Enzymes

Catalysts of living world

Outline
• Classification and properties
• Nature and mechanism of enzyme activity
• Factor affecting activity
• Determination of activity
• Inhibition
• Mechanism and regulation of activity
• Mechanism and regulation of enzyme synthesis
Enzymes
• A catalyst is defined as a Substance that increases the velocity or rate of the chemical
reaction without itself undergoing any change in the overall process
Two types 1. chemical catalyst 2. biological catalyst
Protein hydrolysis in lab by strong acid at 100°C takes couples of days. Same
protein in gastrointestinal tract at 37°C hydrolysed within couple of hours

• Enzymes are biocatalysts – catalysts of life

• Enzymes defined as
 Biocatalysts synthesized by living cells
They are protein in nature, colloidal and thermolabile
Specific in their action
 Historical background
Berzelius (1836) coined term catalysis (to dissolve)
Kuhne (1878) used word enzyme to indicate catalysis taking place in biological system
Buchner (1883) Isolation of enzyme from yeast Named as zymase (mix of enzyme) which
could convert sugar to alcohol
James summer (1926) 1st isolation and crystallization of enzyme urease from jack bean and
identified it as a protein

Urease was 1st enzyme isolated in crystalline form by Sumner who got Nobel prize in 1947
Protein nature of enzyme was accepted by widely after John Northrop et.al. in 1930 who
crystallized pepsin and trypsin and found them to be protein
 Nomenclature of Enzyme
In early days, Given names by their discoverers

Suffix –ase added to substrate for naming enzymes eg. Lipase, nuclease, lactase

International Union of Biochemistry (IUB) appointed as Enzyme Commission for

basic principles for classification and nomenclature

Enzymes are divided into six major classes (OTHLIL)

Each class on its own represents general type of reaction brought by the enzymes
of class
Two types of enzyme on the basis of action

• 1. Intracellular enzyme or endoenzyme :They are functional within cells where

they are synthesized- performed catabolic reaction which provide energy

• 2. Extracellular enzyme or exoenzyme : They are functional outside the cell ; All
digestive enzyme belong to this group
 Classification of Enzyme
• 1. Oxidoreductases: Involved in oxidation-reduction reactions eg: alcohol
dehydrogenase E.C. 1.1.1.1

2. Transferases: Transfer of functional group eg: hexokinase E.C. 2.7.1.1

3. Hydrolases: Bring about hydrolysis of various compounds eg: lipase E.C. 3.1.1.3
• 4. Lyases: Addition or removal of water, ammonia, CO2 etc. eg: Aldolase E.C.
4.1.2.7

• 5. Isomerase: Isomerization reaction :eg triose phosphate isomerase E.C. 5.3.1.1

• 6. Ligases: Synthetic reaction where two molecule joined together and ATP is
used Eg. Glutamine synthetase (glutamate ammonia ligase) E.C. 6.3.1.2
 Chemical and physical properties of enzymes
All enzyme are protein or combined with chemical group
Contain properties of protein :
Denature by heat,
Precipitated by ethanol or inorganic salts

Consist of protein combination with low-molecular weight organic molecule called a

coenzyme. Protein portion is referred to as apoenzyme. When united, complete enzyme,
identified as holoenzyme

Holoenzyme Apoenzyme + Coenzyme

(Active enzyme) (Protein part) (Non Protein part)
 High Molecular weight Low molecular weight
Coenzyme

Integral part of some coenzyme is vitamin and metal. Eg. Iron in enzyme catalase

Many enzymes require addition of metal ions (mg+2, mn+2, fe+2, zn+2, etc.) to activate

These metal ions function in combination with enzyme protein, and they are regarded as
inorganic coenzymes or cofactors.

