EnzymesTrypsin

Enzymes• Enzymes are biological catalysts.• Essential for almost all of the chemical reactions that

maintain life. [Essential for biological reactions and hence life]

• They are macromolecules, generally protein in nature .• Enzymes are found in all tissues and fluids of the body.• The substance on which the enzymes act are known as

Substrates. • Enzymes bind substrates at the active site and change

it to product.

Enzyme Catalysed Reactions- During enzyme action, there is a temporary combination between enzyme and its substrate forming enzyme by relatively weak forces.

- This occur at the active site of the enzyme (most substrates are bound to the enzyme by relatively weak forces (hydrogen bonds, hydrophobic, ionic and van der Wals bonds)

E + S ES complex

This is followed by dissociation of this complex into enzyme and product.

ES E + P

Active site of enzymes

E + S ES E+ P

Enzyme Reaction

Active Site

ES complexES complex

+ Substrate

+

Product

• Enzymes increase the rate [speed] of reactions

• Themselves do not changes in the reaction.

• Specific for the substrate and type of reaction catalysed.

• Any change in a single enzyme can have very harmful effects. e.g. albinism, glycogen storage disease etc.

Cofactors• Some enzymes require a non-protein part for their activity.

This is known as a cofactor.• Cofactor. Cofactor may be metal ion or an organic molecule

called Coenzyme. Some enzymes require both:Apoenzyme + Cofactor Haloenzyme(Catalytically inactive) (active

enzyme)i) Metal as cofactor

Alcohol dehydrogenase - Zn ++

Kinases (phosphotransferase) - Mg++

Cytochromes - Fe++ or Fe+++

Cytochrome oxidase - Cu++

ii) Co-enzymes• Organic molecules, heat stable

• Derived from water soluble vitamins.

• Usually function as intermediate carriers of functional groups of specific molecules

Pyridoxal phosphate - Amino transferase

NAD+ , NADP - in H+ transfer

FAD, FMN - “ “

COQ - “ “

Coenzyme A - Acyl group transfer

Biotin - Addition of CO2

Thiamine pyrophosphate - Removal of CO2

• If the cofactor or coenzyme is tightly bond to the enzyme molecule, it is called a prosthetic group

Apoenzyme + Coenzyme Halo Enzyme

• Enzymes are highly specific both• Very important characteristic of all enzymes.• Enzymes are specific:

• for the reactions they catalyze.• and in their course of reaction, which are called

substrates.(a) Absolute specificity:

The enzyme can act only on one specific substrate.

GlucokinaseGlucose Glucose 6 -

ATP ADP

P

Enzyme Specificity

Absolute Specificity

GlucokinaseNo Reaction

(b) Group specificity:

• Broad specificity• Enzyme act on a group of related substrates.• The substrates have a common group on

which the enzyme acts:e.g. - esterase can act on

different esters - proteases can act on

different protein[proteases: chymotrypsin, trypsin, pepsin, act on

proteins to give amino acids]

HexokinaseHexoses Hexose - 6 -

ATP ADP

P

- The specificity is due to substrate binding site (active site) which lies on the enzyme surface.

- The specificy is due to the specific arrangement of a.a. in the active site that participate in the bond making and bond breaking (These residues are called catalytic groups).

Models to explain substrate specificity of enzymes:

i. Lock and Key Model

ii. Induce Fit Model

i. Lock and Key Model:

The active site of the unbound enzyme is complementary in shape to the substrate.

(The enzyme active site is rigid and fixed → does not change)

ii. Induce Fit Model:

The active site of the enzyme has no fixed shape.The active site of the enzyme changes shape on binding with the substrate . The active site forms a shape complementary to the substrate only after the substrate has been bound.

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Nomenclature and Enzyme Classification

- Many enzymes have been named by adding the suffix “ase” to the name of the substrate or to a word describing the action or activity: e.g.protease, sucrase, lipase, amylase, dehydrogenase, oxidase, carboxylase etc

- Some enzymes were given trivial names: e.g. trypsin, chymotrypsin, pepsin, thrombin etc.

