Enzymes pdf - Dr.Muzhim Alkabban

ENZYMES

PROFESSOR

MUZHIM

ALKABBAN

• Enzymes are protein catalysts,

increase the velocity of chemical

reactions. They are not consumed

during the catalytic reactions.

• Nomenclature:
• Two types of names are given for

each enzyme.

• 1. Recommended name which is short.

• 2. Systematic name which is more

complete.

• Recommended name:

Most commonly used, have the suffix [-

ase], attached to the substrate of the

reaction. Ex: Glucosidase; Urease;

Sucrase.

The suffix may attached to the

description of the action performed.

• Ex: Lactate dehydrogenase; adenylate

cyclase.

• Some enzymes retain their original names

which has no hint of the associated

enzyme reaction.

• Ex: Trypsin; pepsin.
• Systematic name:
• The enzymes are divided to six major

classes in this system, each with

numerous subgroups.

• The suffix [-ase] in this system is attached to

the complete description of the chemical

reaction catalyzed. Ex: D-glyceraldehyde 3-

phosphte: NAD oxidoreductase.

•

1. Oxidoreductase:

Catalyze oxidation-reduction reaction. These

are enzymes that catalyze reactions in which one

substrate is oxidized, acting as a proton donor,

and another substrate is reduced, acting as a

proton acceptor.

• Ex: Dehydrogenase, which convert single

bond to double bond.

• Oxidase, which use O

as oxidant.

• Peroxidase, which use H

as oxidant.

• Hydroxylase, which introduce hydroxyl

groups.

• Oxygenases, which introduce molecular

oxygen in place of a double bound in the

substrate.

• Lctate + NAD

←--------→ Pyruvate + NADH + H

-CH

OH-COO

+ NAD

↔ CH

-C

O-COO

+NADH+H

•

2. Transferases:

Enzymes that catalyze transfer of C, N or P

containing groups. Ex: Transfer of one carbon group

(methyl group), aldehydic or ketotic groups or

phosphoryl groups from one substrate to another.

UDP-galactose + glucose

←----------------→ UDP +

galactosylglucose (lactose)

Enzyme: UDP galactose-glucose galactosyl

transferase enzyme.

• Serine + Tetrahydrofolate

←------→ Glycine + Methyl

tetrahydrofolate

CH2-OH-CH-NH3+-COO

+THF

↔CH

-NH3+-COO

THFCH

• Enzyme: serine hydroxymethyl transferase.

•

3. Hydrolases:

• Enzymes that catalyze cleavage of bonds by

addition of water as C-O, C-N, P-O, C-C and other

single bonds.

• Ex: Esterase: R-C=O-R + H

←---→ R-C=O-OH + R-

• Glycosidases and phosphatases:

• Urea + H

O -------

urease

--------

→ CO

+ 2NH

• NH

-C=O-NH

+ H

O ----

urease

----

→ CO

+ 2NH

•

4. Lyases:

• Enzymes that catalyze cleavage bonds as C-C C-S

and certain C-N bonds without the addition of

water, but by elimination reactions to form double

bonds or rings.

• Ex: Decarboxylases as aldolase:

• Fructose 1,6-diphosphate

←------

Aldolase

----

→

Dihydroxyacetonephosphate + glceraldehyde-3-

phosphate.

Pyruvate-

-pyruvate

decarboxylase

→Acetaldehyde+CO

-C=O-COO

---

pyruvate decarboxylase

→CH

C=O

H+

•

5. Isomerases:

• Enzymes that catalyze the rearrangement of the

atoms of a molecule (catalyze racemization of

geometric isomers).

• Dihydroxyacetone phosphate

←

triose phosphate

isomerase

→ Glyceraldehyde 3-phosphate.

•

6. Ligases:

• Enymes that catalyze the joining of two

compounds so, catalyze formation of bonds

between carbon and O,S,N coupled to hydrolysis of

high-energy phosphatases.

• Also called or known as synthetase. They

couple the hydrolysis of a pyrophosphate in

ATP or other nucleoside triphosphate to a

second reaction in which two molecules are

joined.