Sometimes both cofactor and coenzyme are required before an enzyme becomes active
Characteristics of enzyme

• 1. High catalytic efficiency

• May transform 102 to 106 molecule of substance to product per minute

• 2. High degree of specificity for substance

Single enzyme + single substrate
Cell produce different enzyme for different substrate they metabolized
A B C D E F
a b c d e
Every single enzymes work in sequence, Last reaction yield final
product
Nature and mechanism of enzyme action (activity)
Enzyme reaction represented by following reaction
Enzyme is not used up in reaction but released for further reaction with
another substrate molecule
Repeated many times until all available substrate molecules consumed
 4

1
2
3

1. Substrate bind to active site

2. Enzyme-substrate complex form
3. Substrate transform into the product
4. Product release
5. Enzyme recycle
Enzyme lower activation energy
Activation energy: amount of energy required to bring substance to reactive state.
Enzyme combine with substrate molecule to produce transition state requiring less
activation energy for chemical reaction to proceed
Active site
• Small region of enzyme at which the substrate has high affinity to binds and
participates in the catalysis
1. It is also knows as substrate binding site or catalytic site
2. Existence of active site is due to the tertiary structure of protein
3. Made of amino acids- E.g Lysozyme has 129 amino acids and active site
contain amino acid No. 35, 52, 62, 63, 101
4. It is regarded as pocket occupying a small region in big enzyme
5. It is flexible to promote the specific substrate binding
6. Substrate binds at the active site by weak non covalent bonds
7. Enzyme are specific in their function due to the existence of active site
8. Commonly found amino acid are serine, aspartate, histidine, cysteine, lysine,
arginine, glutamate, tyrosine
Product has low affinity for active sites and hence it released from active site
thereby the active sites is free to repeat the process with another two molecules of
substrate

Intracellular enzymes have more than one active site per molecule
eg. Lactate dehydrogenase have 4.
Extracellular like chymotrypsin has only one active site

Active site on enzyme surface is usually very small area i.e. large regions of enzyme
protein do not contribute to enzyme specificity or enzyme action

Thus only few amino acids are directly involved in catalytic process
Factors affecting enzyme activity

• 1. Concentration of enzyme
• 2. Concentration of substrate
• 3. Effect of temperature
• 4. Effect of pH
• 5. Effect of product concentration
• 6. Effect of activators
• 7. Effect of time
• 8. Effect of light and radiation
 1) Concentration of enzyme
As enzyme concentration increased, velocity of
reaction proportionately increases
At low enzyme concentration there is great
competition for active site, so rate of reaction is
low
At high enzyme concentration there is more
active site, so rate of reaction is faster
Substrate concentration must be excess i.e.
reaction must be independent to substrate
concentration
 2) Concentration of substrate
If the amount of enzyme is kept constant and
substrate concentration is then gradually increased,
velocity of reaction will increase until it reaches a
maximum
i.e. increase in substrate concentration gradually
increases the velocity of enzyme reaction within
limited range of substrate levels
Further increases in substrate concentration will not
increase the reaction velocity
 With only a few bricks With more bricks available, Bob Adding more Bobs (enzymes)
(substrates), Bob (the enzyme) works faster. The increased helps build the building faster.
works slowly. The reaction rate substrate concentration allows The reaction rate increases as
is low due to the limited Bob to build more quickly, more enzymes are available to
substrate availability. increasing the reaction rate. handle the surplus of
substrates.

When there are lots of bricks, Bob

works at his maximum speed. At
this point, Bob is saturated with
substrates, and the reaction rate
has reached its peak.
 Michaelis-menten equation
V= Vmax*[S]/ Km+[S]
Where ,
V= velocity
S= substrate concentration
Km= michaelis-menten constant
Vmax= maximum velocity

Km: Michaelis-menten constant : Substrate concentration to

produce half-maximum velocity in an enzyme catalyzed reaction
3) Effect of temperature
Velocity of enzyme reaction increase with increase in
temperature up to maximum and then declines

Bell-shaped curve is observed

Temperature coefficient Q10: Increase in enzyme velocity

when temperature is increase by 10° C

Majority of Enzyme is between 0 to 40° C

Increase in temperature result in higher activation energy

of the molecules and more molecular (enzyme and
substrate) collision and interaction for reaction to proceed
faster
Optimum temperature for most of the enzyme is between 35°C - 40°C

Few enzyme are active even at 100 °C

Some plant enzymes like urease have optimum activity around 60 °C. may be due to
very stable structure and conformation of enzymes

When enzymes are exposed to temperature above 50 °C, denaturation leading to

loss of native structure of protein and active site.

