Enzyme Catalysis

Science of

Living System

Soumya DeSchool of Bio Science

Email: [email protected]

Tel: 03222-260514

BS20001

mailto:[email protected]

Lecture Date Topic

1 20/7/16 Nucleic acids

2 27/7/16 Transcription and Translation

3 3/8/16 Protein structure

4 10/8/16 Enzymes

5 17/8/16 Photosynthesis

6 24/8/16 Respiration

** 31/8/16 CLASS TEST-1

** 7/9/16 DISCUSSION AND REVISION

** 14/9 to 21/9 MID-SEM EXAM

7 28/9/16 Cellular architecture

8 5/10/16 Cell division and apoptosis

** 12/10/16 Autumn Break

9 19/10/16 Host defense/Disease biology/vaccines/antibiotics

10 26/10/16 Responses of living systems/scaling factors

12 2/11/16 Recombinant DNA Technology & its impact

** 9/11/16 CLASS TEST-2

** 16/11/16 DISCUSSION AND REVISION

Books Followed:

• How Proteins Work (Mike Williamson)

• Introduction to protein structure (Carl

Branden & John Tooze)

• Biochemistry (Lubert Stryer)

Protein Structure, Function, Kinetics

and Energetics

Hierarchy of Protein Structure

Protein classification by function

Enzymes: catalyze chemical reactions.

Regulatory proteins: bind to protein receptors, e.g. hormones such as

insulin.

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

Hemoglobin – O2 carrier




insulin.


Storage proteins: e.g. casein in milk, ovalbumin in eggs.

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

muscle.

The major proteins of muscle are

myosin and actin




insulin.


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.

Antibodies recognize antigens

Enzymes – Biological catalysts

• First discovered by Eduard Buchner in 1897 whoobserved that yeast extracts can ferment sugar toalcohol. Nobel Prize 1907.

• This proved that fermentation was promoted bymolecules that continued to function when removedfrom cells.

• The first enzyme to be purified and crystallized wasurease in 1926 by James Sumner at Cornell University;these crystals consisted entirely of protein. Nobel Prize1946.

• Later, pepsin, trypsin and other digestive proteins wereisolated and determined to be purely protein as well.

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.

Enzymes

Enzymes

Most enzymes are proteins.

Simple Enzymes: composed of whole proteins.

Complex Enzymes: composed of protein plus a relatively

small organic molecule.

holoenzyme = apoenzyme + prosthetic group or coenzyme

Enzymes

Most enzymes are proteins.

Simple Enzymes: composed of whole proteins.

Complex Enzymes: composed of protein plus a relatively

small organic molecule.

holoenzyme = apoenzyme + prosthetic group or coenzyme

A prosthetic group describes a small organic or metalloorganic

molecule bound to the apoenzyme by covalent bonds.

When the binding between the apoenzyme and non-protein

components is non-covalent, the small organic molecule is

called a coenzyme.

Coenzymes serve as transient carriers of specific functional

groups.

They often come from vitamins (organic nutrients required in

small amounts in the diet)

Oxidoreductases Add or remove hydrogen atoms (transfer of electrons).

Transferases

Transfer functional groups between donor and

acceptor molecules. E.g. Kinases transfer phosphate

group from ATP to other molecules.

HydrolasesCatalyze hydrolysis i.e. breaking of a bond by the

addition of a water molecule.

Lyases

Add water, ammonia or carbon dioxide across double

bonds, or remove these elements to produce double

bonds.

Isomerases

Carry out many kinds of isomerization. E.g. L to D

isomerizations, mutase reactions (shifts of chemical

groups) and cis-trans isomerization of peptide bonds.

LigasesCatalyze reactions in which two chemical groups are

joined (or ligated) with the use of energy from ATP.

International Classification of Enzymes

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

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

Chymotrypsin

Substrate Binding site (Active site)

Complementarity

• Geometric

• Electronic (electrostatic)

• Stereospecificity (enzymes

and substrates are chiral)

1. Lock and Key model

2. Induced Fit 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.

Lock and Key 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.

Induced Fit Model

Substrate Binding site (Active site)

Enzyme-Substrate Interaction

Factors that influence enzyme activity -

temperature

Factors that influence enzyme activity - pH

ΔGǂS→Pfor uncatalyzed reaction = 107 kJ

ΔGǂcat for catalyzed reaction = 47 kJ

kuncat e-107000/8.314x298

kcat e-47000/8.314x298

kcat/kuncat = ~5x1010

How enzymes work?

k e-ΔG /RTǂ

1 sec ~1500 years

How can an enzyme reduce the

activation energy?

