2. Overview
• Properties of enzyme catalyst
• Classes of enzymes
• Enzyme cofactors
• Thermodynamics law and enzyme
• Enzyme and the formation of transition state
• Enzyme active sites
• Enzyme-substrate binding model
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3. Introduction
• Enzyme = catalyst of biological systems.
• Catalyst is a substance that speeds up chemical reactions
• Most striking characteristics of enzymes: catalytic power &
specificity.
• Catalysis take place at a particular site on the enzyme =
active site.
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4. Introduction
• Enzymes act by converting starting molecules (substrates)
into different molecules (products).
• Enzyme can specifically bind a very wide range of molecules
highly effective catalysts for an enormous diversity of
chemical reaction.
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Most enzymes are protein,
although some catalytic DNA
(deoxyribozyme) and RNA
(ribozyme) have been identified.
Ribozyme
5. Enzymes differ from ordinary
chemical catalysts
1. Higher reaction rates: The rates of enzymatically catalyzed reactions
are typically 106 to 1012 times greater than those uncatalyzed
reactions and are at least several orders of magnitude greater than
those chemically catalyzed reactions.
2. Milder reaction conditions: occur under relatively mild conditions:
temperatures below 100°C, atmospheric pressure, and nearly neutral
pH. In contrast, efficient chemical catalysis often requires elevated
temperatures and pressures as well as extremes of pH.
3. Greater reaction specificity: Enzymes have a greater degree of
specificity to their substrates (reactants) and their products than do
chemical catalysts; enzymatic reactions rarely have side products.
4. Capacity for regulation: The catalytic activities of many enzymes vary
in response to the concentrations of substances other than their
substrates. The mechanisms of these regulatory processes include
allosteric control, covalent modification of enzymes, and variation of
the amounts of enzymes synthesized.
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6. Properties of a true catalyst
1. Catalyst lowers the activation energy barrier of a chemical
reaction
2. Catalyst remains unchanged during the catalytic process
3. Catalyst can be reused.
4. Catalyst does not alter the equilibrium of the chemical
reaction, but alters the rate at which the chemical reaction’s
equilibrium is attained.
5. Catalyst acts by forming a transient complex with the
substrate.
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7. Substrate specificity
• Enzymes are highly specific in:
– the reactions that they catalyze
– their choice of reactants (substrates).
• The specificity of an enzyme is due to the precise interaction of the
substrate with the enzyme.
• Enzyme usually catalyzes a single chemical reaction or set of
closely related reactions.
e.g.: proteolytic enzymes: hydrolysis of a peptide bond.
• Proteolytic enzymes differ in their degree of substrate specificity
– Trypsin: digestive enzyme, catalyzes the splitting of peptide
bonds only on the carboxyl side of lysine & arginine.
– Thrombin: more specific than trypsin, catalyzes the hydrolysis
of Arg-Gly bonds in particular peptide sequences only.
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8. Trypsin cleaves on the
carbonyl side of arginine
and lysine residues.
Thrombin cleaves Arg-Gly bonds in
particular sequences only.
9. Substrate specificity
• The noncovalent forces (van der Waals,
electrostatic, hydrogen bonding, and
hydrophobic interactions) for substrates-
enzyme binding are similar to the forces that
dictate the conformations of the proteins.
• In general, a substrate-binding site consists
of a cleft on the surface of an enzyme that is
complementary in shape to the substrate
(geometric complementarity).
• Moreover, the amino acid residues that form
the binding site are arranged to specifically
attract the substrate (electronic
complementarity).
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10. Classes of enzyme
• Enzymes are classified on the basis of:
• The types of reactions that they catalyze. (e.g.: peptide hydrolase:
hydrolyzes peptide bonds)
• name of their substrates (e.g. fumarase)
• the reactions that they catalyze (e.g.: ATP synthase: synthesizes ATP)
with the suffix “ase” added.
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11. Enzyme Commission number
The Enzyme Commission number (EC number) is a numerical
classification scheme for enzymes, based on the chemical reactions
they catalyze.
