2

E + S ⇄ ES ⇄ ES* ⇄ EP ⇄ E + P

ES*= transition state

How do enzymes Work?

Enzymes work by
weakening bonds
which lowers
activation energy

4
ACTIVATION ENERGY
 Svante Arrhenius (1889)

 It is defined as the energy that must be overcome in order for a

chemical reaction to occur.

 Minimum energy required to start a chemical reaction.

 Ea (kilojoules/kilocalories per mole)

Enzymes function by lowering the activation
energy of reactions
 By bringing the
reactants closer
together,
chemical bonds
may be
weakened and
reactions will
proceed faster
than without the
catalyst
ACTIVATION ENERGY CONCEPT
Quantitative basis of the relationship between the activation
energy and the rate at which a reaction proceeds
 According to Arrhenius equation, the activation energy can be expressed as

 where A is the frequency factor for the reaction

 R is the universal gas constant
 T is the temperature (kelvins)
 k is the reaction rate coefficient.

While this equation suggests that the activation energy is dependent on

temperature, in regimes in which the Arrhenius equation is valid this is
cancelled by the temperature dependence of k.

Thus Ea can be evaluated from the reaction rate coefficient at any temperature
(within the validity of the Arrhenius equation).
HOW ENZYMES LOWER ACTIVATION
ENERGY
 Enzymes carry out their function of lowering activation
energy by temporarily combining with the substrate.

 Enzymes are specific for their substrate: A particular

substrate molecule will combine temporarily with one
enzyme type, and the active site of a particular enzyme
will fit only one kind of substrate.

 For example, the enzyme sucrase will attach only to the

substrate sucrose.
 Transition state

It is the transitory of molecular structure in which the molecule is no

longer a substrate but not yet a product.
Transition state contd.

 As an example, the transition state shown below occurs during the SN2
reaction of bromoethane with a hydroxyl anion.
transition state contd.
 Highest energy along the
reaction coordinate.

 It has more free energy in and is

the least stable state.

 Depends on the mechanisms of

the particular reaction.

 In the equation S → X → P, X is
the transition state, which is
located at the peak of the curve
on the Gibbs free energy
transition state contd.
 The enzyme's ability to make the reaction faster depends on the
fact that it stabilizes the transition state.

 The transition state's energy or, in terms of a reaction, the

activation energy is the minimum energy that is needed to break
certain bonds of the reactants so as to turn them into products.

 Enzymes decreases activation energy by shaping its active

site such that it fits the transition state even better than
the substrate.
Imaginary enzyme ("stickase") designed to catalyze "cleavage" (breaking)
of a metal stick
Enzymes active sites are complimentary to the transition state
Transition state analogs
 Transition-state analogs are compounds that resemble the transition
state of a catalyzed reaction.

 These compounds make for especially potent enzyme inhibitors

Importance of Transition State Analogs
 Very powerful inhibitors.

 Very important in understanding the kinetics and inner

workings of enzyme catalysis. The analogs can “function as
antimetabolites”.

 Plays a role in determination of the actual structure and

actual transformation of the original substrate.

 Help in identifying the “binding determinants at the active

site”.

 Able to generate immunogens which displays catalytic

nature.
Mechanism of enzyme catalysis
 By providing an alternative reaction route and by stabilizing
intermediates the enzyme reduces the energy required to reach the
highest energy transition state of the reaction.

 These conformational changes also bring catalytic residues in the active

site close to the chemical bonds in the substrate that will be altered in
the reaction.

 After binding takes place, one or more mechanisms of catalysis lowers

the energy of the reaction's transition state, by providing an alternative
chemical pathway for the reaction.
Mechanism of enzyme
action
 The catalytic efficiency of enzymes is explained by two
perspectives:

Thermodynamic Processes at the

changes active site
Thermodynamic changes
 All chemical reactions have energy barriers between reactants and
products.

 The difference in transitional state and substrate is called activational

barrier.
Thermodynamic changes overview
Processes at the active site

Electrostatic
Covalent catalysis
catalysis

Quantum
tunnelling
Acid base
Catalysis catalysis
by strain
Catalysis
by
proximity
Mechanism of enzyme catalysis
 There are six possible mechanisms of "over the
barrier" catalysis as well as a "through the barrier"
mechanism :
1 Catalysis by bond strain
2 Catalysis by proximity and orientation
3 Catalysis involving proton donors or acceptors
(Acid/Base Catalysis)
4 Electrostatic catalysis
5 Covalent catalysis
6 Quantum tunneling
 Lock and key model
 EMIL FISCHER (1894)

 Active site of an enzymes are rigid in its shape.