Majority of enzymes become inactive at higher temperature 70°C

 4) Effect of pH

Increase in pH influences enzyme activity and bell-

shaped curve is normally Obtained

Optimum pH at which velocity is maximum.

Most of the enzymes of higher organisms show

optimum activity around neutral pH (6-8). Many are
exceptions like pepsin, acid phosphate (4-5) and
alkaline phosphatase (10-11)

Hydrogen ions influence the enzyme activity by

altering ionic charges on amino acids, substrate, ES
complex etc.
5) Effect of product concentration

Accumulation of reaction products decreases enzyme velocity

Product combine with active site and form loose complex and thus
inhibit enzyme activity

In living system, this type of inhibition is prevented by quick removal

of products formed
 6) Effect of activators
Some enzymes require certain metallic cations ( Mg+2, Mn+2, Zn+2, Ca+2, Co+2, Cu+2, Na+,
K+ etc) for their optimum activity
Rarely, anions also needed for activity i.e. Cl- for amylase

Metal function as activators of enzyme velocity through various mechanism- combining

with substrate, formation of ES-metal complex, direct participation in reaction bringing
conformational change in enzyme

2 categories of enzyme requiring metals for their activity

1. metal –activated enzymes
2. metalloenzymes
1) Metal-activated enzymes
Metal is not tightly held by enzyme and can be exchanged easily with other
ions Eg. ATPase (Mg2+, Ca2+ ), Enolase (Mg 2+)

2) Metalloenzyme
Hold metals tightly which are not readily exchanged.
Eg. Alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase,
carboxypeptide and aldolase contain Zn

Phenol oxidase Cu
Pyruvate oxidase Mg
Xanthine oxidase Mo
Cytochrome oxidase Fe, Cu
 7) Effect of time
Under ideal and optimal condition (pH, temperature etc.), the time required for
enzyme reaction is less.

Variations in time of reaction generally related to alterations in pH and

temperature

8) Effect of light and radiation

Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain

enzymes due to formation peroxides

Eg. UV rays inhibit salivary amylase activity

Determination of enzyme activity
Can be determine by variety of techniques

Some require special, elaborate instruments, other require only test

tube and few reagents

All are based on few simple principles

• To carry out assay of enzyme activity, Necessary to know following

1. Nature of reaction catalyzed

2. what cofactors and coenzyme are required
3. Require concentrations of both substrate and coenzyme or cofactor
4. optimum pH
5. optimum temperature
6. simple analytical method for determining disappearance of substrate
or appearance of product of the reaction
Substrate concentration should be above saturation level.

Coenzyme and cofactor should be added in excess

Doing this ensures that true limiting factor is enzyme

concentration(at optimum pH and temperature)

Measurements of reaction product formation is more accurate than

measurements of disappearance of substrate
Enzyme Inhibition
• Enzyme inhibition: substance or inhibitor which binds with enzyme
and brings about decrease in catalytic activity of that enzyme
• Inhibitor may be organic or inorganic in nature

• Three broad categories of enzyme inhibition

1. Reversible inhibition
• Competitive inhibition
• Non-competitive inhibition
2. Irreversible inhibition
3. Allosteric inhibition
1. Reversible inhibition
• Inhibitor binds non-covalently with enzyme and enzyme inhibition can
be reversed if inhibitor is removed

• Further sub-divided into

• 1.1 competitive inhibition
• 1.2 non-competitive inhibition
1.1 competitive inhibition
Inhibitor which closely similar to real substrate is known as substrate
analogue
Inhibitor compete with substrate and binds at active site but does not
undergo any catalysis
As long as competitive inhibitor holds active site, enzyme is not
available for substrate to bind
During reaction, ES and EI complexes are formed
• Concentration of substrate and inhibitor and their affinity with
enzyme determine degree of competitive inhibition
• Could be overcome by high substrate concentration
• Km value increase, Vmax remain unchanged
• Malonic acid, glutaric acid and oxalic acid have similar structure and compete with
succinic acid for binding at active site of Succinate dehydrogenase (SDH).