12. Example
For example, the tripeptide aminopeptidases have the code "EC
3.4.11.4", whose components indicate the following groups of enzymes:
• EC 3 enzymes are hydrolases (enzymes that use water to break up
some other molecule)
• EC 3.4 are hydrolases that act on peptide bonds
• EC 3.4.11 are those hydrolases that cleave off the amino-terminal
amino acid from a polypeptide
• EC 3.4.11.4 are those that cleave off the amino-terminal end from
a tripeptide
Although the common names are used routinely, the
classification number is used when the precise identity of
the enzyme might be ambiguous.
13. Many enzymes require cofactors for activity
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• The catalytic activity of many enzymes depends on the
presence of small molecules termed cofactors.
• Cofactors are able to execute chemical reactions that
cannot be performed by the standard set of 20 a.a
• Apoenzyme + cofactor = holoenzyme
Catalytically active enzymeEnzyme w/o its cofactor
14. Enzyme cofactors
2 types
• Metals
• Coenzyme = Small organic compound
• Derived from vitamins.
• Can either tightly/loosely bound to the enzyme.
• Tightly bound coenzyme = prosthetic groups.
• Loosely bound coenzyme: act more like cosubstrates, can be
released from enzyme
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15.
16. Thermodynamics law
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• The First Law of Thermodynamics states that energy cannot
be created or destroyed; it can only be converted from one
form to another. In other words, the total amount of energy in
any process remains constant.
• The Second Law of Thermodynamics states that during any
process, the amount of available (or free) energy for work
decreases. In other words, the system loses usable energy as
reactions take place. For example, during any chemical
reaction, some usable energy is lost in the form of heat, which
is a measurement of entropy (disorder). As a result of the First
Law of Thermodynamics, this heat energy cannot be
converted back into usable energy.
17. Free energy is a useful thermodynamic
function for understanding enzymes
• Enzymes speed up the rate of chemical reactions, but the
properties of the reaction— whether it can take place at all—
depends on free-energy differences.
• Gibbs free energy or free energy (G), is a thermodynamic
property of a measure of energy that is capable of doing work.
• To understand how enzymes operate, we need to consider only
two thermodynamic properties of the reaction:
(1) the free-energy difference (ΔG) between the products and the
reactants/substrates
(2) the free energy required to initiate the conversion of
reactants/substrates into products.
The former determines whether the reaction will take place
spontaneously, whereas the latter determines the rate of the
reaction. Enzymes affect only the latter.
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18. Free energy change (∆G)
• G is composed of two components, enthalpy (H, a measure
of heat content) and entropy (S, a measure of disorder in a
system).
• The unit for G are joules/mol or kJ/mol.
• Because we cannot experimentally measure absolute
values of G, we measure the changes in free energy that
occur when a reaction is allowed to proceed to equilibrium
under certain condition.
• Under specific conditions, the free energy change (∆G) is
defined as
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19. Free energy change (∆G)
• ∆G of a reaction = free energy of products (final state) - free
energy of the reactants (initial state)
• ∆G = negative, energy is released; reaction is
spontaneous and exergonic
• ∆G = 0, reaction is at equilibrium
• ∆G = positive, energy is required; reaction is
nonspontaneous and endergonic
• Provides info about the spontaneity of the reaction (i.e. if the
reaction can occur spontaneously), but not the rate of
reaction
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20. Endergonic and exergonic reactions
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Exergonic reactions release free energy while endergonic
reactions consume free energy.
21. Chemical equilibrium
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• Under most conditions, a chemical equilibrium is reached in
which the reaction goes in both directions at the same rate.
• At chemical equilibrium energy is neither lost nor gained.
However, when a reaction departs from chemical equilibrium,
energy is either lost or gained.
22. Free energy change (∆G)
• ∆G of a reaction is independent of the path of the
transformation.
∆G of for the oxidation of glucose to CO2 and H2O is the
same whether it occur by combustion/ by series of enzyme-
catalyzed step in a cell.
• ∆G provides no information about the rate of reaction, e.g.:
A -ve ∆G indicates that a reaction can occur spontaneously,
but it does not signify whether it will proceed at a perceptible
rate.
• Rate of reaction depends on the free energy of activation
(∆G‡), which is unrelated to the ∆G of the reaction.
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23. Standard free energy change (∆G°’) and
equilibrium constant (K’eq)
• To know whether the reaction is spontaneous or requires an input
of energy, we need to determine free energy change (∆G).
