Enzymes

Dr. Saravanan. R
Enzyme (En – in, Zyme – living ) - Greek
• Enzymes may be defined as biocatalysts synthesized by living
cells.
• They are protein in nature(except ribozyme),colloidal, water
soluble and thermo labile in character, and specific in their
action.
• They contain 16% weight as nitrogen
Enzyme + Substrate (Enzy-Subs) Product + Enzyme
NOMENCLATURE OF ENZYMES
• By adding suffix -ase at the end of the name of the substrate,
enzymes are named (all enzyme end with “ase”).
Substrate Enzyme Product
lactose lactase Glucose + galactose
maltose maltase Glucose
cellulose cellulase Glucose
Classification of Enzymes
• Enzymes are classified based on IUBMB(International union of
biochemistry and molecular biology)
• According to IUBMB, enzymes are classified into 6 major
classes according to the reaction they catalyze
• Each enzyme denoted four digits
• First digit : Represents Main class
• Second digit: sub class
• Third digit : sub-sub class
• Fourth digit : number of particular enzyme in the list or serial
number in the list
Ex: Alcohol dehydraganase (Code number EC.1.1.1.1)
*EC – Enzyme Commission
Nomenclature
 The Six Classes
⚫EC 1. Oxidoreductases O
⚫EC 2. Transferases T
⚫EC 3. Hydrolases H
⚫EC 4. Lyases L
⚫EC 5. Isomerases I
⚫EC 6. Ligases L
EC 1. Oxidoreductases
The enzymes which are involved in both oxidation and reduction
in same reaction

AH2 +B → A+ BH2

Ex.Oxidases, Dehydrogenases, Reductases

Ex: Alcohol dehydroganase (1.1.1.1), Succinate dehydrogenase

EC 2. Transferases
The Enzyme which transfer functional group from one substrate
to another substrate
A-X + B ↔ BX + A
Ex: Transaminases (transfer amino group), Kinases (transfer
Phosphategroup) , Hexokinase
EC 3. Hydrolases
Catalyze hydrolytic reactions.
A-X + H2O ↔ X-OH + A-H
Ex: lipases, esterases, Amylases, peptidases/proteases, etc.
(Acetylcholine esterase)
EC 4. Lyases
The enzyme which cleave the bond between substrate and creating
double bond in the product without adding water
A- X +B-Y → A=B + X-Y
Ex: Decarboxylases, Aldolases, Dehydrases, Deaminases,
Synthases, etc.
Fructose 1,6 bis phosphate → G3P + DHAP
EC 5. Isomerases
Catalyzes isomerization reactions, including epimerizations and
cis-trans isomerizations.

Ex: Isomerases (Cis-Trans), Epimerases (D—L) ,

Triose phosphate isomerase.
EC 6. Ligases
The enzyme which link two substrate together with help of
hydrolysis of ATP

Ex: Synthetases, Carboxylases

(Glutamine synthetase)
Co enzymes
• All enzymes are protein
• Non protein part of enzyme “ Prosthetic group” which is otherwise
called “Coenzyme”
• Protein part of enzyme – Apoenzyme
• Non protein part of enzyme – Co enzyme
• Apo enzyme + Co enzyme – Holo enzyme
First group of co enzyme :
Changes occur in the substrate which counterbalanced by co enzyme,
otherwise co substrate

EX: NAD to NADH2 (Pyruvate to Lactate)

Second group of co enzyme:

The coenzyme which transfer groups other than hydrogen
Ex: ATP
Salient features of co enzyme
• Essential for biological activities of enzyme
• It is low molecular weight organics substance and heat stable
• Generally it combines loosely with enzyme molecule. Both
enzyme and co enzyme can easily separated by dialysis
process
• Most of co enzymes are derivative of vitamin B complex
• One coenzyme molecule can work with different enzymes.

Synthetases are ATP dependent enzymes catalyzing linkage of

two molecules; they belong to Ligases (class 6).
Examples are Carbamoyl phosphate synthetase; Argininosuccinate
synthetase; PRPP synthetase and glutamine synthetase.

Synthases are enzymes catalyzing biosynthetic reactions; but they

do not require ATP directly; they belong to classes other than
ligases. Examples are glycogen synthase and ALA synthase.
Metallo enzyme
• The enzyme which require certain metal ions for its activity,
called ‘metallo enzyme’
• Metals are tightly bound with enzyme
• Ex: Carbonic anhydrase (Zinc), Hexokinase(Mg2+),
Enolase(Mn2+), Tyrosinase(Cu), cytochrome oxidase(Fe3+),
Lipase(Ca2+)

• Even without metals, enzyme may active but when metal is

added, the activity is enhanced. They are “metal activated
enzyme”
Ex; Pancreatic lipase(Ca2+)
Active Site
• Small region in enzyme where substrate comes and bind to
catalysis the reaction

