Protein Structures

What Are Enzymes?

• Most enzymes are
Proteins (tertiary
and quaternary
structures)
• Act as Catalyst to
accelerates a reaction
• Not permanently
changed in the process

4
 Timeline of enzyme discovery
1835:
Breakdown of starch to sugar by malt

1877:
Name enzyme coined to describe chemicals in yeast that ferment sugars

1897:
Eduard Buchner extracted enzyme from yeast and showed it could work outside cells

1905:
Otto Rohm exyracted pancreatic proteases to supply enzymes for tanning

1926:
James B Sumner produced first pure crystalline enzyme (urease)
and showed enzymes were proteins
1930-1936:
Protein nature of enzymes finally established when digestive enzymes
crystallised by John H Northrop

1946: Sumner finally awarded Nobel prize

 Enzymes are globular proteins

• Active site has a specific

shape due to tertiary
structure of protein.

• A change in shape of the

protein affects shape of
active site and the function
of the enzyme.
 Examples of specific enzymes
There are thousands of enzymes in the human body, here are just a
few examples:

Lipases – a group of enzymes that help digest fats in the gut.

Amylase – helps change starches into sugars. Amylase is found in
saliva.
Maltase – also found in saliva; breaks the sugar maltose into
glucose. Maltose is found in foods such as potatoes, pasta, and
beer.
Trypsin – found in the small intestine, breaks proteins down into
amino acids.
Lactase – also found in the small intestine, breaks lactose, the
sugar in milk, into glucose and galactose.
 Industrial Applications of Enzymes
Enzyme Industry Application
Palatase Food Enhance cheese flavor

Lipozyme Food Interesterification of vegetable oil

Lipase Pharmaceutical Synthesis of chiral compounds

Lipopan Food Emulsifier

Cellulase Biofuel Class of enzymes that degrade cellulose to glucose monomers

Amylase Food/biofuel Class of enzymes that degrade starch to glucose monomers

Xylose isomerase Food High fructose corn syrup production

Resinase] Paper Pitch control in paper processing

Penicillin amidase Pharmaceutical Synthetic antibiotic production

Amidase Chemical non-proteinogenic enantiomerically pure amino acid production

Novozym-435 Consumer Goods Isopropyl myristate production (Cosmetic)

Bromelain Food Meat tenderizer
Noopazyme Food Improve noodle quality
Asparaginase Pharmaceutical Lymphatic cancer therapeutic
Ficin Pharmaceutical Digestive aid
Urokinase Pharmaceutical Anticoagulant
β-Lactamase Pharmaceutical Penicillin allergy treatment
Subtilisin Consumer Goods Laundry detergent
 Active site

o Active site can be further divided into:

Active Site

Binding Site Catalytic Site

It chooses the substrate It performs the catalytic

and binds it to active site . action of enzyme.

ENZYME
COFACTORS
• Some enzymes require cofactors to be
active

• Cofactors are nonprotein component of

an enzyme. Cofactors can be
• Organic molecules (coenzymes)
• Inorganic molecules ( Ca+2, Zn+2).

• Cofactors may be:

• Permanently attached, in which
case they are called prosthetic
groups
• Temporarily attached coenzymes
Which detached after a
reaction and may participate
with another enzyme in other
reactions.
 Enzymes work by
How do enzymes weakening bonds
which lowers
Work? activation energy

13
Thermo-dynamic changes overview
Energy levels of molecules Enzymes lower the activation energy of a reaction

Activation energy
of uncatalysed
Initial energy state Activation energy reactions
of substrates of enzyme catalysed
reaction

Final energy state of

products

Progress of reaction (time)

 Enzymes lower activation energy by forming an
enzyme/substrate complex

Substrate + Enzyme

Enzyme/substrate complex

Enzyme/product complex

Product + Enzyme
 Enzyme reactions

enzyme + substrate enzyme-substrate complex

E +S ES
 Enzyme reactions
enzyme + substrate enzyme-substrate complex

E +S ES

enzyme-substrate complex enzyme + product

ES E +P
 Mechanism of enzyme
action
• The catalytic efficiency of enzymes is explained by two
perspectives:

Thermodynamic Processes at the

changes active site
Transition State Theory, Activation Energy (Ea),
& The Reaction Coordinate

Thermodynamics Again?
 Transition State Diagram
Consider the bimolecular Rx (a) : HA – HB + HC  HA + HB-- HC