• ATP + acetate + CoA ---

→ AMP + Ppi + acetyl

CoA

Enzyme: acetate CoA Ligase
Pyruvate + CO

+ ATP---

pyruvate carboxylase-

→ Oxaloacetate + ADP + Pi

-C=O-COO

+ CO

+ ATP--

pyruvate

carboxylase-

→ HOOC-CH

-C

-COO

+ ADP +

•

Properties of enzymes:

•

1. Active sites:

• Active site is a special pocket or cleft present on

enzyme molecules. These active sites contain

amino acid side chains which create a three-

dimensional surface complementary site binds to

the substrate.

• The active site binds the substrate forming

enzyme-substrate (ES) complex. This ES is

converted to enzyme-product (EP) which

dissociated to enzyme and product.

•

2. Catalytic efficiency:

• Most enzymes of high efficient in catalytic

activity reaching from 10

to 10

faster than

un catalyzed reactions. Each enzyme

molecule is capable of transforming 100 to

1000 substrate molecules into product each

second. So,

turnover number

is the number of

molecules of substrate converted to product

per enzyme molecule per second.

•

3. Specificity:

• Enzymes are high specific by interaction with one

or few specific substrates and catalyzing one type

of chemical reaction.

•

4. Cofactors:

• Some enzymes needed a non protein cofactors for

their enzymatic activity. These cofactors include

metal ions as Zn

, Fe

and organic molecules,

known as coenzymes, that are often vitamins

derivatives as NAD

, FAD and coenzyme A.

• Holoenzyme: enzyme with its cofactor.

• Apoenzyme: protein portion of the holoenzyme.

No biological activity of apoenzyme in case of

absence of appropriate cofactors.

• Prosthetic group: it is tightly bound coenzyme that

does not dissociate from the enzyme, ex. Biotin of

carboxylases.

• Enzyme activity can be regulated as activated or

inhibited, so, the rate of product formation

responds to the needs.

• Many enzymes are localized in

specific organelles within the cell.

This will lead to isolate the reaction

substrate or product from other

competing reactions, providing good

environment for reaction and to

regulate the thousands of enzymes in

the cells into their purpose pathways.

•

How enzyme work:

• All enzymes are proteins, but not all proteins

are enzymes. They are a biological catalysts,

mediate the synthesis of biological

compounds and catalyze reactions that

supply the cells with energy.

• Therefore, enzymes are responsible for all

the chemical reactions in the cells at which

covalent bonds are formed or broken.

• Enzymes act by lowering the activation

energy of the reaction they catalyze.

•

The action of enzymes have two faces:

• 1.Catalysis in terms of energy changes

which occur during the reaction. Enzymes

provide an alternate energetically

favorable reaction pathway different from

un catalyzed reaction.

• 2. The property of the active site

chemically to facilitate catalytic reaction

of the enzyme.

•

Energy changes during the reaction:

• For all the chemical reactions their will be energy

barrier separating the reactants and the products,

this is called

free energy of activation

. It is the

energy difference between the energy of the

reactants and the high energy intermediate that

occurs during the formation of products.

• A (substrate)

↔ T* ↔ B (product)

• E + S ---

→ ES ---→ EP ---→ P

• The changes in the energy during conversion of a

molecule of reactant A producing B through

transition state T*

•

Effect of enzyme on the activation

energy of reaction:

•

1. Free energy of activation:

• The peak energy represent the transition

state in which a high energy intermediate

is formed during the conversion of

reactant A to product B. Due to the large

activation energy, the rate of un catalyzed

chemical reaction are often slow.

•

2. Rate of reaction:

• The molecules to be react, they must have

sufficient energy to overcome the energy

barrier of the transition state. So, in the

absence of an enzyme, only a small

amount of molecules between the

reactant and product may have enough

energy to reach the transition state

• The rate reaction is decided by the number of

energized molecules. Generally, the lower the free

energy of activation, the more molecules have

sufficient energy to pass over the transition state,

and thus, the faster the rate of the reaction.

•

3. Alternate reaction pathway:

• Enzyme allows to proceed reaction rapidly in the

cell by alternate reaction pathway with a lower free

energy of activation. The enzyme does not change

the free energy of reactants or products and does

not change the equilibrium of reaction.