• ∆G°’ is standard free energy change when a reaction proceeds
from start to equilibrium under standard condition
(Pressure = 1 atm, Temperature = 25°C, pH = 7.0)
∆G°’ is related to its equilibrium constant.
• Consider the reaction: A + B C + D
• The equilibrium constant (K’eq) is defined by the ratio of the
concentrations of products to the concentrations of reactants.
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Under standard condition
Pressure = 1 atm
Temperature = 25°C
pH = 7.0
Initial concentration of A, B, C, D = 1 M
24. Standard free energy change (∆G°’) and
equilibrium constant (K’eq)
ΔG: free energy change
ΔG°: the standard free-energy change
R: the gas constant, 8.315 J/mol. K
T : the absolute temperature, 273 + 25°C = 298K
[A], [B], [C], and [D]: the molar concentrations of the
reactants.
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25. Standard free energy change (∆G°’) and
equilibrium constant (K’eq)
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A simple way to determine the ΔG°′ is to measure the
concentrations of reactants and products when the reaction has
reached equilibrium. At equilibrium, there is no net change in the
concentrations of reactants and products; in essence, the reaction
has stopped and ΔG = 0. At equilibrium, equation becomes
26. Example of calculation:
Isomerization of dihydroxyacetone phosphate
(DHAP) to glyceraldehyde 3-phosphate (GAP).
• At equilibrium, the ratio of GAP to DHAP is 0.0475
at 25°C and pH 7
Keq’ = 0.0475; ∆Gº’ = ?
• ∆Gº’ = + 7.5 kJ/mol (endergonic reaction)
• Initial concentration of DHAP = 2x10-4 M & initial
concentration of GAP = 3x10-6 M; ∆G = ?
• ∆G = -2.9 kJ/mol (exergonic reaction)
Which of the reaction will take place
spontaneously?
27. Example of calculation:
• ∆G (free energy change) for this reaction
is negative, although ∆Gº‘ (standard free
energy changes) is positive.
• It is important to stress that the ∆G for a
reaction can be larger, smaller or equal to
∆Gº’ , depending on the concentrations
of the reactants & products.
• The criterion of spontaneity for a reaction
is ∆G , not ∆Gº‘.
• Reaction that are not spontaneous based
on ∆Gº‘ can be made spontaneous by
adjusting the concentrations of
reactants and products.
28. Enzyme alter only the reaction rate
and not the reaction equilibrium
• An enzyme cannot alter the equilibrium of a chemical reaction, but
it can change the reaction rate.
• Rate of reaction = rate at which the concentrations of reactants
and products change.
• Equilibrium = a position in which there is no further change on
the concentration of reactants & products.
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Time
[product]
The amount of product formed is the same
whether or not the enzyme is present.
In the presence of
enzyme, the [product]
at equilibrium is
reached in a short
period of time.
Rate of product formation with time in the presence & absence of enzyme.
In the absence of
enzyme, longer time is
required to reach the
{product] at equilibrium
29. Enzymes accelerate reactions by facilitating the
formation of the transition state
• A chemical reaction of substrate S to form product P goes
through a transition state X‡ that has a higher free energy
than does either S or P.
• Transitory molecules are no longer the substrate but they
are not yet the product. They are the least-stable & most-
seldom-occupied molecule species along the reaction
pathway because they have the highest free energy
(transitional state energy).
• The difference in free energy between the X‡ & S is called
Gibbs free energy of activation or activation energy, ∆G‡
∆G‡ =GX
‡ - GS Compare: ∆G =GP - GS
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S X‡ P
30. • Enzymes accelerate reactions by reducing activation energy
(∆G‡)
• Substrate combined with enzyme take up reaction pathway
that has lower transition state energy (GX
‡ ).
∆G‡ =GX
‡ - GS , if GX
‡ ↓ ∆G‡ ↓
(Absence of enzyme higher transition state energy).
• ↓ activation energy more molecules have the energy required
to reach the transition state.
• Essence of catalysis: specific stabilization of the transition state.
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Enzymes accelerate reactions by facilitating the
formation of the transition state
32. The formation of an enzyme-substrate complex is
the first step in enzymatic catalysis
• The interaction of the enzyme and substrate at the active site
promotes the formation of the transition state.
• Enzymes selectively bring together substrates in enzyme-
substrates (ES) complexes.