Consider the generic bimolecular Rx (b) : A + B  X‡  P + Q

rate = k ~ e-ΔG‡/RT ; ΔG‡ = Ea

Covalent Acid base
Catalysis catalysis

Processes at the
active site

Catalysis
by Catalysis
proximity by strain
 Covalent
catalysis

o Enzymes form covalent

linkages with substrate
forming transient
enzyme-substrate
complex with very low
activation energy.
o Enzyme is released
unaltered after
completion of reaction.
 ACID-BASE
catalysis

o Mostly undertaken by
oxido- reductases
enzyme.

o Mostly at the active site,

histdine is present which
act as both proton
donor and proton
acceptor.
 Catalysis By
PROXIMITY

o In this catalysis
molecules must come in
bond forming distance.

o When enzyme binds:

o A region of high substrate
concentration is produced
at active site.
o This will orient substrate
molecules especially in a
position ideal for them.
 Catalysis By
BOND STRAIN

o Mostly undertaken by
lyases.
o The enzyme-substrate
binding causes
reorientation of the
structure of site due to in
a strain condition.
o Thus transitional state is
required and here bond is
unstable and eventually
broken.
o In this way bond between
substrate is broken and
converted into products.
Lock and
Key
  Proposed by EMIL FISCHER in 1894.

Lock and  Lock and key hypothesis assumes the active site of
an enzymes are rigid in its shape.

Key model  There is no change in the active site before and

after a chemical reaction.
  More recent studies have revealed that the process
is much more likely to involve an induced fit
Induced model(proposed by DANIAL KOSH LAND in 1958).
 According to this exposure of an enzyme to
fit model substrate cause a change in enzyme, which causes
the active site to change it’s shape to allow enzyme
and substrate to bind.
 • measurement of velocity = reaction rate
• compare enzymes under different
conditions, or from different tissues or
organisms
Enzyme • compare activity of same enzyme with
different substrates (understand
kinetics specificity)
• measure enzyme purity (specific activity
(reaction = amount of activity/amount of
protein)
rates)? • study/distinguish different types of
inhibitors
 development of specific drugs (enzyme
inhibitors)
 Enzyme Kinetics

K1
k2
E+S
K-1
ES  P  E
 Enzyme Kinetics

At this point, an assumption is required to

achieve an analytical solution.
- The rapid equilibrium assumption
Michaelis - Menten Approach.

- The quasi-steady-state assumption.

Briggs and Haldane Approach.
 Michaelis - Menten Approach

The rapid equilibrium assumption:

- Assumes a rapid equilibrium between the enzyme
and substrate to form an [ES] complex.

K1
k2
E+S
K-1
ES  P  E

k1[ E ][ S ]  k 1[ ES ]
 Michaelis - Menten Approach

'
The equilibrium constant K m can be expressed by the
following equation in a dilute system.

K1
k2
E+S ES  P  E
K-1

' k 1 [ E ][ S ]
Km  
k1 [ ES ]
 Michaelis - Menten Approach

Then rearrange the above equation,

[ E ][ S ]
[ ES ] 
Km '

Substituting [E] in the above equation with enzyme

mass conservation equation
[ E ]  [ E0 ]  [ ES ]

yields,
([ E0 ]  [ ES ])[ S ]
[ ES ] 
Km '
 Michaelis - Menten Approach

[ES] can be expressed in terms of [S],

[ E0 ][ S ]
[ ES ] 
Km'  [S ]

Then the rate of production formation v can be

expressed in terms of [S],
d [ P] k 2 [ E0 ][ S ] Vm [ S ]
v  k [ ES ]  
dt 2
Km '  [S ] Km'  [S ]
Where Vm  k [ E0 ]
2
represents the maximum forward rate of reaction (e.g.moles/L-min).
37
 Meaning of K’m of Vm

K1
k2
E+S
K-1
ES  P  E

d [ P] k 2 [ E0 ][ S ] Vm [ S ]
v  k [ ES ]  
dt 2
Km '  [S ] Km'  [S ]
 d [ P] k 2 [ E0 ][ S ] Vm [ S ]
v  k [ ES ]  
dt 2
Km '  [S ] Km'  [S ]

′
𝐾𝑚 is often called the Michaelis-Menten constant, mol/L, mg/L.
- The prime reminds us that it was derived by assuming rapid
equilibrium in the step of enzyme-substrate complex formation.
- Low value of 𝐾𝑚′ indicates affinity of enzyme to the substrate.

