. (A) Trypsin cleaves on the carboxyl
side of arginine and lysine residues, whereas (B) Thrombin cleaves
Arg-Gly bonds in particular sequences specifically.
 Enzymes Are Classified on the Basis of the
Types of Reactions That They Catalyze
• Many enzymes have common names that provide little
information about the reactions that they catalyze
(e.g., trypsin).
• Most other enzymes are named for their substrates and
for the reactions that they catalyze, with the suffix
“ase” added.
• Thus, an ATPase is an enzyme that breaks down ATP,
while ATP synthase is an enzyme that synthesizes
ATP.
An Energy-Transforming Enzyme. Ca2+ ATPase uses the energy
of ATP hydrolysis to transport Ca2+ across the membrane, generating
a Ca2+ gradient.
 Using of ATP
• The enzyme myosin converts the energy of
ATP into the mechanical energy of contracting
muscles.
• Pumps in the membranes of cells, which can be
thought of as enzymes that move substrates,
create chemical and electrical gradients by
using the energy of ATP to transport molecules
and ions
 Enzyme nomenclature
• 1964 the International Union of Biochemistry
established an Enzyme Commission to develop a
nomenclature for enzymes.
• Reactions were divided into six major groups
numbered 1 through 6. These groups were subdivided
and further subdivided, so that a four-digit number
preceded by the letters EC for Enzyme Commission
could precisely identify all enzymes.
• Nucleoside Monophosphate (NMP) kinase EC 2.7.4.4.
Six major classes of enzymes
 Active site
• A specific enzyme catalyzes each cellular reaction.
• A unique 3-D shape of an enzyme determines
which chemical reaction it catalyzes.
• A specific reactant that an enzyme acts on is called
a substrate of the enzyme.
• A substrate fits into a region of the enzyme called
an active site.
• An active site is typically a pocket or groove on
the surface of the enzyme.
 Substrate

Active site

Enzyme-substrate
Enzyme complex

When a substrate binds to an enzyme, the active site changes shape

slightly so that it embraces the substrate more snugly – induced fit.
 How Enzymes work?
• Enzymes speed up the cell’s chemical reactions by
lowering energy barriers.
• There is an energy barrier that must be overcome before
a chemical reaction can begin – the energy of activation
(EA).
• The reactants must absorb EA to become activated – to
contort or weaken bonds so that they can break and
new bonds can form, and start a reaction.
• An enzyme is a protein molecule that functions as a
biological catalyst.
• The enzyme increases the rate of a reaction without itself
being changed.
An enzyme speeds up a reaction by
lowering the EA barrier.
Each reaction has a transition state where the substrate is in
an unstable, short-lived chemical/structural state.

Enzymes act by lowering the free

energy of the transition state
 How Enzymes work?
• Enzymes lower the free energy of activation by
binding the transition state of the reaction better
than the substrate
• The enzyme must bind the substrate in the
correct orientation otherwise there would be no
reaction
• Not a lock & key but induced fit – the enzyme
and/or the substrate distort towards the
transition state
Enzymes Decrease the Activation Energy. Enzymes accelerate
reactions by decreasing ΔG‡, the free energy of activation.
 The Formation of an Enzyme-Substrate Complex Is the
First Step in Enzymatic Catalysis
• The first step in catalysis is the formation of an enzyme-
substrate complex.The substrates are bound to a specific
region of the enzyme called the active site.

• The recognition of substrates by enzymes is

accompanied by conformational changes at active sites,
and such changes facilitate the formation of the
transition state.
Reaction Velocity Versus Substrate Concentration in an Enzyme-
Catalyzed Reaction. An enzyme-catalyzed reaction reaches a
maximal velocity.
 Common Features of Active Sites of
Enzymes

1. The active site is a three-dimensional cleft

2. The active site takes up a relatively small part of the
total volume of an enzyme.
3. Active sites are clefts or crevices.
4. Substrates are bound to enzymes by multiple weak
attractions
5. The specificity of binding depends on the precisely
defined arrangement of atoms in an active site. lock
and key or induced fit
Lock-and-Key Model of Enzyme-Substrate Binding. In this model,
the active site of the unbound enzyme is complementary in shape to the
substrate.
Induced-Fit Model of Enzyme-Substrate Binding. The enzyme
changes shape on substrate binding. The active site forms a shape
complementary to the substrate only after the substrate has been bound.
 Part 2

Enzyme Kinetics and Inhibition

 The Michaelis-Menten Model Accounts for
the Kinetic Properties of Many Enzymes
• To understand how enzymes function, we need a
kinetic description of their activity.
• The Michaelis-Menten equation relates the initial
velocity of an enzyme-catalyzed reaction, Vi, to
the concentration of substrate, S, and two
parameters, Km and Vmax.
• Vmax is the velocity of the reaction extrapolated
to infinite substrate concentration and Km is the
substrate concentration at which the initial
velocity equals ½ Vmax.
Michaelis-Menten Kinetics. A plot of the reaction velocity (V0) as a
function of the substrate concentration [S] for an enzyme that obeys
Michaelis-Menten kinetics shows that the maximal velocity (Vmax ) is
approached asymptotically. The Michaelis constant (KM) is the substrate
 Michaelis-Menten

• In 1913, Leonor Michaelis and Maud Menten proposed a

simple model to account for these kinetic characteristics.