 There is no change in the active site before and after a chemical

reaction.
 Induced fit model
 This study has revealed that the process is much more likely to involve
an induced fit model (DANIAL KOSHLAND in 1958).
 According to this exposure of an enzyme to substrate cause a change in
enzyme, which causes the active site to change it’s shape to allow
enzyme and substrate to bind.
Catalysis by bond strain (Lyases)
 Principle effect of induced fit binding (affinity of the enzyme to the
transition state is greater than to the substrate itself).

 Structural rearrangements strain substrate bonds into a position closer

to the conformation of the transition state, so lowering the energy
difference between the substrate and transition state and helping
catalyze the reaction.

 The strain effect is, a ground state destabilization effect, rather

than transition state stabilization effect.

 In addition to bond strain in the substrate, bond strain may also be

induced within the enzyme itself to activate residues in the active site.
Catalysis by bond strain
Catalysis by proximity and orientation
 This increases the rate of the reaction as enzyme-
substrate interactions align reactive chemical
groups and hold them close together.

 This reduces the entropy of the reactants and thus

makes reactions such as ligations or addition
reactions more favorable, there is a reduction in
the overall loss of entropy when two reactants
become a single product.

 This effect is analogous to an effective increase in

concentration of the reagents.

 The binding of the reagents to the enzyme gives

the reaction intramolecular character, which gives
a massive rate increase.
Catalysis involving proton donors or
acceptors (Acid/Base Catalysis)
 Oxidoreductases

 Proton acceptors and donors, i.e. acids and bases, may

donate and accept protons in order to stabilize
developing charges in the transition state.

 This typically has the effect of activating nucleophile

and electrophile groups, or stabilizing leaving groups.
Histidine is often the residue involved in these
acid/base reactions, since it has a pKa close to neutral
pH and can therefore both accept and donate protons.
Electrostatic catalysis
 Stabilization of charged transition states can also be by residues
in the active site forming ionic bonds (or partial ionic charge
interactions) with the intermediate.

 These bonds can either come from acidic or basic side chains
found on amino acids such as lysine, arginine, aspartic acid or
glutamic acid or come from metal cofactors such as zinc.

 Metal ions are particularly effective and can reduce the pKa of
water enough to make it an effective nucleophile.

 In order to stabilize ionic and charged states, the catalysis is

associated with the fact that the enzyme polar groups are
preorganized
METAL ION OR ELECTROSTATIC CATALYSIS - STABILIZATION OF
TS CHARGE

A metal such as Cu2+ or Zn2+ can also stabilize the TS by binding to the charged
intermediate and hence the TS.
The tetrahedral oxyanion intermediate of the reaction of an electrophilic carbonyl C can
interact with a metal if there is an O on an adjacent atom which can help coordinate the
metal ion.
This charge stabilization of the developing negative in the TS and the full negative in the
intermediate is often called electrostatic catalysis.
A classic example of an enzyme using metal ion catalysis is carboxypeptidase A.
Covalent catalysis
 Covalent catalysis involves the substrate forming a
transient covalent bond with residues in the active site or
with a cofactor.

 This adds an additional covalent intermediate to the

reaction, and helps to reduce the energy of later transition
states of the reaction.

 The covalent bond must, at a later stage in the reaction, be

broken to regenerate the enzyme.
Covalent catalysis
Quantum tunneling
 Traditional "over the barrier" mechanisms have been challenged
in some cases by models and observations of "through the
barrier" mechanisms (quantum tunneling).

 Some enzymes operate with kinetics which are faster than what
would be predicted by the classical mechanisms.

 In "through the barrier" models, a proton or an electron can

tunnel through activation barriers. Quantum tunneling for
protons has been observed in tryptamine oxidation by aromatic
amine dehydrogenase.

 Interestingly, quantum tunneling does not appear to provide a

major catalytic advantage, since the tunneling contributions are
similar in the catalyzed and the uncatalyzed reactions in
solution.
 Quantum tunneling model

 However, the tunneling

contribution typically enhance the
rate constants by a factor of ~1000
compared to the rate of reaction
for the classical 'over the barrier'
route.

 Emphasizes the general

importance of tunneling reactions
in biology.