′
𝑘−1
𝐾𝑚 =
𝑘1

- It
corresponds to the substrate concentration, giving the
reaction velocity. I.E. if 𝐾𝑚′ = [𝑆]

𝑉𝑚 [𝑆] 1
𝑣= = 𝑉
′
𝐾𝑚 + [𝑆] 2 𝑚
 d [ P] k 2 [ E0 ][ S ] Vm [ S ] '  k 1  [ E ][ S ]
v  k [ ES ]   Km
k1 [ ES ]
dt 2 '
K m  [S ] Km'  [S ]

1. It establishes an approximate value for the intracellular level of a substrate.

2. Since it is constant for a given enzyme/substrate, its numerical value provides
a means of comparing enzymes from different organisms (Vmax is not a
constant but depends on k and [E0]).
3. K’m will vary with temperature and pH.
4. K’m can be altered by ligand binding – one mode of enzyme regulation.
5. If K’m is known the assay conditions can be altered so that [S] >> K’m so that
Vmax can be determined which is a measure of [E0].
6. It indicates the relative “suitability” of alternate substrates for an enzyme.
The substrate with the lowest Km has the highest affinity for the enzyme. The
“best” substrate has the highest Vmax/Km ratio.
 ′
𝑣
𝑣 = 𝑉𝑚 − 𝐾𝑚
𝑆

𝑆 ′
𝐾𝑚 1
= + 𝑆
𝑣 𝑉𝑚 𝑉𝑚
Example
 Briggs - Haldane Approach
The Quasi Steady-State assumption:

- In most experimental systems a closed system (batch

reactor) is used in which the initial substrate concentration
greatly exceeds the initial enzyme concentration.
- It was suggesting that since [E0] was small, d[ES]/dt ≅ 0

K1 k2
E+S ES  P  E
K-1
 COMPLEX ENZYME KINETICS

Inhibition
The prevention of an enzyme process as a result of interaction of inhibitors
with the enzyme.

 INHIBITORS:
Any substance that can diminish the velocity of an enzyme
catalyzed reaction is called an inhibitor.
 Types of inhibition

Inhibition

Reversible Irreversible

Non-
Competitive Uncompetitive Substrate
competitive
REVERSIBLE INHIBITION
 Competitive
inhibition

• In this type of inhibition, the inhibitors

compete with the substrate for the
active site. Formation of E.S complex is
reduced while a new E.I complex is
formed.
  competes with the substrate
k2 for the active site of an
enzyme.
K’m
 inhibitor (I) occupies
 the active site it prevents
binding of the substrate to the
enzyme.
 compounds that resemble the
substrate and combine with
the enzyme to form an EI
complex.
 Assuming rapid equilibrium and
with the definition of

k2
K’m
 E S E I
ES = EI =
K′m KI

E S E I
= E + +
K′m KI

S I E0
= E 1+ + E =
K ′m KI S
1+ ′ +
I
Km KI

E S E0 S
ES = =
K′m S
K′m 1+ ′ + K
I
Km I

𝑘2 E0 S 𝑉𝑚 S
𝑣 = 𝑘2 𝐸𝑆 = =
S I I
K ′m 1+ ′ + K K ′m 1 + K + S
Km I I
𝑉𝑚 S
= ′
K m,app + S
 Assuming rapid equilibrium and
with the definition of

k2
K’m
 Lineweaver–Burk plot

• The net effect of

competitive inhibition is an
increased value of Km’ app
and, therefore, reduced
reaction rate. Competitive
inhibition can be overcome by
high concentration of
substrate.
 Noncompetitive
Inhibition

A noncompetitive inhibitor binds to the

enzyme away from the active site,
altering the shape of the enzyme so that
even if the substrate can bind, the active
site functions less effectively.
 Assuming rapid equilibrium and
with the definition of

K’m k2

K’m
 E S E I
ES = EI =
K′m KI

E S E I ES I E S E I E S I
= E + + + = E + + +
K′m KI KI K′m KI K′m KI

S I S I E0
= E 1+ + + E =
K ′m KI K ′m K I S I S I
1+ ′ + + ′
Km KI Km KI

E S E0 S
ES = =
K′m S
K′m 1+ ′ + K + ′
I S I
Km I Km KI

𝑘2 E0 S 𝑉𝑚 S
𝑣 = 𝑘2 𝐸𝑆 = =
S I S I I I
K ′m 1+ ′ + K + ′ K ′m 1 + K + S 1 + K
Km I KmKI I I

𝑉𝑚
=
I K ′m
1+K 1+
I S
 Assuming rapid equilibrium and
with the definition of

K’m k2

K’m
 Lineweaver–Burk plot

• The net effect of noncompetitive

inhibition is in reduction in Vm, therefore,
high concentration of substrate would not
overcome noncompetetitive inhibition.
 Allosteric site

Uncompetitive Some enzymes have more than one substrate binding

site. The binding of one substrate to the enzyme
Inhibition facilitates binding of other substrate molecules. This
behavior is known as allostery or cooperative binding,
and regulatory enzymes show this behavior.