• The ES complex has two possible fates.

– It can dissociate to E and S, with a rate constant k-1 ,
– or it can proceed to form product P, with a rate constant k2.
 Michaelis-Menten

• the catalytic rate is equal to the product of

the concentration of the ES complex and k2.
 Michaelis-Menten
• when [S] is much less than KM, then V0 = (Vmax /KM)[S]; that is,
the rate is directly proportional to the substrate concentration.
• when [S] is much greater than KM, V0 = Vmax ; that is, the rate is
maximal, independent of substrate concentration.
• When [S] = KM, then V0 = Vmax /2.
• Thus, KMis equal to the substrate concentration at which the
reaction rate is half its maximal value.
• KM is an important characteristic of an enzyme-catalyzed
reaction and is significant for its biological function.
 The physiological consequence of KM

• It is illustrated by the sensitivity of some

individuals to ethanol.
• Such persons exhibit facial flushing and rapid
heart rate (tachycardia) after ingesting even
small amounts of alcohol.
• In the liver, alcohol dehydrogenase converts
ethanol into acetaldehyde.
 The physiological consequence of KM
• Acetaldehyde, is processed to acetate by acetaldehyde
dehydrogenase.

• Most people have two forms of the acetaldehyde

dehydrogenase, a low KM mitochondrial form and a high
KM cytosolic form.
• In susceptible persons, the mitochondrial enzyme is less
active due to the substitution of a single amino acid, and
acetaldehyde is processed only by the cytosolic enzyme.
• Because this enzyme has a high KM, less acetaldehyde is
converted into acetate; excess acetaldehyde escapes into
the blood and accounts for the physiological effects.
 Kinetic Perfection in Enzymatic
Catalysis
• The kcat /KM Criterion. When the substrate concentration is
much greater than KM, the rate of catalysis is equal to kcat ,
the turnover number
• Most Biochemical Reactions Include Multiple Substrates
• Allosteric Enzymes (display sigmoidal plots in which the
binding of substrate becomes cooperative ) Do Not Obey
Michaelis-Menten Kinetics
• cooperativity results in a sigmoidal plot of V0 versus [S].
Kinetics for an Allosteric Enzyme. Allosteric enzymes display a
sigmoidal dependence of reaction velocity on substrate concentration.
 Enzymes Can Be Inhibited by
Specific Molecules
• Specific small molecules, ions, drugs and
toxic agents act by inhibiting enzymes
• Enzyme inhibition can be either reversible
or irreversible.
 Irreversible inhibitor
• An irreversible inhibitor dissociates very slowly
from its target enzyme because it is tightly bound
to the enzyme, either covalently or noncovalently.
• Penicillin acts by covalently modifying the enzyme
transpeptidase, thereby preventing the synthesis of
bacterial cell walls and thus killing the bacteria.
• Aspirin acts by covalently modifying the enzyme
cyclooxygenase, reducing the synthesis of
inflammatory signals.
 Reversible inhibition
• Reversible inhibition, in contrast with irreversible
inhibition, is characterized by a rapid dissociation of the
enzyme-inhibitor complex.
• In competitive reversible inhibition, an enzyme can bind
substrate (forming an ES complex) or inhibitor (EI) but
not both (ESI).
• The competitive inhibitor resembles the substrate and
binds to the active site of the enzyme.
• The substrate is thereby prevented from binding to the
same active site.
• A competitive inhibitor diminishes the rate of catalysis
by reducing the proportion of enzyme molecules bound
to a substrate.
Distinction between a Competitive and a Noncompetitive
Inhibitor. (Top) enzyme-substrate complex; (middle) a competitive
inhibitor binds at the active site and thus prevents the substrate from
binding; (bottom) a noncompetitive inhibitor does not prevent the
substrate from binding.
 Methotrexate
• Methotrexate is a structural analog of
tetrahydrofolate, a coenzyme for the enzyme
dihydrofolate reductase, which plays a role in the
biosynthesis of purines and pyrimidines.
• It binds to dihydrofolate reductase 1000-fold more
tightly than the natural substrate and inhibits
nucleotide base synthesis.
• It is used to treat cancer.
Figure 8.16. Enzyme Inhibitors. The cofactor tetrahydrofolate and its
structural analog methotrexate. Regions with structural differences are
shown in red.
• In noncompetitive reversible inhibition, the inhibitor and
substrate can bind simultaneously to an enzyme molecule at
different binding sites.
• A noncompetitive inhibitor acts by decreasing the turnover
number rather than by diminishing the proportion of enzyme
molecules that are bound to substrate.
• Noncompetitive inhibition, in contrast with competitive
inhibition, cannot be overcome by increasing the substrate
concentration.
• A more complex pattern, called mixed inhibition, is produced
when a single inhibitor both hinders the binding of substrate
and decreases the turnover number of the enzyme.
Competitive and Noncompetitive Inhibition Are
Kinetically Distinguishable
• In competitive inhibition, the inhibitor competes
with the substrate for the active site.
• Because increasing the amount of substrate can
overcome the inhibition, Vmax can be attained in the
presence of a competitive inhibitor.
• The hallmark of competitive inhibition is that it can
be overcome by a sufficiently high concentration of
substrate.
• However, the apparent value of KM is altered; the
effect of a competitive inhibitor is to increase the
apparent value of KM
 Competitive and Noncompetitive Inhibition
Are Kinetically Distinguishable