 In 1971-1972 the first quantum-

mechanical model of enzyme
catalysis was formulated
(Volkenshtein et al., 1972)
 Quantum tunneling model

 In quantum tunnelling, a particle

passes through a barrier that,
according to the laws of classical
mechanics, it should not be able to
get over.

 Many reactions that involve transfer

of particles as small as hydrogen
atoms.
Chemical Kinetics
 Chemical kinetics, also known as reaction kinetics,
is the study of rates of chemical processes.

 In 1864, Peter Waage and Cato Guldberg pioneered the

development of chemical kinetics by formulating the
law of mass action, which states that the speed of a
chemical reaction is proportional to the quantity of
the reacting substances.
Chemical Kinetics
 Chemical kinetics deals with the experimental determination of
reaction rates from which rate laws and rate constants are
derived.

 In consecutive reactions the rate-determining step often

determines the kinetics.

 The main factors that influence the reaction rate include:

 the physical state of the reactants
 the concentrations of the reactants
 the temperature at which the reaction occurs
 whether or not any catalysts are present in the reaction.
Order of reaction
 Chemical reactions can be classified based on their
reaction kinetics. The general reaction form is:
aA + bB → cC + dD
 Reactions are categorized:
 zero-order,
 first-order
 second-order
 mixed-order (higher-order) reactions.
 Zero-Order Reactions (order = 0) have a constant rate.
This rate is independent of the concentration of the
reactants. The rate law is:
rate = k, (units M/sec.)
Order of reaction contd.
 First-Order Reactions (order = 1) has a rate proportional to the
concentration of one of the reactants. A common example of a first-order
reaction is the phenomenon of radioactive decay. The rate law is:
rate = k[A] (or B instead of A), with k having the units of sec-1

 Second-Order Reactions (order = 2) has a rate proportional to the

concentration of the square of a single reactant or the product of the
concentration of two reactants:
rate = k[A]2 (or substitute B for A or k multiplied by the concentration of A
times the concentration of B), with the units of the rate constant M-1sec-1

 Mixed-Order or Higher-Order Reactions have a fractional order for their

rate:
e.g., rate = k[A]1/3
Order of reaction contd.
 Michaelis-Menten kinetics for enzyme-catalysis:
 first-order in substrate (second-order overall) at low substrate
concentrations
 zero order in substrate (first-order overall) at higher substrate
concentrations
Enzyme kinetics
 Enzyme kinetics is the study of the chemical
reactions that are catalysed by enzymes.

 In enzyme kinetics, the reaction rate is measured and

the effects of varying the conditions of the reaction
investigated.

 Enzyme kinetics:
 reveal the catalytic mechanism of this enzyme
 its role in metabolism
 how its activity is controlled
 how a drug or a inhibitor might inhibit the enzyme.
How to obtain kinetic data ?
Continous measurment Discontinous measurment

 Spectrophotometric •Radiometric
 Fluorometric •Chromatographic
 Colorimetric
 Chemiluminescent
Enzyme Kinetics Equation
Initial Velocity (vo) and [S]
 The concentration of substrate [S] present will greatly influence
the rate of product formation, termed the velocity (v) of a reaction.

 Studying the effects of substrate concentration [S] on the velocity

of a reaction is complicated by the reversibility of enzyme
reactions, e.g. conversion of product back to substrate.

 To overcome this problem, the use of initial velocity (vo)

measurements are used.

 At the start of a reaction, [S] is in large excess of [P], thus the initial
velocity of the reaction will be dependent on substrate
concentration.
Initial Velocity (vo) and [S]
 When initial velocity is plotted against [S], a
hyperbolic curve results, where Vmax represents the
maximum reaction velocity.
 At this point in the reaction, if [S] >> E, all available
enzyme is "saturated" with bound substrate, meaning
only the ES complex is present.
 Michaelis-Menten Curve

Leonor Michaelis

Maud Leonora Menten

Michaelis-Menten Equation
 Steady State Assumption
 The Michaelis-Menten equation was derived in part by making
several assumptions.

 An important one was: the concentration of substrate must

be much greater than the enzyme concentration. In the
situation where [S] >> [E] and at initial velocity rates, it is
assumed that the changes in the concentration of the
intermediate ES complex are very small over time (vo).