• In this type of inhibition,

inhibitor does not compete with
the substrate for the active site
of enzyme instead it binds to
another site known as allosteric
site.
 Assuming rapid equilibrium and
with the definition of

k1
k-1 k2
 Lineweaver–Burk plot

• The net effect of uncompetitive

inhibition is in reduction of both Vm and
Km’ , therefore, net result in reduction of
reaction rate.
 Assuming rapid equilibrium and
with the definition of
Uncompetitive
SUBSTRATE Inhibition
 Lineweaver–Burk plot

• The net effect of uncompetitive

inhibition is in reduction of both Vm and
Km’ , therefore, net result in reduction of
reaction rate.
• The net effect of uncompetitive
inhibition is in reduction of both Vm and
Km’ , therefore, net result in reduction of
reaction rate.
Example
3.3
 Irreversible
inhibition

• This type of inhibition involves

the covalent attachment of the
inhibitor to the enzyme.
• The catalytic activity of enzyme
is completely lost.
 IMMOBILIZED ENZYME SYSTEMS

IMMOBILIZED : enzyme mobility gets restricted in a fixed space

Advantages of immobilized enzymes:
- Easy separation from reaction mixture, providing the ability to control
reaction times and minimize the enzymes lost in the product

- Re-use of enzymes for many reaction cycles, lowering the total

production cost of enzyme mediated reactions

- Ability of enzymes to provide pure products

- Possible provision of a better environment for enzyme activity

Disadvantages of immobilized enzymes:

- Problem in diffusional mass transfer
- Enzyme leakage into solution
- Reduced enzyme activity and stability
- Lack of controls on micro environmental conditions
Methods of immobilization
Methods of immobilization
1. Adsorption
Immobilization
Adsoption
 is an oldest method of enzyme immobilization
 Simplest method of enzyme immobilization
 Nelson & Griffin use charcoal to adsorb
inverase
 Enzymes are adsorbed to external surface called
support
 Support or carrier may be:
 Mineral support (Aluminum oxide, clay
etc.)
 Organic support (Starch)
 Modified Sepharose and ion-exchange
resins
2. Entrapment
Immobilization
-Matrix entrapment (matrices used are
polysaccharides, proteins, polymeric
materials, activated carbon, porous
ceramic and so on)

-Membrane entrapment
(microcapsulation or trapped between
thin, semi-permeable membranes)
3. Covalent
Attachment
Immobilization
Single point Covalent Attachment
 Covalent attachment of enzymes to an
insoluble support is an often-used method of
enzyme immobilization.

 Typically it is more expensive and complex to

covalently immobilize an enzyme compared to
the other methods due to the higher costs of
the support.

Multipoint Covalent Attachment

 Multipoint covalent attachment of each

immobilized enzyme molecule may promote a
very interesting stabilizing effect.

 The immobilization of enzyme through the

region having the highest amount of amino
groups (Lys residues) is key for a successful
stabilization.
4. Cross-Linking
Immobilization
 Based on intermolecular
reactions, enzymes are
cross-linked to the
support matrices using
bifunctional reagents.
 In this mode, with
covalent bonds,
enzymes are
immobilized firmly to
improve the reusability
and stability.
 Glutaraldehyde,
isocyanate and N, N′-
ethylene bis maleimide
are the commonly used
bifunctional reagents
Comparison between the methods
Characteristics Adsorption Covalent Entrapment Membrane
coupling confinement
Preparation Simple Difficult Difficult Simple
Cost Low High Moderate High
Binding force Variable Strong Weak Strong
Enzyme leakage Yes No Yes No
Applicability Wide Selective Wide Very wide
Running problems High Low High High
Matrix effects Yes Yes Yes No

Large diffusional No No Yes Yes

barriers

Microbial protection No No Yes Yes

Immobilized enzyme reactor (example)

- Allow the reactor to

operate at high fluid
velocities
- An immobilized enzyme
tends to decompose upon
physical stirring.