Active Site

Binding Site Catalytic Site

Salient Features
• Three dimensional in nature
• Made up aminoacids Ex : Lysozyme – 129 Aas
• Active site regarded as cleft or pockets occupying a small
region in a big enzyme molecule
• Not rigid in nature , flexible
• Active site posses two site 1. substrate binding site 2. catalytic
site
• The substrate binds at the active site by weak covalent bond
• Commonly found aminoacid at the active site are serine,
aspargine, histidine, cysteine, lysine, arginine and tyrosine.
Among the above, serine is most frequently bound
Intracellular enzyme:
Enzyme synthesized in cell and catalysis the reaction within cell
called intra cellular enzyme
They are found in cytoplasm, mitochondria
Ex: Oxido reductase

Extra cellular enzyme:

Synthesized in cell and catalysis the function in somewhere else
in cell
Ex; Digestive enzyme
Enzyme Kinetics
 Con. Of
Enzyme
Time Con. Of
Substrate

Factors
Light & affecting
Radiation Temperature
enzyme
activities
Activators
Product PHpphV
pH
Concentration
 1. Concentration of enzyme
Increase the con of enzyme, velocity of reaction (Vmax) is
also increased. Knowing the concentration of serum enzyme
in a particular reaction is useful in diagnosis of complications

LDH – Increased in liver disease, myocardial infarctions and hemolysis

CPK – Myocardial infarctions and skeletal muscle disorder
ALP – Bone disorder and hepatitis
 2. Concentration of Substrate
The rate of reaction (Velocity Vmax) increases as substrate
concentration (Km) increases with in limited range of substrate
level. A rectangular hyperbola is obtained when velocity is plotted
against substrate concentration

LB Plot
Dixon Plot

The substrate concentration is known as Km or Michaelis–Menten

constant
V0 : initial velocity
Vmax : maximum velocity
Km : 1/2 Vmax = Michaelis Menten constant
[S] : substrate concentration
Salient Features of Km
1. Km value is substrate concentration (expressed in moles/ L) at
half-maximal velocity (1/2 Vmax).
2. It denotes that 50% of enzyme molecules are bound with
substrate molecules at that particular substrate concentration.
3. Km is independent of enzyme concentration. If enzyme
concentration is doubled, the Vmax will be double. But the Km will
remain exactly same. In other words, irrespective of enzyme
concentration, 50% molecules are bound to substrate at that
particular substrate concentration.
4. Km is the signature of the enzyme. Km value is thus a constant for
an enzyme. It is the characteristic feature of a particular enzyme
for a specific substrate.
5. Km denotes the affinity of enzyme for substrate. A low Km value
indicates a strong affinity between enzyme and substrate
3. Effect of Temperature
• Increase the temperature, velocity of reaction of enzyme also
increase upto certain limits and declines.
• The temperature at which an enzyme show its maximum
activity is called optimum temperature
• A bell-shaped curve is usually observed
• Optimum temperature for most of enzyme around 37 to
40°C. Beyond 500C there is denaturation of enzyme
• Temperature coefficient Q10 : Every 100C, velocity of
enzyme is also increase.
• Certain bacteria living in hot springs will have enzymes with
optimum temperature near to 1000 C

Venom phospho kinase& Muscle adenylate kinase - 1000 C

Urease - 600 C
4. Effect of pH

• At low and high pH , velocity is reduced and its activity is

raised at particular pH called “Optimum pH”
• Optimum pH of most of enzyme is 6-8 except Pepsin (1-2),
acid phosphatase(4-5) and alkaline phosphatase(4-5)
• Bell shape curve is obtained while plotting

5. Effect of Product concentration

Increase the concentration of product reduce the activity of an
enzyme and velocity of reaction is slowed or stopped
6. Effect of Activators
Some of enzyme require metals for its activation
Ex: Mg2+, Ca2+, Na+, K+
Salivary amylase - Chloride
Lipase - Calcium
There are 2 categories of enzyme requiring metals for their
activity
• Metal activated enzyme: Hold the enzyme loosely and can
be exchanged easily. Ex: ATPAase (Mg and Ca) and Enolase
• Metalloenzyme: Hold the metal tightly. Ex: alcohol
dehydrogenase, carbonic anhydrase, alkaline phosphatase,
carboxypeptidase
7. Effect of time
Under ideal and optimal pH and temperature, time require for an
enzyme action is less
8. Light and radiation: exposure to UV, beta- gamma and X-rays
inactivates certain enzyme ex: UV rays inhibit salivary amylase
activity

Mechanism of Enzyme Action

There are 3 theories that explain the mechanism of action on
enzyme
1. Lock and Key theory or Fisher’s template theory
2. Induced fit theory or Koshland’s theory
3. Substrate strain theory
1. Lock and key theory or Fisher’s template theory
• Proposed by Emil Fisher
• According to this theory, enzyme is rigid and pre shaped where
only specific substrate can bind
• Enzyme and substrate are complimentary to each other
• Substrate binds to enzyme similar to lock and key
• Since theory failed to explain the flexibility of the enzyme, not
accepted.
2. Induced fit theory or Koshland’s theory
• It was proposed by koshland in 1958
• The important feature of this theory is flexibility of the region
of the active site of an enzyme
• According to this exposure of an enzyme to substrate cause a
change in enzyme, which causes the active site to change it’s
shape to allow enzyme and substrate to bind.
• Substrate induce the conformational changes in active site of
an enzyme for its binding with it. Ex: Hands in glove
• The active site is flexible, not rigid.
• The shapes of the enzyme, active site, and substrate adjust
to maximum the fit, which improves catalysis.
• There is a greater range of substrate specificity.
Inhibitions of Enzyme
Inhibitors:
• They are defined as substrate which binds with enzyme and
decrease the velocity and its catalytic activities
• Inhibitors may be organic or inorganic
Competitive Inhibition