 This condition is termed a steady-state rate, and is referred to

as steady-state kinetics. Therefore, it follows that the rate
of ES formation will be equal to the rate ES breakdown.
 Michaelis-Menten Equation
Derivation

 Rate of ES formation = k1 [E][S] = k1([ET] - [ES])[S]

(where [ET] is total concentration of enzyme E and
k-2 is considered neglible)
 Rate of ES breakdown to product = k-1[ES] +
k2[ES]
 Michaelis-Menten Equation
Derivation (cont)
 Thus for the steady state assumption:

 k1([ET] - [ES])[S] = k-1[ES] + k2[ES]

 This equation is the basis for the final Michaelis-Menten

following algebraic rearrangement and substitution of Km
and Vmax terms.
Michaelis-Menton Equation
 Relates rate and [S]
 Rate = V0 = k2[ES]
 V0 = initial velocity or rate
 Neglects complications from inhibition, deactivation,
etc.
 To determine, look at rate of formation and
disappearance of [ES]
[ES]
k1 k2
E + S ES E + P
k-1

 Rate of formation = k1[E][S]

 Rate of breakdown = k-1[ES] + k2[ES]
 Assume steady state
 As ES is produced, it reacts
 [ES] remains constant
 Rate formation = rate breakdown

 So, k1[E][S] = k-1[ES] + k2[ES]

 k1[E][S] = k-1[ES] + k2[ES]
k1[E][S] = (k-1 + k2)[ES]
 Rearrange

[E][S] / [ES] = (k-1 + k2) / k1

 Where (k-1 + k2) / k1 = KM (Michaelis constant)

 Define total enzyme concentration [E]T = [E] + [ES]

 Substitute for [E]

([E]T – [ES])[S] / [ES] = KM

 Solve for [ES]

[ES] = [E]T[S] / KM + [S]

 Solve for [ES]

([E]T[S] – [ES])[S]) / [ES] = KM

Multiply on both sides by [ES]
[ES]([E]T[S] – [ES])[S]) / [ES] = [ES]KM
Cancel [ES] by [ES] on left hand side
[ES] KM=([E]T[S] – [ES][S])
[ES] KM+[ES][S]= [E]T [S]
Divide both sides by (KM+[S])
[ES]=[E]T [S]/KM+[S]
 [ES] = [E]T[S] / KM + [S]
 Since V0 = k2[ES]
V0 = k2[E]T[S] / KM + [S]
 Define maximum velocity Vmax
 Occurs at high [S]

 Enzyme is all in ES form

 [ES] = [E]T

Vmax = k2[E]T
 Therefore
V0 = Vmax[S] / KM + [S]
 Meaning of Km
 An important relationship that can be derived from the Michaelis-
Menten equation is the following:

 If vo is set equal to 1/2 Vmax, then the relation

Vmax /2 = Vmax[S]/Km + [S]

Simplify

Km + [S] = 2[S],

or Km = [S]

 This means that at one half of the maximal velocity, the substrate
concentration at this velocity will be equal to the Km.
Meaning of Km (cont)
 The significance of Km will change based on the different rate
constants and which step is the slowest (also called the rate-
limiting step).

 In the simplest assumption, the rate of ES breakdown to product

(k2) is the rate-determining step of the reaction, so k-1 >> k2 and
Km = k-1/k1.

 This relation is also called a dissociation constant for the ES

complex and can be used as a relative measure of the affinity of a
substrate for an enzyme (identical to Kd).

 However if k2 >> k-1 or k2 and k-1 are similar, then Km remains

more complex and cannot be used as a measure of substrate
affinity.
KM
 Unique for each E-S pair
 Magnitude varies
 Depends on E and S

 Function of Temperature
 Rate increases with T
 Optimum T ~ 37°C for most
enzymes
 Function of pH
 Change catalytic activity
 Most enzymes active in narrow
pH range close to pH of
environment of enzyme
 Uses of Km
 For characterizing the number and/or types of substrates that
a particular enzyme will utilize.

 For comparing similar enzymes from different tissues or

different organisms.

 Also, it is the Km of the rate-limiting enzyme in many of the

biochemical metabolic pathways that determines the amount
of product and overall regulation of a given pathway.

 Clinically, Km comparisons are useful for evaluating the effects

mutations have on protein function for some inherited genetic
diseases.
 There are some enzymes that have been shown to have the following
reaction sequence:

 In this situation, the formation of product is dependent on the

breakdown of an enzyme-product complex, and is thus the rate-limiting
step defined by k3.
 Meaning of Vmax
 The values of Vmax will vary widely for different enzymes

 can be used as an indicator of an enzyme’s catalytic efficiency.

 It does not find much clinical use.

