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� It is a substance which binds with the

enzyme and brings about decrease in the

catalytic activity of the enzyme.

�Enzyme inhibitors are molecules that

interact in some way with the enzyme to

prevent it from working in the normal

manner.

�Organic or inorganic

�Reversible or irreversible

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�Reversible

• Competitive

• Non- competitive

• uncompetitive

� Irreversible

�Allosteric

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Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

E

E

Different siteCompete for

active siteInhibitor

Substrate

Ca

rto

on

Gu

ide

Equ

atio

n an

d D

escr

iptio

n

[II] binds to free [E] only,

and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES]

complex; Increasing [S] can

not overcome [II] inhibition.

[II] binds to [ES] complex

only, increasing [S] favors

the inhibition by [II].

E + S→ES→E + P

+II

↓

EII

←

↑

E + S→ES→E + P

+ +II II

↓ ↓

EII+ S→EIIS

←

↑ ↑

E + S→ES→E + P

+II

↓

EIIS

←

↑

E

I

S

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Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect P

lots

Do

ub

le R

ecip

roca

l

Vmax Vmax

Km Km’ [S], mM

vo

[S], mM

vo

II II

Km [S], mM

Vmax

II

Km’

Vmax’Vmax’

Vmax unchangedKm increased

Vmax decreasedKm unchanged Both Vmax & Km decreased

II

1/[S]1/Km

1/vo

1/Vmax

II

Two parallellines

II

Intersect at X axis

1/vo

1/Vmax

1/[S]1/Km 1/[S]1/Km

1/Vmax

1/vo

Intersect at Y axis

= Km’

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Reaction of the irreversible inhibitor

diisopropylfluorophosphate (DFP) with a

serine protease

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Iodoacetate is an irreversible inhibitor of all cysteine

peptidases

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� Allosteric protein:-two or moretopological distinct bindingsites which interact functionallywith each other.

� Cooperatability:-modification ofthe binding constant of theprotein for a small molecule bythe prior binding of anothersmall molecule.• +ve:-binding ability or affinity

increases

• -ve:- binding ability or affinitydecreases

� Allosteric effectors(inhibitors &activators):- for speed up & tospeed down

when 2,3-BPG binds to an allosteric site

on hemoglobin, the affinity for oxygen of

all subunits decreases.

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Where n is cooperativity and n>1

,Indicates positive cooperativity.

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High substrate concentrations

may

cause inhibition in some

enzymatic

reactions, known as substrate

inhibition

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� Assumption :-A second substrate molecule

can bind to the enzyme when S binds the ES

complex, an unreactive intermediate results.

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� Unit of enzyme activity:

Used to measure total units of activity in a given

volume of solution.

� Specific activity:

Used to follow the increasing purity of an enzyme

through several procedural steps.

� Molecular activity:

Used to compare activities of different enzymes.

Also called the turn-over number (TON = kcat)

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� Unit of enzyme activity:

µµµµmol substrate transformed/min = unit

� Specific activity:

µµµµmol substrate/min-mg E = unit/mg E

� Molecular activity:

µµµµmol substrate/min- µµµµmol E = units/µµµµmol E

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�Temperature

�pH

�Concentration

�Activators

�Product concentration

�Time

�Radiations

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Temperature / oC

Collision rate of

enzymes and

substrates

Number of

enzymes

remaining

undenatured

Reaction rate

/ a

rbitra

ry u

nits

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Temperature / oC

Increasing kinetic

energy increases

successful collision

rate

Reaction rate

/ a

rbitra

ry u

nits

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Temperature / oC

Permanent disruption

of tertiary structure

leads to loss of active

site shape, loss of

binding efficiency and

activity

Reaction rate

/ a

rbitra

ry u

nits

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Temperature / oC

Optimum temperature

Reaction rate

/ a

rbitra

ry u

nits

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�The precise shape of an enzyme (andhence its active site) depends on thetertiary structure of the protein

�Tertiary structure is held together byweak bonds (including hydrogen bonds)between R-groups (or ‘side-chains’)

�Changing pH can cause these side chainsto ionise resulting in the loss of H-bonding…

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� Temperature: enzymes work best at an optimum temperature.

Below this, an increase in temperature provides more kinetic energy to the molecules involved. The numbers of collisions between enzyme and substrate will increase so the rate will too.

Above the optimum temperature, and the enzymes are denatured. Bonds holding the structure together will be broken and the active site loses its shape and will no longer work

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Even if temperature lowered – enzyme

can’t regain its correct shape

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pH

Reaction rate

/ a

rbitra

ry u

nits

Either side of the

optimum pH, the

gradual ionising of the

side-chains (R-groups)

results in loss of H-

bonding, 3D structure,

active site shape loss of

binding efficiency and

eventually enzyme

activity

Optimum pH

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pH

Reaction rate

/ a

rbitra

ry u

nits

This loss of activity is

only truly denaturation

at extreme pH since

between optimum and

these extremes, the loss

of activity is reversible

Optimum pH

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�pH: as with temperature, enzymes have an optimum pH. If the pH changes much from the optimum, the chemical nature of the amino acids can change.

This may result in a change in the bonds and so the tertiary structure may break down. The active site will be disrupted and the enzyme will be denatured.

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