• Methanol is toxic to body when it is converted to formaldehyde by enzyme alcohol

dehydrogenase (ADH). Ethanol can compete with methanol for ADH. So ethanol can
be used in the treatment of methanol poisoning
• Antimetabolites : chemical compounds that block metabolic reactions by their
inhibitory action on enzymes.
• Antimetabolites are usually structure analogues of substrate and thus competitive
inhibitors
• Antivitamins used for the antimetabolites which block biochemical actions of vitamins
causing deficiencies
1.2 Non- competitive inhibition
Inhibitor binds at a site other than active site on enzyme surface

Binding impairs enzyme function

Inhibitor has no structural resemblance with substrate

Usually exists a strong affinity for inhibitor to bind at second site

Inhibitor does not interfere with enzyme-substrate binding

Catalysis is prevented due to distortion in enzyme conformation

• Inhibitor generally binds with enzyme as well as ES complex.

• Km value is unchanged while Vmax is lowered

• Heavy metal ions can non-competitively inhibit enzymes by binding
cysteinyl sulfhydryl groups

• heavy metals also lead to formation of covalent bonds with carboxyl

groups and histidine, often result in irreversible inhibition
2. Irreversible inhibition
Inhibitor bind covalently with enzymes and inactive them which is irreversible
Usually toxic substance that poison for enzyme

Iodoacetate is irreversible inhibitor of enzymes like papain and glyceraldehyde 3-

phosphate dehydrogenase

Iodoacetate combines with sulfhydryl groups at active site of these enzyme and make
them inactive

Many organophosphorus insecticides (melathion) are toxic to animals as they block the
activity of acetylcholine esterase, resulting in paralysis of vital body functions
Penicillin antibiotics acts as irreversible inhibitors of serine- containing
enzyme, and block bacterial cell wall synthesis

Cyanide inhibits cytochrome oxidase (binding to iron atom) of electron

transport chain
 Suicide inhibition
• Specialized form of irreversible inhibition
• Original inhibitor is converted to more potent form by same enzyme that to be
inhibited
• so the formed inhibitor binds irreversibly with enzyme

• Allopurinol , inhibitor of xanthine oxidase, gets converted to alloxanthine, a

more effective inhibitor of this enzyme

• Use of purine and pyrimidine analogues in cancer therapy

• 5-fluorouracil gets converted to fluorodeoxyuridylate which inhibits enzyme

thymidylate synthase, and thus nucleotide synthesis
3. Allosteric inhibition
Some enzyme have additional sites besides active sites
Such enzymes are known as allosteric enzymes. Allosteric sites are
unique places on enzyme molecule

Allosteric effectors: certain substances referred to as allosteric

modulators (effector or modifiers) binds at allosteric sites and regulate
enzyme activity
Mechanism and Regulation of enzyme activity
• In biological system, it can be occur at different stages in one or more
of following ways to reaches to cellular economy
• 1. allosteric regulation
• 2. activation of latent enzymes
• 3. comapartmentation of metabolic pathways
• 4. control of enzyme synthesis
• 5. enzyme degradation
• 6. isoenzymes
1. Allosteric regulation and allosteric inhibition
Some enzyme have additional sites besides active sites
Such enzymes are known as allosteric enzymes. Allosteric sites are unique places
on enzyme molecule

Allosteric effectors: Certain substances referred to as allosteric modulators

(effector or modifiers) binds at allosteric sites and regulate enzyme activity

Allosteric activator- Enzyme activity increase when positive allosteric effector

binds at allosteric site known as activator site
Allosteric inhibitor- Enzyme activity decrease when negative allosteric effector
binds at allosteric sites called inhibitor site
Conformational changes in allosteric enzymes:
Non-covalent reversible binding of effector molecule at allosteric site bring
conformational change in active site of enzyme, lead to inhibition or activation of
catalytic activity
• In model, allosteric enzyme exist in 2 conformational states-
• T (tense) and R (relaxed)
• T and R states are in equilibrium
Allosteric inhibitors favor T state whereas
activators and substrates favor R state

Substrate can bind only with R form of enzyme.