Definition and Use of kcat
 The constant, kcat (units of sec-1), is also called the turnover
number because under saturating substrate conditions, it
represents the number of substrate molecules converted to
product in a given unit of time on a single enzyme molecule.

 In practice, kcat values (not Vmax) are most often used for
comparing the catalytic efficiencies of related enzyme
classes or among different mutant forms of an enzyme.
Catalytic Constant kcat
kcat = Vmax / [E]T
 Turnover number
 Number of reaction processes each active site catalyzes per
unit time
 Measure of how quickly an enzyme can catalyze a specific
reaction
 For M-M systems kcat = k2
 Rate constant of RDS
 Derivation of kcat
 A more general term has been defined, termed kcat, to describe
enzymes in which there are multiple catalytic steps and possible
multiple rate-limiting steps. The Michaelis-Menten equation can be
substituted with kcat
kcat / kM
 Rate constant of rxn E + S → E + P
 Specificity constant
 Gauge of catalytic efficiency
 Catalytic perfection ~ 108 ─ 109 M-1 s-1
 Two Substrate Reactions
 Many enzyme reactions involve two or more substrates. Though the
Michaelis-Menten equation was derived from a single substrate to product
reaction, it still can be used successfully for more complex reactions (by
using kcat).

Random

Ordered

Ping-pong
 Two Substrate Reactions
(cont)
 In random order reactions, the two substrates do not bind to the
enzyme in any given order; it does not matter which binds first or
second.
 In ordered reactions, the substrates bind in a defined sequence, S1 first
and S2 second.
 These two reactions share a common feature termed a ternary complex,
formed between E, ES1, ES2 and ES1S2. In this situation, no product is
formed before both substrates bind to form ES1S2.
 Another possibility is that no ternary complex is formed and the first
substrate S1 is converted to product P1 before S2 binds. These types of
reactions are termed ping-pong or double displacement reactions
Lineweaver-Burk (double
reciprocal plot)
 If the reciprocal (1/X) of the Michaelis-Menten equation is done, after
algebraic simplification the following equation results:

 This relation is written in the format of the equation for a straight line,
y = mx + b, where y = 1/vo, m (slope) = Km/Vmax, x = 1/[S] and the y-
intercept, b = 1/Vmax. When this relation is plotted, the result is a
straight line graph
Lineweaver-Burk (double reciprocal plot)
Uses of double reciprocal plot
 The x intercept value is equal to -1/Km.

 The biggest advantage to using the double reciprocal plot is a more

accurate determination of Vmax, and hence Km.

 It is also useful in characterizing the effects of enzyme inhibitors and

distinguishing between different enzyme mechanisms.
Eadie-Hofstee plot (Augustinsson Plot)
Eadie–Hofstee plot
 Woolf Eadie and
Augustinsson
Hofstee
 It is a graphical
representation of
enzyme kinetics in
which reaction
velocity is plotted as a
function of the
velocity vs. substrate
concentration ratio
Eadie–Hofstee plot
 V= -km v/[S]+Vmax
Hanes-Woolf plot
Hanes-Woolf plot
 Enzyme Inhibion
 Inhibitors of enzymes are generally molecules which
resemble or mimic a particular enzymes substrate(s).

 Therefore, many therapeutic drugs are some type of enzyme

inhibitor.

 The modes and types of inhibitors have been classified by

their kinetic activities and sites of actions.
Types of Inhibition
 Reversible
 Competitive
 Uncompetitive
 Noncompetitive
 Mixed

 Irreversible
 Suicide inactivators
Definition of Ki
 For reversible inhibitors, a term Ki can be
determined. It can be defind as that value of inhibitor
where it inhibits half of the enzyme activity.

 For competitive inhibitors, the following relation can

be used:

Km + I = Km (1 + [I] / Ki )

(where Km + I is the determined Km in the presence of

[I]).
Uses of Ki
 Ki values are used to characterize and compare the
effectiveness of inhibitors relative to Km.

 This parameter is especially useful and important in

evaluating the potential therapeutic value of inhibitors
(drugs) of a given enzyme reaction.

 For example, Ki values are used for comparison of the

different types of HIV protease inhibitors.

 In general, the lower the Ki value, the tighter the

binding, and hence the more effective an inhibitor is.
Competitive Inhibition
 Competitive inhibitors compete with the substrate for
binding at the active site (as E + I).

