Dr. Deepak K Gupta
• Definition, classification, specificity and active
• Effect of pH temperature and substrate
• Introduction to enzyme inhibitors,
proenzymes and isoenzymes.
• Introduction to allosteric regulation, covalent
modification and regulation by induction /
• Enzymes are biological catalysts synthesized by
living cells that accelerate biochemical reactions.
• The orderly course of metabolic processes is only
possible because each cell is equipped with its
own genetically determined set of enzymes
• It is only this that allows coordinated sequences
of reactions - metabolic pathways
• Involved in many regulatory mechanisms.
• Almost all enzymes are proteins except
catalytically active ribonucleic acids, the ribozymes
• Enzymes are characterized by three distinctive
• Catalytic Power
– Ability to catalyses biochemical reaction
– Accelerating reaction rates as much as 1016 over
uncatalyzed levels - far greater than any synthetic catalysts
– A given enzyme is very selective
– Both in the substances with which it interacts and in the
reaction that it catalyzes
– Metabolic inhibitors and activators
• Traditionally, enzymes often were named by
adding the suffix –ase to the substrate upon
which they acted
• Ex: phosphatase, urease, catalase, proteases
• Confusion arose from these trivial naming.
• So a new system of nomenclature of enzyme was
developed based on nature of reaction it helps
• Six classes of reactions are recognized
– Within each class are subclasses, and under each
subclass are subsubclasses within which individual
enzymes are listed
Classification of Enzyme
• Enzyme are classified on the basis of action it performs
– Oxidoreductases - oxidation–reduction reactions
• Phosphate dehydrogenase
– Transferases - transfer of functional groups
• Methyltransferases, Carboxyltransferases
– Hydrolases - hydrolysis reactions
• Carboxylic ester hydrolases
– Isomerases - isomerization reactions
– Lyases - addition to double bonds
• Carboxy lyases, Aldehyde lyases
– Ligases - formation of bonds with ATP cleavage
• Amino acid–RNA ligases
Intracellular and extracellular
o enzymes are synthesized and retained in the cell for the use of
o They are found in the cytoplasm, nucleus, mitochondria and
Example: Oxydoreductase catalyses biological oxidation,
Enzymes involved in reduction in the mitochondria.
o enzymes are synthesized in the cell but secreted from the cell
to work externally.
Example : Digestive enzyme produced by the pancreas, are
not used by the cells in the pancreas but are transported to the
• Most of enzymes carry out their functions relying solely
on their protein structure
• Many others require non-protein components –
– Usually metal ion or non-protein organic part (Coenzyme)
• Less complex than proteins, tend to be stable to heat
• Many coenzymes are vitamins or contain vitamins as
part of their structure
• Functional unit of enzyme is known as holoenzyme
– Holenzyme = Apoenzyme + Coenzyme
• Apoenzyme : protein without any catalytic activity
• If the enzyme is made of single polypeptide –
monomeric enzyme. Ex: ribonuclease, trypsin
• If the enzyme is made up of more than one
polypeptide – oligomeric enzyme. Ex: lactate
dehydrogenase, aspartate transcarbamoylase
• Multienzyme complex: have multiple enzyme
unit to carry out different reaction in
• The quantitative measurement of the rates of
enzyme-catalyzed reactions and the systematic
study of factors that affect these rates
• Helps in analysis, diagnosis, and treatment of the
enzymic imbalances that underlie numerous
• Levels of particular enzymes serve as clinical
indicators for pathologies
– myocardial infarctions,
– prostate cancer
– damage to the liver
• Any biochemical reaction constitute,
A + B C + D
Where A and B are substrate and C + D are product
• Study of enzyme kinetic has 2 component, i.e.
• Gibs Free Energy : Direction and equilibrium state of
substrate and product
• Activation Energy: Mechanism of reaction and rate
Gibs Free Energy Change ΔG
• Also called either free energy or Gibbs energy
• It describes in quantitative form both the direction in which a
chemical reaction will tend to proceed and the concentrations of
substrate and products that will be present at equilibrium
• Mathematically, ΔG = ΔGp – ΔGs
– ΔGp : sum of the free energies of formation of the reaction
– ΔGs : sum of the free energies of formation of the substrates
• The sign and the magnitude of the free energy change
determine how far the reaction will proceed
• If ΔG is negative then the reaction proceeds in forward direction
• Any reaction doesn’t proceeds directly to product
• There is always a transition state between ground
state and products
• Activation energy: The difference between the
energy levels of the ground state and the
• The function of a catalyst is to increase the rate
of a reaction, it does not affect reaction
• So enzyme just lowers the activation energy.
