October, 2023

Science of Living System

BS10003 (2-0-0)

Dibyendu Samanta
School of Bio Science
Email: [email protected]
Tel: 03222-284576
 Protein Classification by Function

Enzymes: catalyze chemical reactions

Transport proteins: e.g. myoglobin and hemoglobin transport O2

Protective proteins: e.g. immunoglobulin (antibody)

Structural proteins: e.g collagen, keratins (in skin, hair), elastin

(vocal chord, arteries), silk.

Contractile and motile proteins: involved in motion, e.g. myosin

and actin in muscle
 Enzymes – Biological catalysts
• First discovered by Eduard Buchner in 1897 who observed
that yeast extracts can ferment sugar to alcohol. Nobel
Prize 1907.

• This proved that fermentation was promoted by

molecules that continued to function when removed from
cells.

• The first enzyme to be purified and crystallized was urease

in 1926 by James Sumner at Cornell University; these
crystals consisted entirely of protein. Nobel Prize 1946.

• Later, pepsin, trypsin and other digestive proteins were

isolated and determined to be purely protein as well.
 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 dependant upon native

conformation; if it is lost, then catalytic activity is lost as
well

 All levels of protein architecture 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
• 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.
 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)
– Hydrophobicity
– Flexibility
– Reactivity DNA Polymerase
 Substrate Binding Specificity
Enzymes interact with specific substrates via non-covalent Interactions

Complementarity
• Geometric
• Electrostatic
• Stereospecificity (enzymes and
substrates are chiral)

1. Lock and Key model

2. Induced Fit model
 Enzyme-Substrate Interaction

Lock and Key Model

The active site shape is complementary to the substrate i.e. not all
substrates can fit the active site.
 Enzyme-Substrate Interaction

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
 Factors that Influence Enzyme Activity

Effect of temperature Effect of pH

Rate of Reaction

Temperature (C)
 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
How can an Enzyme Reduce the Activation
Energy?

(1) Binding to the substrate can be done in such a way

that the formation of the transition state is favored

(2) Orientation and positioning of substrate

(3) Bonds in the substrate can be ‘activated’ by

functional groups in the catalytic site
How can an Enzyme Reduce the Activation Energy?
 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)
 The turnover number (kcat)
kcat is how many reactions an enzyme can catalyze per second

Vmax
V0 = Vmax [S] kcat   k2
KM + [S] E T
For Michaelis - Menten kinetics k2= kcat
When [S] << KM very little ES is formed and [E] = [E]T
k2 k cat
and
vo  E T S   E S 
KM KM
kcat/KM is a measure of catalytic efficiency
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 K M), 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 as biosensors

Glucose
Oxidase
+ O2 + H 2O2

Glucose Gluconolactone

Measuring Blood Glucose

H 2O2 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.
 Protein Structure, Function, Kinetics
and Energetics

Books Followed:

• How Proteins Work (Mike Williamson)

• Introduction to protein structure (Carl Branden & John
Tooze)
• Biochemistry (Lubert Stryer)
•https://www.ncbi.nlm.nih.gov/books/NBK22430/