 In the double reciprocal plot for a competitive inhibitor

acting at the substrate site for the following reasons, notice
with increasing concentration of inhibitor, the Vmax does not
change; however, the Km of the substrate is increased.

 This also reflects the reversible nature of the inhibitor; there

is always some concentration of substrate which can displace
the inhibitor.
 Vmax [S]
V0 =
αK M + [S]
Michealis-Menton Equation for
Competitive Inhibition
Vmax [S]
V0 =
αK M + [S]

 Where a = 1 + [I]/KI
 Function of inhibitor concentration and affinity for enzyme
 a = 1 when [I] = 0

 As [S] increases, V0 will approach Vmax for any value of a

 S overcome inhibitor effects; saturate

 As [I] increases, KM increases by a

 More S required to reach ½ Vmax
 Rate does not increase rapidly with [S] due to inhibition
Lineweaver-Burk Equation for
Competitive Inhibition
Vmax [S]
V0 =
αK M + [S]

 Slope is larger (multiplied by a)

 Intercept does not change (Vmax
is the same)
 KM is larger (multiplied by a)
Example of Competitive Inhibition
 Methotrexate is a competitive inhibitor of the
enzyme dihydrofolate reductase, which catalyzes the
reduction of dihydrofolate to tetrahydrofolate.

 Allosteric effect (strychnine)

Uncompetitive inhibitors
 Inhibitor binds to a site which is only available after
the substrate binds to enzyme active site (enzyme
substrate complex).

 Most commonly encountered with multi substrate

reaction where the inhibitor is competitive with
respect to one substrate( e.g. S2) but uncompetitive
with respect to another (S1).

 A special case of uncompetitive inhibition is substrate

inhibition (e.g. invertase is inhibited by sucrose).
M-M and L-B Equations for
Uncompetitive Inhibition
Vmax [S] 1  KM  1 α'
V0 = =   +
K M + α' [S] V0  Vmax  [S] Vmax

 Increasing [I]
 Lowers Vmax (y-intercept
increases)
 Lowers KM (x-intercept
decreases)
 Ratio of KM/Vmax is the
same (slope)
Non-Competitive Inhibition
 Non-Competitive Inhibition
 Special case of mixed
inhibition where
 a = a’

 KI = KI’

 Vmax lowers as increase [I]

 Lines intersect on x-axis
 KM does not change

Vmax DECREASES - inhibitor affects rate of reaction

by binding to site other than substrate active-site
Km - No change
Mixed inhibition
Mixed Inhibition
 Inhibitor binds to either E or ES
 M-M and L-B Equations
for Mixed Inhibition
Vmax [S] 1  αK M  1 α'
V0 = 
= 
 +
αK M + α' [S] V0  Vmax  [S] Vmax

 Increasing [I]
 Lowers Vmax (y-intercept
increases)
 Raises KM (x-intercept
increases)
 Ratio of KM/Vmax is not the
same (slope changes)
Summary of Reversible Inhibition
 Irreversible Inhibitors
 Destruction or modification of an essential amino acid required
for enzyme activity.

 Covalent link between enzyme and inhibitor.

 These types of inhibitors range from fairly simple, broadly

reacting chemical modifying reagents (like iodoacetamide that
reacts with cysteines)

 to complex inhibitors that interact specifically and irreversibly

with active site amino acids. (termed suicide inhibitors).
Irreversible Inhibitors
 These inhibitors are designed to mimic the natural substrate in
recognition and binding to an enzyme active site.

 Upon binding and some catalytic modification, a highly

reactive inhibitor product is formed that binds irreversibly and
inactivates the enzyme.

 Use of suicide inhibitors have proven to be very clinically

effective
Irreversible Inhibition
 Inhibitor
 Binds covalently, or
 Destroys functional group
necessary for enzymatic activity,
or
 Very stable noncovalent binding

 Suicide Inactivators
 Starts steps of chemical
reaction
 Does not make product
 Combines irreversibly with
enzyme
Diisopropyl Phosphofluoridate:
Irreversible Acetylcholinesterase Inhibitor
Irreversible Inhibitor:
Allopurinol
Irreversible Inhibitor: Penicillin
Uses of inhibitors
 Since inhibitors modulate the function of enzymes
they are often used as drugs.

 A common example of an inhibitor that is used as a

drug is aspirin, which inhibits the COX-1 and COX-2
enzymes that produce the inflammation messenger
prostaglandin, thus suppressing pain and
inflammation.