Factors effecting enzyme activity
• The contact between enzyme and substrate is the
most essential pre-requisite for enzyme activity.
• The important factors that influence the enzyme
– Concentration of Substrate
– Concentration of Enzyme
– Product concentration
– Light and radiation
Concentration of Substrate
• The frequency with which molecules
collide is directly proportionate to their
Rate ∝ [A]n[B]m , Rate = k[A]n[B]m
– where, nA + mB → P; k = rate constant
• The sum of the molar ratios of the reactants defines
the kinetic order of the reaction
• In the example above, reaction is said to be of (n+m)
order overall but n order with respect to A and m
order with respect to B
Michaelis-Menten Constant Km
• Also known as Haldane’s Constant
• Substrate concentration to produce half
maximum velocity in an enzyme catalyst
• Km is constant and a chracterstic feature of a
given enzyme – strength of Enzyme Substrate
• Low Km value indicates a strong affinity between
enzyme and substrate
• Majority of Enzyme Km value – 10-5 to 10-2
Michaelis-Menten Constant Km
Concentration of Enzyme
• Reaction velocity is directly proportional to
concentration of enzyme
• Serum enzyme for diagnosis of disease
– Known volume of serum and substrate taken at
optimum pH and temperature
– Enzyme is assayed in laboratory
• Velocity of an enzyme reaction increase with the
increase in temperature up to a maximum and then
• Increase in temperature causes increases the kinetic
energy of molecules
• A bell-shaped curve is usually observed
• Temperature coefficient Q10 : increase in enzyme
velocity when the temperature is increased by 100C
• Optimum temperature for most of enzyme – 40 – 45 0C
• Beyond 500C there is denaturation of enzyme
• Most intracellular enzymes exhibit optimal activity at
pH values between 6 - 8.
• Balance between enzyme denaturation at high or low
pH and effects on the charged state of the enzyme, the
substrates, or both
• Exception – pepsin (1-2), acid phosphatase (4-5),
alkaline phophatase (10-11)
• Certain metallica cations – Mn, Mg, Zn, Ca, Co, Cu,
• It acts in a various ways
– Combining with substrate
– Formation of E-S metal complex, direct participation in
the reaction and bringing a conformational changes in
• There are 2 categories of enzyme requiring metals
for their activity
• Metal activated enzyme:Not tightly held by the enzyme 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
• Product concentration: Accumulation of
reaction products generally decreases the
• Light and radiation: exposure to UV, beta-
gamma and X-rays inactivates certain enzyme
– Formation of peroxides, ex: UV rays inhibit salivary
Mechanism of Enzyme Action: Active
• The active site of an enzyme is the region that
binds substrates, co-factors and prosthetic groups
and contains residue that helps to hold the
• Active sites generally occupy less than 5% of the
total surface area of enzyme.
• Active site has a specific shape due to tertiary
structure of protein.
• A change in the shape of protein affects the
shape of active site and function of the enzyme.
This model (above) is an enzyme called
Ribonuclease S, that breaks up RNA
molecules. It has three active sites (arrowed).
The active site contains both binding
and catalytic regions. The substrate
is drawn to the enzyme’s surface and
the substrate molecule(s) are
positioned in a way to promote a
reaction: either joining two molecules
together or splitting up a larger one.Enzyme molecule:
The complexity of the
active site is what makes
each enzyme so specific
(i.e. precise in terms of the
substrate it acts on).
Substrate molecules are the
chemicals that an enzyme
acts on. They are drawn into
the cleft of the enzyme.
o Active site can be further divided into:
it chooses the substrate It performs the catalytic
and binds it to active site. action of enzyme.
Binding Site Catalytic Site
Mechanism of enzyme action
• The catalytic efficiency of enzymes is explained by two
Processes at the
• All chemical reactions have energy barriers between reactants and
• The difference in transitional state and substrate is called activational
Processes at the active site
o Enzymes form covalent linkages with substrate forming transient enzyme-
substrate complex with very low activation energy.
o Enzyme is released unaltered after completion of reaction.