• The substrates are bound to a specific region of the enzyme called
the active site.
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33. The active sites of enzymes
• Active site:
– region that binds the substrates (and the cofactor, if any)
to convert into products.
– Contains the residues that directly participate in the
making & breaking of bonds = catalytic groups.
– Most directly lowers the ∆G‡ of the reaction, thus
providing the rate enhancement characteristic of enzyme
action.
• The interaction of the enzyme & substrate at the active site
promotes the formation of the transition state molecule.
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34. The active sites of enzyme have some
common features
Proteins are not rigid structures, but are flexible and exist in
different conformations. Thus, the interaction of the enzyme
and substrate at the active site and the formation of the
transition state is a dynamic process.
1. Active site is a 3-D cleft or crevice.
2. Active site takes up a relatively small part of the total
volume of an enzyme.
3. Active site is unique microenvironments.
4. Substrate 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.
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35. Active site is a 3-D cleft or crevice
• Active site is a 3-D cleft or
crevice.
– Formed by groups that
come from different parts
of the a.a seq.
– Residues far apart in the
a.a seq may interact more
strongly than adjacent
residue in the seq.
e.g.: lysozyme (a.a. 35, 52,
62, 63, 101, and 108)
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36. Active site takes up a relatively small
part of the total volume of an enzyme
• Most of the a.a residues in an enzyme are not in contact
with substrate.
• The “extra” a.a serve as scaffold to create the 3-D active
site.
• The remaining a.a also constitute regulatory sites, site of
interaction with other proteins or channels to bring the
substrates to the active sites.
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37. Active site is unique
microenvironments
• Substrate molecules are bound to a cleft
• Water is usually excluded unless it is a reactant
• Nonpolar microenvironment of the cleft enhances the
binding of substrates & catalysis
• However, cleft may also contain polar molecules, which
acquire special properties essential for substrate
binding/catalysis.
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38. Substrate are bound to enzymes by
multiple weak attractions
• Interactions between ES complexes: non covalent
interactions = electrostatic interactions, hydrogen bonds,
van del Waal forces, hydrophobic interactions.
• van del Waal forces become significant in binding only when
numerous substrate atom simultaneously come close to
many enzyme atoms.
• Hence, the enzyme & substrate should have complementary
shapes.
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39. The specificity of binding depends on the
precisely defined arrangement of atoms in
an active site
• Since the enzyme & substrate interact by means of short-range
forces that require close contact, a substrate must have a
matching shape to fit into the site
Emil Fischer’s analogy of the lock & key expressed in 1890
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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
40. • Although this model explains enzyme specificity, it fails to
explain the stabilization of the transition state that enzymes
achieve.
• In 1958, Daniel Koshland suggested a modification to the
lock and key model: since enzymes are rather flexible
structures, the active site is continuously reshaped by
interactions with the substrate as the substrate interacts with
the enzyme.
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The specificity of binding depends on the
precisely defined arrangement of atoms in
an active site
41. • Active site of some enzymes assume a shape that is
complementary to that of the substrate only after the substrate is
bound. This process of dynamic recognition = induced fit
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The specificity of binding depends on the
precisely defined arrangement of atoms in
an active site
Induced-fit model of
enzyme-substrate binding.
In this model, the enzyme
changes shape on
substrate binding. The
active site forms a shape
complementary to the
substrate only after the
substrate has been bound.
42. Enzymes lower the activation energy, but where does the
energy to lower the activation energy come from?
• Free energy is released by the binding between
complementary enzyme and substrate. The free energy
released on binding is called the binding energy.
• Furthermore, the full complement of such interactions is
formed only when the substrate is in the transition
state. Thus, the maximal binding energy is released
when the enzyme facilitates the formation of the
transition state. The energy released can lower the
activation energy.
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The Binding Energy Between Enzyme and
Substrate Is Important for Catalysis
44. Study questions
1. What are the properties of a catalyst?
2. What is a holoenzyme?
3. What is the active site of an enzyme?
4. What is the free energy difference and free energy of activation?
5. What is the endergonic and exergonic reactions?
6. What is the first step in enzymatic catalysis?
7. How does enzyme accelerate reaction?
8. What are the common features of enzyme active sites?
9. What is the difference between “lock-and-key” and ”induced-fit”
models of enzyme-substrate binding?
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