- The batch system is

generally suitable for the
production of rather small
amounts of chemicals.
 Fluidized bed reactor
- A high viscosity substrate
solution

- A gaseous substrate or
product in a continuous
reaction system

- Care must be taken to avoid

the destruction and
decomposition of immobilized
enzymes
External Surface (Enzyme can Stick on External walls)
Internal Pores (Enzyme can Stick on internal walls)
 Diffusion Limitations in Immobilized ENZYME SYSTEMS

Mass transfer resistance is present

- due to the large particle size of the immobilized enzymes
- due to the inclusion of enzymes in polymeric matrix
Mass transfer resistance are divided into the following:
- External mass transfer resistance
(during transfer of substrate from the bulk liquid to
the relatively unmixed liquid film surrounding the
immobilized enzyme and
during diffusion through the relatively unmixed
liquid film)
- Intra-particle mass transfer resistance
(during diffusion from the surface of the particle to
the active site of the enzyme in an inert support)
External mass-transfer resistance:
Surface bound Enzyme Immobilize on nonporous support
Ss
S s
Sb Sb
Assumption:
- Enzymes are evenly distributed on
the surface of a nonporous support
material.
- All enzyme molecules are equally
active.
- Substrate diffuses through a thin
liquid film surrounding the support
surface to reach the reactive surface.
- The process of immobilization has Enzyme
Enzyme
not altered the enzyme structure and
the M-M kinetic parameters (Vm, Km)
are unaltered. Liquidfilm
Liquid Film thickness,
Thickness, L L
External mass-transfer resistance:
Diffusional mass transfer across
the liquid film: Ss
Ss
Sb Sb
JS = kL (Sb – Ss)

kL liquid mass transfer

coefficient (cm/s)
Sb substrate concentration in
the bulk solution (mol/cm3)
Ss substrate concentration at
the immobilized enzyme Enzyme
Enzyme
surface (mol/cm3)

Liquid Film
Liquid filmThickness,
thickness,L L
External mass-transfer resistance:

At steady state, the reaction rate is Ss

equal to the mass-transfer rate: Ss
Sb
V’m Ss
JS = kL (Sb –Ss) =
Km + Ss

V’m maximum reaction rate per

unit of external surface area
(e.g. mol/cm2.s)
Km is the M-M kinetic constant
(e.g. mol/cm3) Enzyme
Enzyme

Liquidfilm
Liquid Filmthickness,
Thickness, L L
Example 3.4

Consider a system where a flat sheet of polymer coated with enzyme is

placed in a stirred beaker. The intrinsic maximum reaction rate of the
enzyme is 6 x 10-6 mols/s.mg enzyme. The amount of enzyme bound to the
surface has been determined to be maximum 1 x 10-4 mg enzyme/cm2 of
support. In solution, the value of Km has been determined to be 2 x 10-3
mol/l. The mass-transfer coefficient can be estimated from standard
correlations for stirred vessels. We assume in this case a very poorly mixed
system where kL = 4.3 x 10-5 cm/s. What is the reaction rate, when the bulk
concentration of the substrate ([Sb]) is (a) 7 x 10-3 mol/l and (b) 1 x 10-2
mol/l?
 Solution
Data provided:
V’m = 6 x 10-6 x 1 x 10-4 mols/s.cm2
= 6 x 10-10 mols/s.cm2
Km = 2 x 10-3 mol/l = 2 x 10-6 mol/cm3
kL = 4.3 x 10-5 cm/s
Sb = 7 x 10-3 mol/l OR 1 x 10-2 mol/l
= 7 x 10-6 mol/cm3 OR 1 x 10-5 mol/cm3

Equation to be solved:

V’m [Ss]
JS = kL ([Sb] –[Ss]) =
Km + [Ss]

where Ss should be solved for, which can then be used to calculate JS.
 V’m [Ss]
V =JS = kL ([Sb] –[Ss]) =
Km + [Ss]

The solution is given in Figure. The

key is to note that the mass-
transfer rate equals the reaction
rate at steady state, and as a
consequence the right side of
must equal the left side. In case
(a), this occurs at a substrate
surface concentration of about
0.0015 mol/l with a reaction rate
of 2.3x10-10 mol/s-cm2. By
increasing the bulk substrate
concentration to 0.01 mol/l, the
value of [Ss] increases to
0.0024 mol/l with a reaction rate
about 3.3 x 10-10 mol/s-cm2.
 External mass-transfer resistance

V’m [Ss]
JS = kL ([Sb] –[Ss]) =
Km + [Ss]
Non dimensionalizing the above equation, we get

1 – [Ss]’ β [Ss]’
=
Da 1 + β [Ss]’

where
[Ss]’ = [Ss] / [Sb]

Da = V’m / (kL [Sb] ) is the Damköhler number

β = [Sb] / KM is the dimensionless substrate

concentration
Damköhler number (Da)

Maximum rate of reaction V’m

Da = =
Maximum rate of diffusion kL [Sb]

If Da >> 1, rate of diffusion is slow and therefore the limiting

mechanism
Vp = JS = kL ([Sb] –[Ss])

If Da << 1, rate of reaction is slow and therefore the limiting

mechanism
V’m [Ss]
Vp =
Km + [Ss]
If Da = 1, rates of diffusion and reaction are comparable.