Velocity of enzyme molecule in R state increases

as more substrate is added, therefore binding of
substrate to allosteric enzymes give sigmoidal
curve; when velocity verses substrate
concentration are plotted
Feedback regulation
Inhibiting 1st step by final product, in a series of enzyme catalyzed
reactions of metabolic pathway is referred to as feedback regulation

Very 1st step is most effective for regulating pathway, by final end
product.
This type of control is called negative feedback regulation since
increased levels of end product will result in its decreased synthesis

Cellular economy to save cell from wasteful synthesizing compound

Feedback inhibition or end product inhibition is special type of

allosteric inhibition to control metabolic pathway for cellular
efficiency
E.g.- Feed back inhibition
2. Activation of latent enzymes
Latent enzyme are inactive
Some enzyme are synthesized as proenzymes or zymogens which undergo
irreversible covalent activation by breakdown of one or more peptide bonds

Proenzyme- Chymotrypsinogen - chymotrypsin

 Pepsinogen – pepsin
 Plasminogen - plasmin

Enzyme exist in active and inactive forms which are interconvertible,

depending on needs of body

Interconversion by reversible covalent modifications, like phosphorylation

and dephosphorylation, and oxidation and reduction of disulfide bonds
Glycogen phosphorylase is muscle enzyme That break down glycogen to
provide energy.
This enzyme is homodimer and exists in two Interconvertible forms
Some enzymes which are active in dephosphorylated state and become
inactive when phosphorylated
Eg. Glycogen synthase, acetyl CoA carboxylase
Few enzyme active only with sulfhydryl group. Eg. Succinate
dehydrogenase, urease
Substance like glutathione bring stability of these enzymes

2G-SH GS-SG
• E-S-S-E E-SH + E-SH
• Oxidized inactive enzyme Reduced active enzyme
3. Compartmentation
Certain substance in body which are synthesis and also degraded

No point for simultaneous occurrence of both pathways-anabolic and

catabolic.

Synthesis and breakdown pathways are operative in different cellular

organelles to achieve maximum economy

Enzyme for fatty acid synthesis are found in cytosol whereas enzymes for
fatty acid oxidation are present in mitochondria

Depending upon needs of body fatty acids are either synthesized or oxidized
4. Control of enzyme synthesis

Amount of enzyme directly controls velocity of reaction, catalyzed by

that enzyme
Many rate limiting enzymes have short half lives
2 Types of enzyme
1. Constitutive enzyme: levels of which are not controlled and remain
fairly constant.
2. Adaptive enzyme: concentration increase or decrease as per body
needs and are well-regulated.

Enzyme synthesis is regulated by genes

Induction and repression
Induction: used to represent increased synthesis of enzyme.
Eg. Hormone insulin induces synthesis of glycogen synthetase,
glucokinase, phosphofructokinase and pyruvate kinase which are
involved in utilization of glucose

Repression: use to represent decreased synthesis of enzyme.

Pyruvate carboxylase used for synthesis of glucose from non-
carbohydrate source like pyruvate and amino acids.

Induction or repression ultimately determines enzyme concentration at

gene levels through mediation of hormones or other substances
5. Enzyme degradation
Not immortal
it will create series of problems
Lots of variability in half-lives of individual enzymes
For some, it is half-lives of individual enzymes. For some, it is in days
while for other in hours or in minutes eg. LDH4- 5 to 6 days, LDH1-8 to 12
hours; amylase- 3 to 5 hours
Key and regulatory enzymes are most rapidly degraded.
If not needed, they immediately disappear and when required, they are
quickly synthesized
Not always true, an enzyme with long half-life is usually sluggish in its
catalytic activity
6. Isoenzymes
Multiple forms of the same enzyme will also help in regulation of enzyme activity

Many of isoenzymes are tissue-specific

Although isoenzymes of a given enzyme catalyze the same reaction, they differ in
Km, Vmax or both.

eg. Isoenzymes of LDH (Lactate dehydrogenase ) and CPK(creatine

phosphokinase)
Mechanism and regulation of enzyme
synthesis
• When product of an enzymatic pathway is no longer required to be in
cell, enzymes that catalyze the reactions of pathway become
unnecessary
• Regulation is effected at gene expression level
• enzymes can be divided into 2 groups as follows
1. Constitutive enzyme: continuous produced by cells
• Found in essential same amounts of concentration of their substrates
in the medium
2. Inducible enzymes: produce by cell only in response to presence of
particular substance; they are produced only when needed
• Process referred to as enzyme induction.