EnzymesO -

C

Hi, Everybody!Hi, Everybody!

Intended learning outcomes(ILO)

Intended learning outcomes(ILO)

1. Compare reversible competitive to non competitive enzyme inhibitors and others acting as irreversible enzyme inhibitors.

2. List some examples of drugs acting by competitive enzyme inhibition and others acting as irreversible enzyme inhibitors.

Objectives Objectives Enzymes as Biological Catalysts

The Properties of

Enzymes

Enzyme classification

Enzyme Kinetics

Enzyme Inhibition

Regulation of enzyme activity.

Applications of Enzyme

Action

Enzymes Inhibitors(reduce the reaction rate via a non substrate

molecule)

Enzymes inhibitors

Enzyme inhibitors

Reversible

Competitive Non competitive

Irreversible

Non competitive

Reversible inhibitors

• They bind to enzymes non covalent bonds.• Characterized by rapid dissociation of the

enzyme inhibitor complex.

Enzyme inhibitors

Competitive

• Compete with the substrate for the active site.

Non competitive

• Bind to the enzyme at sites other tan the active site.

Competitive inhibitors

No product is formed

The inhibitor binds to E & forms an [EI] complex at the active site.

Competes with the substrate for the active site of the enzyme.

The Inhibitor is structurally similar to the substrate .

Competitive

Competitive inhibitorsInhibition can be

overcome if [S] is very high i.e. [S] is more than

[I]

No effect on Vm as excess substrate will displace the inhibitor and the enzyme will

work at its

maximum rate

Competitive inhibitors

Increased Km because it takes more substrate to half

saturate the enzyme.

For Lineweaver-Burk plots, Y intercept is the same regardless of whether the inhibitor is

present or absent, but the slope differ between the two lines(different Km)

ExamplesClassical example is malonic acid inhibition of (succinate dehydrogenase enzyme) of Kreb’s cycle.

Examples and clinical use

DRUG SIMILAR TO(inhibit) Uses

sulfonamides Para-amino benzoic acid

Antibacterial drug

Dicumarol Vitamin K Anticoagulant

Examples and clinical use

DRUG SIMILAR TO(inhibit) Uses

Methotrexate Folic acid

-( Dihydrofolate (reductase

Anticancer

Ethanol MethanolAlcohol

dehydrogenase

Methanol toxicity

Statin Inhibit HMG-COA reductase

Inhibit cholesterol

Folic acid cannot be synthesized which is essential for bacterial growth.

Methorexate (Anticancer drug)Structural

analogue of folic acid

Inhibit dihydrofolate

reductase

Inhibit DNA ,RNA synthesis

One problem of this drug is that affect other rapidly dividing cells as bone marrow and mucosal cells causing severe anemia and mucosal ulceration.

Cancer cells which are rapidly dividing are very sensitive to treatment by methotrexate(antitumor

drug)

Methorexate (Anticancer drug)

Ethanol

Converted into acetaldehyde which is

less toxic

Ethanol injected intravenously to treat methanol toxicity to prevent damaging

effects of formaldehyde

Methanol

Converted into formaldehyde which is

toxic

Causes tissue damage and blindness

Competitive inhibitors

NON-COMPETITIVE  INHIBITION...

Inhibitor binds to the E, forms an [EI] complex not at the active site(Not affect the affinity of the enzyme to substrate) .

Inhibitor often have no structural

similarity to substrate

Inhibition NOT reversed by increasing [S].

Inhibitor can bind the enzyme before or after substrate binding

E+S

E+I

Km not changed ,Vm decreased

Reversible Non competitive

inhibitorsInhibition can be reversed by dialysis of the

inhibited enzyme.

For Lineweaver –Burk plot, lines for inhibited reaction converge on the X

axis with those for the uninhibited

reaction

Reverse non

competitive inhibition is

rare.

IRREVERSIBLE INHIBITOR (Enzyme poison)

Inhibitor combines or destroy

a functional group on

the enzyme that is

essential for the activity

The binding

is so tight

usually covalent that they are not

dissociatedE+I

The kinetics of irreversible inhibitors are similar

to reversible

non competitiv

e inhibitors(Decrease

Vmax)

IRREVERSIBLE INHIBITOR

Km is Same & Vmax are Lowered

It resembles enzyme Kinetics of non competitive inhibitors

No competition between substrate and inhibitor because the inhibitor has no structural resemblance to the substrate.

Increase of substrate not relieve the inhibition.

Examples of irreversible non competitive inhibitors

1-Alkylating agents like iodoacetamide (bind to -

SH’s).

2-heavy metals (silver & mercury) bind to -SH’s.

3-cyanide inhibit mitochondrial

cytochrome oxidase.

4-Fluoride inhibit enolase enzyme

5- Penicillin is an antibiotic , inhibits

bacterial transpeptidase.

6-Nerve gas and organo- phosphorus on cholinesterase.

7-Aspirin as anti-platelet aggregator on cyclo-

oxygenaseInhibit prostaglandins

and thromboxane synthesis.

Antimetabolites • Block one or more of the metabolic

pathway involved in DNA synthesis

• Used in treatment of cancer.

Organophosphorus Compounds(diisopropylfluorophosphate=

DIPF)

Form covalent bond with a serine residue in the active site of choline esterase

Cause neurotoxicity

When choline esterase is inactivated, acetyl choline, the neuromuscular

transmitter, persists and this leads to muscle paralysis and death.

Poison nerve gas

Suicide inhibitors

• A special class of irreversible inhibitorInhibitor is structural

analogue to the substrate on which the enzyme act giving

product

The products bind irreversibley with the enzyme

The product inactivate the enzyme

Hypoxanthine xathine oxidase Xanthine

xathine oxidase

Uric acid

-

Allopurinol(suiccide subtrate):

used in treatment of GOUT that result from increased uric acid level in blood

N

N

Allopurinol as suicide enzyme inhibitor

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Reversible Non-competitive Irreversible Non-competitive

EE

Different siteCompete for active siteInhibitor

Substrate

Car

toon

Gui

deEq

uatio

n an

d De

scrip

tion

[I ]binds to free ]E[ only,and competes with ]S[;

increasing ]S[ overcomesInhibition by ]I[ .

[I ]binds to free ]E[ or ]ES[ complex; Increasing ]S[ can

not overcome ]I[ inhibition.

[I ]binds to ]E[ tightly only, destroy functional

group increasing ]S[ favorsthe inhibition by ]I[.

E + S → ES → E + P+

I↓

EI

←

↑

E + S → ES → E + P+ +

I I↓ ↓

EI + S →EIS

←

↑ ↑

E + S → ES → E + P+

I↓

EIS

←

↑

EI

S X

Km

Enzyme Inhibition (Plots)

I II Competitive

D

irect

Plo

tsD

oubl

e R

ecip

roca

l

Vmax Vmax

Km Km’ [S ,]mM

vo

[S ,]mM

vo

I IVmax’

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Vmax decreased

I

1[/S]1/Km

1/vo

1/ Vmax

I

Intersect at X axis

1/vo

1/ Vmax

1[/S]1/Km

Intersect at Y axis

= Km’

Reversible Non-competitive Irreversible Non-competitive