Classification of enzymes [on the basis of the nature of reaction catalysed]

This system divide enzymes into six major classes:(1) Oxido-reductases- catalyse oxidation

and reduction reactions(2) Transferases- transfer one group from

donor to acceptor(3) Hydrolases- break bonds by adding

water(4) Lyases- break bond but do not use

water(5) Isomerases- change one isomer to

another(6) Ligases- bring about formation of a

bond (biosynthetic reactions)

Number Classification Biochemical Properties

1.Oxidoreduct

asesAct on many chemical groupings to add or remove hydrogen atoms or electrons [oxidation-reduction ]

2. Transferases

Transfer functional groups between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules.

3. HydrolasesCleavage of bond between C and other group by addition of water. [ hydrolyzing it].

4. LyasesNonhydrolytic cleavage of C-C, C-S, C-N bond. Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds.

5. Isomerases

Convert on isomer to another, by transfering a group from one position to another within the same molecule: L to D isomerizations, mutase reactions (shifts of chemical groups) and others.

6. LigasesCatalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP. Formation of C-C, C-S, C-N bonds etc

How?

Enzymes increase the rate of reaction

How do enzymes increase the rate reaction

How do enzymes increase the rate of reaction?

• Enzymes increase reaction rates by decreasing the amount of energy required to form a complex of reactants that is competent to produce reaction products. This complex is known as the activated state or transition state complex for the reaction.

• Enzymes and other catalysts accelerate reactions by lowering the energy of the transition state.

How do enzymes increase the rate of a reaction? Enzymes increase rate of reaction by decreasing energy of activation (∆G#)

E + S ES EP E + P

Such a reaction S P can be given by the above figure. • This is picture of the energetic course of the reaction.• The free energy of the system is plotted against the progress of the

reaction.• In its normal stable form or (ground state), any molecule (such as S or

P) contains characteristic amount of free energy.• Normal stable form of S or P contains a characteristic amount of free

energy.

Reaction rates can be increase by:• ↑ Temperature• ↓ activation energy

(1) Raising the temp.resulting in → increasing the number of molecules

with sufficient energy to overcome this energy barrier.

(2) Catalysts (enzymes) → 10- 10+15 times faster than the same uncatalysed.

- Enzymes speed up the rate of which a reaction approaches equilibrium.

- Enzymes increase rate of reaction by decreasing energy of activation (∆G#).

A Chemical Reaction Progress Curve

A chemical reaction progress curve:

velocity

• Enzymes affect the rate of chemical reaction.• It is expressed in terms of change in concentration of the

[S] or [P] per unit time.*Rate and velocity are the same.

0.1

0.2

0.3

0.4

0.5

0.6

0.7

10 20 30 40 50

Time (min.)

Linear region of the curve

It is defined as the number of moles of substrate transformed into product per second at 25°C.

Enzyme activity

.

Turnover number:It is defined as the number of moles of substrate transformed per minute per mole of enzyme

This tells how many S molecules are converted to product by each enzyme molecule.

It tells us how fast an enzyme work or turnovers S into P.

e.g. for catalase:

turnover number is 5 x 106

for α-amylase → it is 1.9 x 104

This indicates that catalase is ~ 250 times more active than amylase.

Turnover number

Factors affecting the rate of enzyme catalysed reactions

• Enzyme concentration• Substrate concentration• Temperature• pH• Activators• Inhibitors

Activation Energy:Activation Energy:

Definition of Active energy:Definition of Active energy:

It is the amount of energy require to rais all the molecules in one mole of It is the amount of energy require to rais all the molecules in one mole of a substrate to the transition state .The presence of enzymes lower the a substrate to the transition state .The presence of enzymes lower the activation energy.activation energy.

Factors affecting the rate of enzyme reaction:Factors affecting the rate of enzyme reaction:

(kinetic properties of enzyme)(kinetic properties of enzyme)

1\Temperature:1\Temperature:

Temp. increase rate of E reaction untill a certain temp. reach the E Temp. increase rate of E reaction untill a certain temp. reach the E acts maximally (optimum temp. and ranges from 40-60 c° denatured.acts maximally (optimum temp. and ranges from 40-60 c° denatured.

2\ PH:2\ PH:

Strong acid & base denature the enzyme. There is an optimum PH at Strong acid & base denature the enzyme. There is an optimum PH at

which enzyme act maximally.which enzyme act maximally.

3\Concentration of substrate:3\Concentration of substrate:

The rate of enzyme reaction is increased with increase substrate conc. The rate of enzyme reaction is increased with increase substrate conc.