(1) Binding to the substrate can be done such that the

formation of the transition state is favored

(2) Orientation and positioning of substrate(s)

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

groups in the catalytic site

How can an enzyme reduce the

activation energy?

- the activation energy for the formation of the intermediate state, and its

conversion to the final product are each lower than the activation energy for the

uncatalyzed reaction

-intermediate state- resembles transition state but with lower energy, (due to

interaction with a catalyst)

- transition state defines free energy maximum state

Binding energy helps reduce

the activation energy

How enzymes work? Example: Chymotrypsin

• Chymotrypsin is a digestive enzyme component of pancreatic juice.

• It performs proteolysis i.e. the breakdown of proteins and polypeptides.

• Chymotrypsin preferentially cleaves peptide amide bonds where the

carboxyl side of the amide bond is an aromatic amino acid (tyrosine,

tryptophan, and phenylalanine).

• Chymotrypsin facilitates the nucleophilic (OH-) attack from the correct

orientation.

• It lowers the transition state energy.

• Chymotrypsin favors reaction ‘b’ after formation of the intermediate.

N N NN

N

N

How enzymes work? Example: Chymotrypsin

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

Enzyme Kinetics

E S

k1

k1

ESk

2 E P

ES

P2k

dt

dv

ESESSE

ES211 kkk

dt

d

1

E S

k1

k1

ESk

2 E P

Rate of formation of product P:

Rate of formation of enzyme-substrate complex ES:

ES

SE

1

1

k

kKD

KD is the dissociation constant for the ES complex.

Formation of enzyme-substrate complex

E S

k1

k1

ESk

2 E P

At equilibrium:

forwards reaction rate = reverse reaction rate

i.e. k1[E][S] = k-1[ES]

Assumption of steady-state

Transient phase where in the course of a reaction the

concentration of ES does not change

0

ES

d t

d

2

E S

k1

k1

ESk

2 E P

ES E E T 3

Combining 1 + 2 + 3

ESk k SES-Ek 21-T1

SEk Sk k kES T1121-

rearranging

Divide by k1 and solve for [ES] Where

1

21-

k

k k K

M

S K

SE ES T

M

SK

SEE S

P T22

0

Mt

o

kk

d t

dv

vo is the initial velocity when the reaction is just starting out.

And is the maximum velocity T2max Ek V

SK

SV max

M

ovThe Michaelis - Menten

equation

Michaelis – Menten Kinetics

The Km is the substrate concentration

where vo equals one-half Vmax

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

high [S], v is independent of [S] - zero order

Michaelis – Menten Kinetics

The KM widely varies among different enzymes

The KM

can be expressed as: 1

2

1

2

1

1 KKk

k

k

k

k

kDM

As KD decreases, the affinity for the substrate

increases. The KM can be a measure for substrate

affinity if k2<<k-1

The parameters vmax and KM are used to

compare enzyme activities

V0 = Vmax [S]

Km + [S]

Lineweaver-Burk plot transforms the Michaelis-

Menten equation into linear form.

1 = Km + [S]

V0 Vmax [S]Lineweaver-Burk Plot

The double reciprocal plot

Km 1 1

Vmax [S] Vmax

= +1

V0

(y = mx + c)

For small errors in at low [S]

leads to large errors in 1/vo

For Michaelis - Menton kinetics k2= kcat

When [S] << KM very little ES is formed and [E] = [E]T

and SE

K

kSE

K

k

M

catT

M

2 ov

kcat/KM is a measure of catalytic efficiency

V0 = Vmax [S]

KM + [S] kcatk 2T

max

E

V

The turnover number (kcat)

kcat is how many reactions an enzyme can catalyze per second

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.

A high kcat/KM ratio implies an efficient enzyme

This could result from: Large kcat

Small KM

• kcat = turnover number; kcat = Vmax/[ET]

• kcat/Km is a measure of activity, catalytic efficiency

KM is a useful indicator of the affinity of an enzyme

for the substrate

A low KM indicates a high affinity for the substrate

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


(y = mx + c)

Km 1 1

Vmax [S] Vmax

= +1

V0


(y = mx + c)

Km 1 1

Vmax [S] Vmax

= +1

V0

α = 1 + [I]/Ki


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

(y = mx + c)

α’ = 1 + [I]/K’i

Km 1 1

Vmax [S] Vmax

= +1

V0

Inhibitor can bind at a site distinct from the substrate active site to either E or ES.