• Chemistry of the active site:

• Active site is a complex molecular machine posses

a diversity of chemical mechanisms to facilitate

the conversion of substrate to product.

•

Factors responsible for the catalytic efficiency of

enzymes:

•

1. Transition state stabilization:

• The active site acts as a flexible molecular

template that binds the substrate in a geometry

structurally resembling the activated transition

state of the molecule (T*).

• By stabilizing the substrate in its transition

state, the enzyme greatly increases the

concentration of the active intermediate

which converted to product, so accelerates

the reaction.

•

2. The active site provide catalytic groups

which enhance the formation of transition

state:

Some time active site involve in general

acid-base catalysis when the amino acid

residues provide or accept protons.

•

3. Visualization of transition state:

• Enzymes catalyzed conversion of

substrates to products. This spend high

energy of activation, first enzyme come in

contact with the substrate (ES), then

guiding the reaction analogous to the ES

transition state. The ES present at lower

energy than unbound substrate(S).

• Enzyme represent a parent who need to

remove sweater from his or her infant who is

unable to do that. The parent come in

contact with the infant as (ES), guiding the

infant’s arms into extended in vertical

position, which facilitates the removal of the

sweater, forming the disrobed baby, which

represent product. This will lead to spend

energy, so, substrate bound enzyme have

slightly low energy than unbound substrate

(S).

•

Factors affecting reaction velocity:

• Different enzymes shows different response

to changes in substrate concentration;

temperature and pH.

•

1. Substrate concentration:

•

The rate or velocity of a reaction (V)

is the

number of substrate molecules converted to

product per unit of time.

• It is expressed as µmoles product formed

per minute. The rate of enzyme reaction

increases with substrate concentration

until reach a maximal velocity (V

max

• The reaction velocity off at high substrate

concentration, and this reflects the

saturation with substrate of all available

binding sites on the enzyme.

• Most enzymes shows hyperbolic shape

similar to oxygen dissociation curve of

myoglobin when draw reaction velocity

against substrate concentration. While

allosteric enzymes shows sigmoid curve

similar in shape to the oxygen

dissociation curve of hemoglobin.

•

2. Temperature:

• Reaction velocity increases with temperature

until a peak of velocity is increased. This

increase occur due to increasing number of

molecules which have sufficient energy to pass

the energy barrier forming the product. More

increasing in temperature result in a decrease

in reaction velocity, as more temperature will

reduce denaturation of the enzyme.

•

3. pH:

• Effect of pH on ionization of active site:
• Hydrogen ion concentration [H

] affects reaction

velocity in several ways, as the catalytic activity of

an enzyme requires the enzyme and substrate

should have specific ionized or unionized chemical

groups inorder to interact; ex: The catalytic activity

require the amino group of the enzyme in a

protonated form (-NH

). At alkaline pH this group

is deprotonated, so, the rate of reaction therefore

declines.

• Extremes of pH will lead to denaturation of

the enzyme due to the structure of the

catalytic active protein molecule depends on

the amino acid character of the side chains.

The maximal enzyme activity reached at

different pH and differ at different enzymes

and this is reflect the [H

] at which the

enzyme function in the body, ex: pepsin, the

digestive enzyme in the stomach maximally

active at pH 2. Some designed to work at

neutral pH which denaturized at high acidic

pH.

•

Enzyme unit:

• The amount of an enzyme that will catalyze the

transformation of a specified amount of substrate

into product under defined conditions.

• International unit (I.U) of enzyme corresponds to

the amount of enzyme that causes the loss of 1

µmole of substrate/ minute under specified

conditions, usually a saturating condition of

substrate.

• Katal (kat): The amount of enzyme that cause the

loss of one mole of substrate/ second Under

specified condition.

•

Enzyme Inhibitors:

• A great variety of naturally occurring and

synthetic compounds have the ability to

bind reversibly or irreversibly to specific

enzymes and alter their activity.

• Enzyme inhibitors reduce or eliminate the

catalytic activity of the enzymes.

• Ex: Drugs, Antibiotics, Toxins and

Antimetabolites.

•

Two general classes of inhibitors:

• 1.