Untill certain point when the active sites of enzyme was saturated by Untill certain point when the active sites of enzyme was saturated by

substrate.substrate.

Substrate inducation:Substrate inducation:

Addition of substrate increase activity & formation of enzyme Addition of substrate increase activity & formation of enzyme

(substrate inducation). It is example of positive feed back.(substrate inducation). It is example of positive feed back.

Michaelis constant k m value:Michaelis constant k m value:

The substrate conc. That produce half maximum velocity is termed k m The substrate conc. That produce half maximum velocity is termed k m

value or Michaelis constant . k m indicate the amount of substrate to be value or Michaelis constant . k m indicate the amount of substrate to be

used.used.

Km is constant for every enzyme.Km is constant for every enzyme. substrate present in the physiological fluid in amount=Kmsubstrate present in the physiological fluid in amount=Km The smaller Km value the more active the enzyme.The smaller Km value the more active the enzyme. Small Km reflect high effenity of E for substrate so,decrease conc. Of Small Km reflect high effenity of E for substrate so,decrease conc. Of

substrate(is needed to half saturate the enzyme).substrate(is needed to half saturate the enzyme).

4\ Concentration of enzyme:4\ Concentration of enzyme:

The rate of E reaction is directly proportional to the increase in enzyme The rate of E reaction is directly proportional to the increase in enzyme

conc.conc.

5\Effect of end product or feed back inhibition:5\Effect of end product or feed back inhibition:

The activity of same E is effected by low M.w. allosteric effectors The activity of same E is effected by low M.w. allosteric effectors

which have little or similarity to substrate or Co enzyme. So feed back which have little or similarity to substrate or Co enzyme. So feed back

inhibition inhibit enzyme .inhibition inhibit enzyme .

A enzyme 1 A enzyme 1 B enz.2 B enz.2 C enz.3 D C enz.3 D

D can inhibit enzyme (negative allosteric effector inhibitor)D can inhibit enzyme (negative allosteric effector inhibitor)

Effect of Substrate on Enzyme Catalysed Reaction

Km

v/2

Velocity

Michaelis-Menten Kinetics

• E + S <---> ES <---> ES* <---> EP <---> E + P • The Michaelis-Menten equation:

• Lineweaver-Burk equation:

Km of enzymes in physiological systems

Lineweaver and Burk Plot

Allosteric Enzymes

• Control [regulatory] enzymes• Have quaternary structure• Have active site and modulatory site

– Active site binds substrate to give product– Modulatory site binds +ve or – ve modulator to increase or

decrease the activity of the active site

• Catalyse an irreversible reaction• Inhibited by end product

– A B C D E F G H

• Activated by substrate and other positive modulators• Do not obey Michaelis Menten Kinetics

Feedback inhibition

Positive and negative modulator

Activation of PFK by ADP

Structure of PFK

Activation of Pyruvate dehydrogenase

Enzyme Inhibitors

Substances that decrease the activity of enzymes

Enzyme inhibitors

ReversibleIrreversible

E+ I EI E+I EI

UncompetitiveNoncompetitiveCompetitive

Inhibitor Type

Binding Site on Enzyme Kinetic effect

Competitive Inhibitor

Specifically binds at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

Vmax is unchanged; Km, as defined

by [S] required for 1/2 maximal activity, is increased.

Noncompetitive Inhibitor

Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

Km appears unaltered; Vmax is

decreased proportionately to inhibitor concentration.

Uncompetitive Inhibitor

Binds only to ES complexes at locations other than the catalytic site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

Apparent Vmax decreased; Km, as

defined by [S] required for 1/2 maximal activity, is decreased.

Un-competitive

Non-competitiveCompetitive

Competitive

Effect of Inhibitors on Michaelis Menten curve

Competitive Inhibitor

Non-Competitive Inhibitor

Enzymes in the Diagnosis

Serum Enzymes Used in Clinical DiagnosisSerum Enzymes Used in Clinical Diagnosis

Enzymes Major Diagnostic Use

Acid phosphatase Prostate cancer

Alkaline phosphatase Liver and bone disease

Amylase Acute pancreatitis

Aspartate aminotransferase Liver and heart disease

Alanine aminotransferase Viral hepatitis

Creatinine kinase Muscle disorders and

myocardial infarction

Lactate dehydrogenase Myocardial infarction

Lipase Acute pancreatitis

Serum Enzymes in DiseaseSerum Enzymes in Disease

• Acid phosphatase: a tumour marker in prostatic carcinoma.• Alanine aminotransferase (ALT): an indicator of hepatocellular

damage.• Alkaline phosphatase: increase in cholestatic liver disease and

is a marker of osteoblast activity in bone disease.• Amylase: an indicator of cell damage in acute pancreatitis.• Aspartate amino transferase (AST): an indicator of

hepatocellular damage, or as a marker of muscle damage, such as a myocardial infarction (MI).