Mixed (Non-competitive) Inhibition

• Vo

= Vmax[S]/(KM + ’[S])

• Vmax decreases; KM can

go up or down.

Non-competitive Inhibition

(y = mx + c)

Km 1 1

Vmax [S] Vmax

= +1

V0

Mixed inhibition refers to a combination of two different types of reversible enzyme

inhibition – competitive inhibition and uncompetitive inhibition. The term 'mixed' is used

when the inhibitor can bind to either the free enzyme or the enzyme-substrate complex.

In mixed inhibition, the inhibitor binds to a site different from the active site where the

substrate binds. Mixed inhibition results in a decrease in the apparent affinity of the

enzyme for the substrate (Kmapp > Km, a decrease in apparent affinity means the Km

value appears to increase) and a decrease in the apparent maximum enzyme reaction

rate (Vmaxapp < Vmax).

Mathematically, mixed inhibition occurs when the factors α and α’ (introduced into the

Michaelis-Menten equation to account for competitive and uncompetitive inhibition,

respectively) are both greater than 1.

In the special case where α = α’, noncompetitive inhibition occurs, in which case

Vmaxapp is reduced but Km is unaffected. This is very unusual in practice

Non-competitive inhibition models a system where the inhibitor and the substrate

may both be bound to the enzyme at any given time. When both the substrate and the

inhibitor are bound, the enzyme-substrate-inhibitor complex cannot form product and

can only be converted back to the enzyme-substrate complex or the enzyme-inhibitor

complex. Non-competitive inhibition is distinguished from general mixed inhibition in

that the inhibitor has an equal affinity for the enzyme and the enzyme-substrate

complex.

Lineweaver-Burke plots - Summary

Allosteric regulation of enzymes

A small molecule can bind an enzyme and act as an effector

or regulator to activate or inactivate its function.

In such case, the protein is said to be under allosteric control.

If the binding of the small molecule (ligand) is distant from the

protein’s active site and regulation is a result of a

conformational change in the protein when the ligand is

bound, the regulation is called allosteric regulation.

Many types of proteins show allosteric control:

- hemoglobin (NOT myoglobin)

- various gene-regulating proteins (transcription factors)

e.g. Lac repressor (see lecture 2)

Phosphofructokinase (PFK-1) and ATP

Substrate: Fructose-6-phosphate

fructose-6-phosphate + ATP fructose-1,6-bisphosphate + ADPphosphofructokinase-1

Allosteric regulation of PFK-1

• PFK-1 catalysed reaction lies near the beginning of the respiration pathway in cells.

• The end product of respiration is synthesis of ATP, the energy currency.

• If there is a lot of ATP in the cell PFK-1 is inhibited. Then respiration slows down and less ATP is produced.

• As ATP is used up PFK-1 inhibition stops and the reaction speeds up again.

The switch: Allosteric inhibition

Allosteric means “other site”

E

Active site

Allosteric

site

Allosteric inhibition: Switching off the enzyme

• These enzymes have two receptor sites

• One site fits the substrate like other enzymes

• The other site fits an inhibitor molecule Inhibitor fits

into allosteric

site

Substrate

cannot fit

into the

active site

Inhibitor

molecule

E

Active

site

Allosteric

site empty

Substrate fits into the active site

The inhibitor

molecule is

absent

Conformational

change

Inhibitor fits into

allosteric site

Substrate

cannot fit

into the

active

site

Inhibitor molecule

is present

E

Allosteric inhibition: Switching off the enzyme

Conformational change: Change in shape of

the protein

• When the inhibitor is present it fits into its site

and there is a conformational change in the

enzyme molecule

• The enzyme’s molecular shape changes

• The active site of the substrate changes

• The substrate cannot bind with the substrate

• Irreversible inhibitors are those that combine with or

destroy a functional group on an enzyme that is

essential for activity

• They usually form covalent linkages to the enzyme

Diisopropylfluorophosphate binds irreversibly with

chymotrypsin at the Ser195 residue; this gives info

justifying this as the primary active site of the enzyme

Irreversible inhibition: Kill the enzyme

• A special class of irreversible inhibitors is the suicide

inactivators

• These are unreactive until bound to the active site

• They are designed to carry out the first few steps of a

normal enzyme reaction, but instead of forming a

product, they form a highly reactive compound that

binds irreversibly to the enzyme

• They are sometimes called mechanism-based

inactivators, because they use the normal enzyme

mechanism to lead to the inactivation

• These are often used in drug design

Enzyme Catalysis

Education

Transcript of Enzyme Catalysis