E+S [ES] E+P

E+I EI No Product will form

• Inhibitors are closely resembles the substrate (Substrate

analogue)
• Inhibitors compete with substrate to bind with active site of an
enzyme
• Competitive inhibition is usually reversible.
• Km value (concentration of substrate) is increase, where as
Vmax (Velocity of reaction ) remain unchanged
• Clinical importance : Many drugs are designed based on principle
of competitive inhibition.
• Ex: Sulfonamide – antibacterial agents
Methotrexate – Anticancer drug
Dicumerol – Anticoagulant (Structural analog of vitamin K)
Isonicotinic acid hydrazide (INH) - antituberculous drug

Enzyme kinetics of competitive inhibitor

Vmax is unaltered
Km is increased

• Statin drugs (to treat hypercholesterolemia) such as lipitor

compete with HMG-CoA(substrate) and inhibit the active site of
HMG CoA-REDUCTASE (that bring about the catalysis of
cholesterol synthesis).
Clinical importance of competitive inhibition
1. Sulfonamides: They are commonly employed antibacterial
agents. Bacteria synthesize folic acid by combining PABA with
pteroyl glutamic acid. Sulfa drugs, being structural analog of PABA,
will inhibit the folic acid synthesis in bacteria, and they die.
2. Methotrexate is a structural analogue of folic acid, and so can
competitively inhibit folate reductase enzyme. Therefore,
methotrexate is used as an anticancer drug.
3. Dicoumarol: It is structurally similar to vitamin K and can act
as an anticoagulant by competitively inhibiting the vitamin K
activity.
4. Isonicotinic acid hydrazide (INH) is an antituberculous drug. It is
structurally similar to pyridoxal, and prolonged use of INH may
cause pyridoxal deficiency and peripheral neuropathy.
 Isoniazid is an anti-tuberculosis drug, inhibits the biosynthesis of
NAD and restrict the growth of the organisms that cause
tuberculosis.
 Medical therapy for methanol poising based on competitive
inhibition
 Ethanol competes effectively with methanol as an alternative
substrate for alcohol dehydrogenase.
Non Competitive Inhibition

E+S ES E+P

I
ESI

• No competition between inhibitor and substrate in this type

• Inhibitor binds on site other than substrate binding active site
(allosteric site) on the enzyme
• inhibitors have no structural resembles to the substrate
• Inhibitors does not interfere with enzyme substrate complex but
the catalysis of reaction is stopped.
• Km value (substrate concentration ) is unchanged while Vmax
(velocity of reaction) is lowered.
• It is irreversiable
• Increasing substrate concentration will stop the competitive
inhibition but will not stop non competitive inhibition
EX:
• Cyanide inhibits cytochrome oxidase of ETC (Respiratory chain)
• Fluoride will remove magnesium and manganese ions and so
will inhibit the enzyme, enolase, and consequently the
glycolysis
• Iodoacetate would
inhibit enzymes having -
SH group in their active
centers
• BAL (British Anti
Lewisite; dimercaprol) is
used as an antidote for
heavy metal poisoning.
Comparison of Two Types of Inhibition

Competitive Non competitive

inhibition inhibition
Acting on Active site May or may not
Structure of Substrate analogue Unrelated molecule
inhibitor
Excess substrate Inhibition is stopped No effect

Km Increased No change

Vmax No change Decreased

Significance Drug action Toxicological

Uncompetitive Inhibition
E+S ES I ESI

• Inhibitors binds to enzyme substrate complex but not with free

enzyme. In such case both Vmax and Km are decreased
• Inhibition of placental alkaline phosphatase
(Regan isoenzyme) by phenylalanine is an example of
uncompetitive inhibition.

(-)
*Alloxanthine - betrayer
The anti-inflammatory action of Aspirin is also based on the
suicide inhibition. Arachidonic acid is converted to
prostaglandin by the enzyme Cyclo-oxygenase. Aspirin
acetylates a serine residue in the active center of cyclo-
oxygenase, thus prostaglandin synthesis is inhibited, and so
inflammation subsides.

Arachidonic acid
(-)
Cyclo-oxygenase Aspirin
prostaglandin
Penicillin
Inactivates bacterial enzyme glycopeptidyl transpeptidase
involved in the formation of bacterial cell wall

Feed back Inhibition (Allosteric feedback inhibition)

The process of inhibiting the first step by final product whenever
the end product of such metabolic reaction produced in excess of
the cells needs
The end product, heme
will allosterically inhibit
the ALA synthase