• Creatine kinase: a marker of muscle damage and acute MI. -glutamyl transpeptidase: a sensitive marker of liver cell

damage.• Lactate dehydrogenase: a marker of muscle damage.

ISOENZYMES

• Multiple forms of the same enzyme.

• Catalyse the same reaction. Act on the same S and give the same P.

• Differ in molecular weight or structure or change. Can be separated by electrophoresis.

• Have different Km for the same S.

• Important in diagnosis of disease.

e.g. Creatine Phospho Kinase (CPK)

Creatine + ATP Creatine Phosphate + ADPCPK

• Has 3 isoenzyme. Each isoenzyme has 2 subunits (polypeptides)

• Two types of polypeptides: M & B.

• Isoenzymes: MB : Mainly in Heart

BB : Mainly in Brain

MM : Mainly in Muscles

Lactate dehydrogenase

• LDH occurs in 5 closely related, but slightly different forms (isozymes)

• LDH 1 - Found in heart and red-blood cells

• LDH 2 - Found in heart and red-blood cells

• LDH 3 - Found in a variety of organs • LDH 4 - Found in a variety of organs • LDH 5 - Found in liver and skeletal

muscle

Lactate Dehydrogenase (LDH)

Lactate + NAD+ Pyruvate + NADH +H+

• It is a tetramer. (4 subunits)• Composed of 2 types of polypeptide chains (M & H).• Has 5 isoenzymes, due to different combination of M & H chains.

M4 ………………………… LDH5 In skeletal muscles and liver

M3H ……………………… LDH4 In many tissues

M2H2 …………………….. LDH3 In lungs

MH3 ……………………… LDH2 In Heart

H4 ……………………….. LDH1 In Heart muscles

• These forms have different charge and can be separated on electrophoresis.1 3 4 52+ -

• Useful in differential diagnosis.

e.g. LDH 1 and 2 …………… Myocardial infarction (MI)

Normally LDH1 = 30% : 3 : 1 LDH2 10%

In MI LDH1 : 1 : 1 Due to in LDH2LDH2

LDH2 > LDH1

LDH5; (No change LDH2) – Liver disease (Viral Hepatitis)

LDH5 & LDH2 – Infectious mononucleosis

LDH3 – In lung disease (pumonary infection)

CPK isoenzymes

• Exists as Three isoenzymes: MM, MB, BB• CK-1 (BB) is the characteristic isozyme in brain and is in

significant amounts in smooth muscle. • CK-3 (MM) is the predominant isozyme in muscle. • CK-2(MB) accounts for about 35% of the CK activity in

cardiac muscle, but less than 5% in skeletal muscle. • Since most of the released CK after a myocardial

infarction is MM, an increased RATIO of CK-MB to total CK may help in diagnosis of an acute MI, but an increase of total CK in itself may not.

BB MB MM

MMMMBB BB MBMB

24 48 hr.

NormalHeart Diseases Brain Damage

MI

CPK: in serum in Heart diseases, brain disease and muscle disease.

CPK isoenzymes help in different diagnosis:

CPK BB: Brain injury, stroke.

- CPK MM: Muscle disease.

- CPK MB: Heart disease (Myocardial infarction.

ZYMOGENS

Inactive Enzymes (pre enzymes).

Many E synthesised as zymogens, and are activated when needed. e.g.Zymogen Active E

Trypsinogen Trypsin + PeptideEnterokinase

Autocatalysis

Chymotrypsinogen Chymotrypsin + peptideTrypsin

Pepsinogen Pepsin + PeptideHCl

Prothrombin Thrombin + peptideclotting

factors

This provides protection to the body. As the active E may destroy body substances if activated in absence of S.

e.g. if thormbin is formed in the body, it will convert fibrinogen Fibrin. This will form clot in blood: Stroke

Heart attach