18th January, 2021

Science of Living System

BS10003 (2-0-0)

Dibyendu Samanta
School of Bioscience
Email: dibyendu.samanta@iitkgp.ac.in
Tel: 03222-260295
Hierarchy of Protein Structure
 Protein classification by function
Enzymes: catalyze chemical reactions.1300 enzymes in Human body.
Regulatory proteins: bind to protein receptors, e.g. hormones such as
insulin.
Transport proteins: e.g. myoglobin and hemoglobin transport O2.
Storage proteins: e.g. casein in milk, ovalbumin in eggs.
Contractile and motile proteins: involved in motion, e.g. myosin and actin in
muscle.
Structural proteins: e.g collagen, keratins (in skin, hair), elastin (vocal
chord, arteries), silk.
Binding/Interaction proteins: proteins bind one another only when a signal
is received, e.g. phosphorylation of Insulin Receptor Substrate (IRS) protein.
Protective proteins: e.g. immunoglobulins, proteins of blood clotting system.
 Enzymes
➢ Enzymes are the catalysts of nature.
➢ With the exception of catalytic RNA, all enzymes are
proteins.
➢ Catalyst alter the rate of a chemical reaction without
undergoing a permanent change in structure.
➢ Catalytic activity is dependent upon native (i.e. folded)
conformation of the enzyme; if it is lost, then catalytic
activity is lost as well.
➢ All levels of protein architecture (i.e. primary to
quaternary structure) must be intact and correct for
enzymes to perform their functions.
➢ They range in molecular weights from 12,000 to over 1
million daltons.
 How enzymes work
• Enzymes catalyze chemical reactions that do not normally proceed under conditions
such as neutral pH, mild temperature, and aqueous solvent.
• The site of catalytic activity on the enzyme is known as the active site.
• The molecule that binds to the active site and is acted upon by the enzyme is called
the substrate.
• The two together form what is known as the enzyme-substrate complex
• The function of an enzyme is to increase the rate of a chemical reaction without
affecting its equilibrium.
• Therefore, enzymes don’t make more product, they just make product faster.

enzyme-substrate complex
 Chymotrypsin
Active Site
• The area of an enzyme where the substrate
binds.
• Structure has a unique geometric shape that
is designed to fit the molecular shape of the
substrate.

• Active sites contain residues that bind the

substrate and also participate in catalysis.
• Active sites sometimes contain a co-factor.
• Active site residues have several important
properties:
– Charge (partial, dipoles, helix dipole)
– pKa
– Hydrophobicity
– Flexibility

DNA Polymerase
 Substrate Binding site (Active site)

Complementarity
• Geometric
• Electronic (electrostatic)
• Stereospecificity (enzymes
and substrates are chiral)

1. Lock and Key model

2. Induced Fit model
 Substrate Binding site (Active site)

Lock and Key Model

• An enzyme binds a substrate in a region called the active site.
• The active site shape is complementary to the substrate i.e. not all
substrates can fit the active site.
• Amino acid sidechains in the active site bind the substrate.

Induced Fit Model

• Enzyme structure is flexible, not rigid.
• Enzyme and active site adjust their shape to bind the substrate.
• Increases range of substrate specificity.
• Shape changes also improve catalysis during reaction
- by stabilizing the transition-state.
Enzyme-Substrate Interaction
Factors that influence enzyme activity -
temperature
Factors that influence enzyme activity - pH
 How enzymes work?

ΔGǂ S→Pfor uncatalyzed reaction = 107 kJ kuncat  e-107000/8.314x298

ΔGǂ cat for catalyzed reaction = 47 kJ
kcat  e-47000/8.314x298

ǂ
kcat/kuncat = ~5x1010
k  e-ΔG /RT
1 sec ~1500 years
 Enzyme Kinetics

⎯⎯ →
k1
E + S ⎯⎯ ES ⎯⎯→ E + P
k2
k −1

E = Enzyme S = Substrate P = Product

ES = Enzyme-Substrate complex
k1 rate constant for the forward reaction
k-1 = rate constant for the breakdown of the ES to
substrate
k2 = rate constant for the formation of the products
 ⎯⎯ →
k1
E + S ⎯⎯ ES ⎯⎯→ E + P
k2
k −1

Rate of formation of product P:

d P 
v= = k 2 ES 
dt

Rate of formation of enzyme-substrate complex ES:

d ES 
1

= k1 E S − k −1 ES  − k 2 ES 

dt
 Formation of enzyme-substrate complex

⎯⎯ →
k1
E + S ⎯⎯ ES ⎯⎯→ E + P
k2
k −1

At equilibrium:
forwards reaction rate = reverse reaction rate
i.e. k1[E][S] = k-1[ES]

k −1 E S
KD = =
k1 ES
KD is the dissociation constant for the ES complex.
 Assumption of steady-state
Transient phase where in the course of a reaction the
concentration of ES does not change