Irreversible inhibitors:

• Form covalent bonds with specific functional

groups, amino acid side chain.

• Can not be released by dilution or dialysis, its

effects can not be reversed simply by increasing

the [S].

• Occurs when the inhibited enzyme does not regain

activity upon dilution of the enzyme- inhibitor

complex. Ex: The neurotoxic effects of certain

insecticides are due to their irreversible binding at

the catalytic site of the enzyme acetylcholine

esterase.

• 2.

Reversible inhibitors (RI):

• Binding to enzymes through non covalent

bonds.

• Dilution of the enzyme inhibitor complex

result in dissociation of the reversibly-

bound inhibitor and recovery of enzyme

activity.

• Have only a transient association with the

enzyme.

•

Two important types of inhibition:

•

1.Competitive inhibition (CI):

• In CI compounds that may or may not be

structurally related to the natural substrate

combine reversibly with the enzyme at or

near the active site.

• Both the substrate and the inhibitor

compete for the same site which leads to

a series of reactions.

• E + S

↔ ES → E + P

I
k

↕k

(inactive)

= k

is dissociation constant for

the EI complex).

• In CI, ES and EI complexes are formed but EIS

complexes never produced.

• In conclusion, the high concentrations of substrates

will overcome the inhibition by shifting the reaction to

the right.

COOH COOH

ɪ ɪ

CH2 2H CH

←--↗--→ ɪ

CH2 HC

ɪ ɪ

COOH COOH

Succinic acid Fumaric acid

Malonic acid is a (CI) of the enzyme

succinic dehydrogenas

COOH
ɪ

CH2
ɪ

COOH

• Ex. of (CI), succinate dehydrogenase enzyme

catalyzes the oxidation of succinate to

fumarate. Malonate is structurally similar to

the substrate (succinate) and competes for

binding at the active site of the enzyme.

Increasing succinate concentration, the

probability that the active site is occupied by

the substrate molecules rather than an

inhibitor.

•

2. Non competitive inhibition (NCI):

• Compounds that are reversibly bind with

either the enzyme or the enzyme substrate

complex are designated as NCI.

• E + S

↔ ES → E + P

+ +

I I

↕ ↕

↔ ESI

↘ (inactive)↙

• Ex: Reagent that can combine reversibly

with the –SH groups of cystein residues

that are essential for the catalytic activity

of some enzymes. ex. (Cu

, Hg

and

• E-SH + Ag

↔ E-S-Ag + H

• This type of inhibition is not completely

reversed by high [S], since the cyclic

sequence of the above equation will occur

regardless of the [S].

• NCI occurs when the inhibitor and

substrate bind at different site on the

enzyme.

• NCI can bind either free enzyme or ES

complex so prevent occurring of reaction.

• NCI can not be overcome by increasing

the substrate concentration.

• NCI does not interfere with the binding of

substrate to enzyme.

• Many drugs act as inhibitors of enzyme, Ex. Many

antibiotics as penicillin and amoxicillin, act as

inhibiting one or more of the enzymes of bacterial

cell wall synthesis.

• Drugs may also act by inhibiting extracellular

reactions as angiotensin converting enzyme (ACE)

inhibitors, which lower blood pressure by blocking

the enzyme that cleaves angiotensin 1 to form the

potent vasoconstrictor angiotensin 11. These

drugs as captopril, enalopril and lisinopril cause

vasodilation and reduce blood pressure.

•

Uncompetitive inhibition (UCI):

• Compounds that reversibly combine with only

ES complex, but not the free enzyme are

called (UCI). The inhibition is not overcome

by high substrate concentration, Ex:

• Succinate + CoA + GT

↔ Succinyl CoA +

GDP + Pi

• Enzyme: Succinyl CoA synthetase.
• Pi is the UCI

•

Enzyme theories proposed to explain

the specificity of enzyme action:

•

1. Lock and key theory:

• The active site of the enzyme is

complementary in conformation to the

substrate, so that enzyme and substrate

recognize one another.

•

2. Induced-fit theory:

• The enzyme changes its shape upon

binding the substrate, so, the

conformation of substrate are only

complementary after the binding reaction.