• Competitive increase the Km of the enzyme, but

not the Vmax.
• A noncompetitive inhibitor will change the Vmax
of the enzyme.
• Uncompetitive inhibitors decrease both Km and
Vmax. An uncompetitive inhibitor binds only to
the enzyme-substrate complex.
Lineweaver-Burk Transformation

• The Km and Vmax for an enzyme can be

visually determined from a plot of 1/v
versus 1/S, called a lineweaver-Burk or a
double reciprocal plot.
1 = Km 1 + 1
Vi Vmax [S] Vmax
Enzyme Inhibition by Diisopropylphosphofluoridate (DIPF).
1) DIPF can inhibit an chymotrypsin by covalently modifying a crucial serine
residue. DIPF modifies only 1 of the 28 serine residues in chymotrypsin.
2) DIPF also revealed a reactive serine residue in acetylcholinesterase, an enzyme
important in the transmission of nerve impulses. Thus, DIPF and similar
compounds that bind and inactivate acetylcholinesterase are potent nerve gases.
 Summary
1) Enzymes are Powerful and Highly Specific
Catalysts
2) Free Energy Is a Useful Thermodynamic Function
for Understanding Enzymes
3) Enzymes Accelerate Reactions by Facilitating the
Formation of the Transition State
4) The Michaelis-Menten Model Accounts for the
Kinetic Properties of Many Enzymes
5) Enzymes Can Be Inhibited by Specific Molecules
6) Vitamins Are Often Precursors to Coenzymes
Part 3
 Enzymes Cofactors
• Many enzymes require cofactors to be more
active.
• Enzyme without its cofactor is referred to as an
apoenzyme; the complete, catalytically active
enzyme is called a holoenzyme.
• Cofactors can be subdivided into two groups:
1) metals (coenzymes),derived from vitamins,
either tightly or loosely bound to the enzyme.
2) small organic molecules
 Vitamins Are Often Precursors to Coenzymes

• Water-Soluble Vitamins Function As

Coenzymes
• Ascorbate, the ionized form of ascorbic acid,
serves as a reducing agent (an antioxidant), as
will be discussed shortly. The vitamin B series
comprises components of coenzymes
• Vitamin deficiencies are capable of causing a
variety of pathological conditions
Structures of Some Water-Soluble Vitamins.
Structures of Some Fat-Soluble Vitamins
 Factors Affecting Enzyme
Action: Temperature
• Little activity at low temperature
• Rate increases with temperature
• Most active at optimum temperatures
(usually 37°C in humans)
• Activity lost with denaturation at high
temperatures
 Factors Affecting Enzyme
Action: Substrate Concentration
• Increasing substrate concentration
increases the rate of reaction (enzyme
concentration is constant)
• Maximum activity reached when all of
enzyme combines with substrate
 Factors Affecting Enzyme
Action: pH
• Maximum activity at optimum pH
• R groups of amino acids have proper
charge
• Tertiary structure of enzyme is correct
• Narrow range of activity
• Most lose activity in low or high pH
• The cellular environment affects enzyme activity.
• An enzyme is most effective under an appropriate
condition.
• Temperature affects molecular motion – an enzyme’s
optimal temperature produces the highest rate.
• Most human enzymes work best at 35-40 ºC.
• Salt concentration and pH influence enzyme activity.
• The salt ions interfere with some of the chemical bonds
that maintain protein structure. The same is true of the
extra hydrogen ions at very low pH.
• The optimal pH for most enzymes is near neutrality.
• Allosteric regulation controls an enzyme’s
activity.
• Allosteric regulation is the term used to describe
cases where a protein’s function at one site is
affected by binding of a regulatory molecule at
another site.
• Allosteric regulation may either inhibit or
stimulate an enzyme’s activity by changing an
enzyme into its active or inactive forms.
• Not all vitamins function as coenzymes. The fat-
soluble vitamins, which are designated by the letters A,
D, E, and K , have a diverse array of functions.
• Vitamin K, which is required for normal blood clotting,
participates in the carboxylation of glutamate residues
to γ-carboxyglutamate, which makes modified
glutamic acid a much stronger chelator of Ca2+.
• Vitamin A (retinol) is the precursor of retinal, the light-
sensitive group in rhodopsin and other visual pigments.
• A deficiency of this vitamin leads to night blindness.
 Fat-Soluble Vitamins
• Vitamin D is regulates the metabolism of
calcium and phosphorus. A deficiency in
vitamin D impairs bone formation.
• Infertility in rats is a consequence of vitamin E
(α-tocopherol) deficiency. This vitamin reacts
with and neutralizes reactive oxygen species
such as hydroxyl, radicals before they can
oxidize unsaturated membrane lipids, damaging
cell structures.