⎯⎯ →
k1
E + S ⎯⎯ ES ⎯⎯→ E + P
k2
k −1
 ET = E + ES 3

Combining 1 + 2 + 3

k1 (ET - ES)S = (k -1 + k 2 )ES

rearranging

ES(k-1 + k 2 + k1S) = k1ET S

Divide by k1 and solve for [ES] Where

ES  = E T S
KM =
k -1 + k 2
K M + S k1
vo is the initial velocity when the reaction is just starting out.
And Vmax = k 2 ET is the maximum velocity

The Michaelis - Menten

equation
 Michaelis – Menten Kinetics

The Km is the substrate concentration

where vo equals one-half Vmax
 Michaelis – Menten Kinetics

low [S], v is proportional to [S] - first order

high [S], v is independent of [S] - zero order
 The double reciprocal plot
Lineweaver-Burk plot transforms the Michaelis-
Menten equation into linear form.

V0 = Vmax [S] 1 = Km + [S]

Lineweaver-Burk Plot Km + [S] V0 Vmax [S]

1 = Km 1
+
1
V0 Vmax [S] Vmax

(y = mx + c)
KM
Relates to how strongly an enzyme binds its substrate.
High KM means strength of binding is low.

kcat
Relates to how rapid a catalyst the enzyme is.
High kcat means high speed of catalysis.

Vmax
Related to kcat and [ET] by: Vmax=kcat[ET]
High Vmax means high rate of catalysis.
 Enzyme Inhibition
• Inhibitors: compounds that decrease or eliminate activity of an
enzyme.
• Can decrease binding of substrate (affect KM), or turnover number
(affect kcat) or both.
• Most drugs are enzyme inhibitors.
• Inhibitors are also important for determining enzyme mechanisms
and the nature of the active site.

Some examples of enzyme inhibitors:

• Antibiotics inhibit enzymes by affecting bacterial metabolism.
• Nerve Gases cause irreversible enzyme inhibition.
• Insecticides – choline esterase inhibitors.
• Many heavy metal poisons work by irreversibly inhibiting enzymes,
especially cysteine residues.
 Types of Enzyme Inhibition

• Reversible inhibition
reversibly bind and dissociate from enzyme,
activity of enzyme recovered on removal of
inhibitor - usually non-covalent in nature
– Competitive
– Uncompetitive
– Noncompetitive (Mixed)

• Irreversible inhibition
irreversibly associate with enzyme. Activity of
enzyme not recovered on removal - usually
covalent in nature.
 Competitive Inhibition

• Inhibitor competes for the substrate binding site

• most look like substrate
• substrate mimic / substrate analogue
Competitive Inhibition

1 = Km 1
+
1
V0 Vmax [S] Vmax

(y = mx + c)
Competitive Inhibition

1 = Km 1
+
1
V0 Vmax [S] Vmax

(y = mx + c)

α = 1 + [I]/Ki
 Competitive Inhibition

No Reaction

• Methanol poisoning is treated with ethanol; the formation of

formaldehyde is slowed and spread out over a longer period of time,
lessening its effects on the body
 Uncompetitive Inhibition
Uncompetitive inhibitors bind at a site distinct from the substrate active site
and bind only to the ES complex

• Active site distorted after binding of S (usually

occurs in multisubstrate enzymes) Decreases both
KM and kcat
• Vo = Vmax[S]/(KM + ’[S]) K’I = [ES][I]/[ESI]
• Cannot be reversed by increasing [S] – available
enzyme decreases
Uncompetitive Inhibition

1 = Km 1
+
1
V0 Vmax [S] Vmax

(y = mx + c)

α’ = 1 + [I]/K’i
 Enzymes we have already seen

• DNA Polymerase, Helicase, Primase, RNA Polymerase,

Taq Polymerase, Ribosome, beta-galactosidase.
• Several antibiotics are inhibitors of ribosomes.
Enzymes with important industrial applications
 Enzymes as biosensors

Glucose
Oxidase
+ O2 + H2O2

Glucose Gluconolactone

Measuring Blood Glucose

H2O2 O2 + 2H+ + 2e- Detect by electrode

Current measures the rate of product

formation i.e.v0, which is proportional to
substrate concentration [S] i.e.
concentration of glucose in blood.