 However, other enzyme inhibitors are poisons. For

example, the poison cyanide is an irreversible enzyme
inhibitor that combines with the copper and iron in
the active site of the enzyme cytochrome c oxidase
and blocks cellular respiration.
 Inhibitor Summary
 REMEMBER - The types of enzyme inhibitors described
have been for relatively simple, single substrate-product
reactions that obey Michaelis-Menten kinetics.

 However, not all enzyme inhibitors will necessarily be one

type of inhibitor. Especially for some multi-substrate
reactions, a particular inhibitor can be competitive for one
substrate and non-competitive with a second or third
substrate.
 Also, suicide inhibitors by design are generally competitive
inhibitors of a substrate, and therefore must first bind in the
active site.
Effect of pH, temperature, substrate concentration,
ionic strength and pressure on enzyme activity
Georgia et al., 2008
Effect of pH

Changes in pH will denature

the enzyme by changing the
shape of the enzyme.

Enzymes are also adapted to

operate at a specific pH or pH
range.

•Effects
•Activity
•Structural stability
•solubility
 pKa of the ionizing groups commonly found
in enzymes
The pKa (defined as -Log10(Ka)) is the pH at which half the groups are ionised.
Group Usual pKa range Approximate
charge at pH 7

Carboxyl (C-terminal, glutamic acid, 3-6 -1

aspartic acid)
7-9
Amino (N-terminal) (lysine) +1

Imidazolyl (histidine) 5-8 +0.5

Guanidyl (arginine) 11 - 13 +1
Phenolic (tyrosine) 9 - 12 0
Thiol (cysteine) 8 - 11 0
pH optimum Enzyme pH Optimum
Lipase (pancreas) 8.0
Lipase (stomach) 4.0 - 5.0
Lipase (castor oil) 4.7
Pepsin 1.5 - 1.6
Trypsin 7.8 - 8.7
Urease 7.0
Invertase 4.5
Maltase 6.1 - 6.8
Amylase (pancreas) 6.7 - 7.0
Amylase (malt) 4.6 - 5.2
Catalase 7.0
 Increasing hydrogen ion concentration will, additionally, increase the
successful competition of hydrogen ions for any metal cationic binding sites
on the enzyme, reducing the bound metal cation concentration.

 Decreasing hydrogen ion concentration, on the other hand, leads to

increasing hydroxyl ion concentration which compete against the enzymes'
ligands for divalent and trivalent cations causing their conversion to
hydroxides and, at high hydroxyl concentrations, their complete removal
from the enzyme.

 These charge variations, plus any consequent structural alterations,

may be reflected in
 changes in the binding of the substrate,

 the catalytic efficiency and

 the amount of active enzyme.

 In alkaline solution (pH > 8),
 partial destruction of cystine residues due to base catalysed β-
elimination reactions
 whereas, in acid solutions (pH < 4),
 hydrolysis of the labile peptide bonds, sometimes found next to aspartic
acid residues, may occur.
 Reducing the dielectric constant of an aqueous solution by the addition
of a co-solvent of low polarity (e.g. dioxan, ethanol), or by
immobilisation , increases the pKa of carboxylic acid groups.

 This method is sometimes useful but not generally applicable to

enzyme catalysed reactions as it may cause a drastic change on an
enzyme's productivity due to denaturation .

 The pKa of basic groups are not similarly affected as there is no

separation of charges when basic groups ionise. However, protonated
basic groups which are stabilised by neighbouring negatively charged
groups will be stabilised (i.e. have lowered pKa) by solutions of lower
polarity.
Enzyme and isoelectric point (pI)
 At which the net charge on the
molecule is zero.

 At which the enzyme generally has

minimum solubility in aqueous
solutions.

 The variation of 2-3 units each side of

the pI: reversible process.

 Extremes of pH: time- and

temperature-dependent, essentially
irreversible, denaturation.
Temperature
Temperature
 Increases in the temperature of a system results
from increases in the kinetic energy of the system.
This has several effects on the rates of reactions.
1) More energetic collisions
2) The number of collisions per unit time will increase.
3) The heat of the molecules in the system will increase

 The half-life (t1/2) of an enzyme is the time it takes for

the activity to reduce to a half of the original activity (i,e.
Af = 0.5).
 Derivation:
t1/2=0.693/kd
kd= Deactivation constant
Thermostability of Enzymes
Effect of temperature
 The temperature also has a marked effect on ionisations, the extent of which
depends on the heats of ionisation of the particular groups concerned.

 The relationship between the change in the pKa and the change in
temperature is given by a derivative of the Gibbs-Helmholtz equation.

where T is the absolute temperature (K), R is the gas law constant (8.314 J M-1 K-1), ∆H is the heat of
ionisation and the numeric constant (2.303) is the natural logarithm of 10, as pKa's are based on logarithms
with base 10.

 This variation is sufficient to shift the pI of enzymes by up to one unit

towards lower pH on increasing the temperature by 500C
Effect of tempreture
 Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as
the temperature is raised.
 A ten degree Centigrade rise in temperature will increase the activity of most
enzymes by 50 to 100%.
 Variations in reaction temperature as small as 1 or 2 degrees may introduce changes
of 10 to 20% in the results.
 In the case of enzymatic reactions, this is complicated by the fact that many
enzymes are adversely affected by high temperatures, the reaction rate increases
with temperature to a maximum level, then abruptly declines with further increase of
temperature.
 Because most animal enzymes rapidly become denatured at temperatures above
40°C, most enzyme determinations are carried out somewhat below that
temperature.
 Over a period of time, enzymes will be deactivated at even moderate temperatures.
Storage of enzymes at 5°C or below is generally the most suitable. Some enzymes
lose their activity when frozen.
Effect of temperature, pH and
substrate concentration
Substrate Saturation
 Increasing the substrate concentration increases the rate of
reaction (enzyme activity). However, enzyme saturation
limits reaction rates.
 An enzyme is saturated when the active sites of all the
molecules are occupied most of the time.
 At the saturation point, the reaction will not speed up, no
matter how much additional substrate is added. The graph
of the reaction rate will plateau.
 Substrate Saturation of an Enzyme

A. Low [S] B. 50% [S] or Km C. High, saturating [S]

Substrate Saturation of an Enzyme
Effect of ionic strength

 Most enzymes cannot tolerate

extremely high salt
concentrations.

 The ions interfere with the weak

ionic bonds of proteins.

 Typical enzymes are active in salt

concentrations of 1-500 mM.

 Enzymes from halophilic (salt

loving) algae and bacteria:
Exceptions
Ionic strength
 The ionic strength is defined as half of the total sum of the concentration (ci) of
every ionic species in the solution times the square of its charge(zi);
 i.e. I = 0.5∑(cizi2).

For example, the ionic strength of a 0.1 M solution of CaCl2 is 0.5 x (0.1 x 22 + 0.2 x 12) =
0.3 M.
 Where catalysis depends on the movement of charged molecules relative to each
other, both the binding of charged substrates to enzymes and the movement of
charged groups within the catalytic 'active' site will be influenced by the ionic
composition of the medium.
 If the rate of the reaction depends upon the approach of charged moieties the
following approximate relationship may hold:

where k is the actual rate constant, k0 is the rate constant at zero ionic strength, zA
and zB are the electrostatic charges on the reacting species, and I is the ionic strength
of the solution
Ionic strength
 If the charges are opposite then there is a decrease in the reaction rate
with increasing ionic strength whereas if the charges are identical, an
increase in the reaction rate will occur.

 E.g. the rate controlling step in the catalytic mechanism of chymotrypsin

involves the approach of two positively charged groups, 57histidine+ and
145arginine+ causing a significant increase in k
cat on increasing the ionic
strength of the solution.

 Even if a more complex relationship between the rate constants and the
ionic strength holds, it is clearly important to control the ionic strength of
solutions in parallel with the control of pH.
Effects of pressure
 To elucidate the effects of pressure on
the function of Escherichia coli
dihydrofolate reductase (DHFR), the
enzyme activity and the dissociation
constants of substrates and cofactors
were measured at pressures up to
250 MPa at 25 °C and pH 7.0. (Eiji
Ohmae et al., 2008)
 The enzyme activity decreased with
increasing pressure.
 The values of the Michaelis constant
(Km) for dihydrofolate and NADPH
were slightly higher at 200 MPa than at
atmospheric pressure.
 The dissociation constants of substrates
and cofactors increased with pressure.
References
 Nicholas C. Price and Lewis Stevens (1999) Fundamentals of
Enzymology: The cell and molecular biology of catalytic proteins. Oxford
University Press.3rd edition

 Chaplin MF and Bucke C. Enzyme Technology

 Enzyme From Wikipedia, the free encyclopedia

 Michaelis L and Menten ML (1913) Die kinetik der invertinwirkung.

Biochem. Z. 49:333-369.