• Mostly undertaken by oxido- reductases enzyme.
• Mostly at the active site, histdine is present which act as both proton
donor and proton acceptor.
Catalysis by proximity
• In this catalysis molecules must come in bond forming distance.
• When enzyme binds:
A region of high substrate concentration is produced at active site.
This will orient substrate molecules especially in a position ideal for them.
Catalysis by bond strain
• Mostly undertaken by lyases.
• The enzyme-substrate binding causes reorientation of the structure
of site due to in a strain condition.
• Thus transitional state is required and here bond is unstable and
• In this way bond between substrate is broken and converted into
Lock and key model
• Proposed by EMIL FISCHER in 1894.
• Lock and key hypothesis assumes the active site of an enzymes are rigid in
• There is no change in the active site before and after a chemical reaction.
Lock and Key Model
The lock and key model of enzyme action, proposed earlier this century,
proposed that the substrate was simply drawn into a closely matching
cleft on the enzyme molecule.
Symbolic representation of the lock and key model of enzyme action.
1. A substrate is drawn into the active sites of the enzyme.
2. The substrate shape must be compatible with the enzymes active site in
order to fit and be reacted upon.
3. The enzyme modifies the substrate. In this instance the substrate is
broken down, releasing two products.
Induced fit model
• More recent studies have revealed that the process is much more likely to
involve an induced fit model(proposed by DANIAL KOSH LAND in 1958).
• 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.
Induced Fit Model
More recent studies
have revealed that the
process is much more
likely to involve an
The enzyme or the reactants
(substrate) change their shape
The reactants become bound to
enzymes by weak chemical bonds.
This binding can weaken bonds
within the reactants themselves,
allowing the reaction to proceed
forcing the substrate
drawn into the cleft
of the enzyme.
The resulting end
product is released
by the enzyme
which returns to its
normal shape, ready
to undergo more
Induced Fit Model
Changing the Active Site
• Changes to the shape of the active site will result
in a loss of function. Enzymes are sensitive to
various factors such as temperature & pH.
• When an enzyme has lost its characteristic 3D
shape, it is said to be denatured. Some enzymes
can regain their shape while in others, the
changes are irreversible.
o The prevention of an enzyme process as a result of interaction of
inhibitors with the enzyme.
Any substance that can diminish the velocity of an enzyme
catalyzed reaction is called an inhibitor.
o It is an inhibition of enzyme activity in which the inhibiting molecular
entity can associate and dissociate from the protein‘s binding site.
TYPES OF REVERSIBLE INHIBITION
o There are four types:
• In this type of inhibition, the inhibitors compete with the substrate for the
active site. Formation of E.S complex is reduced while a new E.I complex is
Examples of competitive
Statin Drug As Example Of Competitive
• Statin drugs such as lipitor compete with HMG-CoA(substrate) and inhibit
the active site of HMG CoA-REDUCTASE (that bring about the catalysis of
• In this type of inhibition, inhibitor does not compete with the substrate for
the active site of enzyme instead it binds to another site known as
• Drugs to treat cases of poisoning by methanol or ethylene glycol
act as uncompetitive inhibitors.
• Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are
uncompetitive inhibitors of liver alcohaldehydrogenase.
o It is a special case of inhibition.
o In this inhibitor has the same affinity for either enzyme E or the E.S
o In this type of inhibition both E.I and E.S.I complexes are formed.
o Both complexes are catalytically inactive.
• This type of inhibition involves the covalent attachment of the inhibitor to
• The catalytic activity of enzyme is completely lost.
• It can only be restored only by synthesizing molecules.
Examples of irreversible
• Aspirin which targets and covalently modifies a key enzyme involved in
inflammation is an irreversible inhibitor.
• SUICIDE INHIBITION :
It is an unusual type of irreversible inhibition where the enzyme converts
the inhibitor into a reactive form in its active site.
• Enzymes are highly specific in nature, interacting with one or few
substrates and catalyzing only one type of chemical reaction.
• Substrate specificity is due to complete fitting of active site and substrate .
Oxydoreductase do not catalyze hydrolase reactions and hydrolase do not
catalyze reaction involving oxidation and reduction.
Types of enzyme
• Enzymes show different degrees of specificity:
Optical or stereo-specificity.
• In this type, enzyme acts on substrates that are similar in structure and
contain the same type of bond.
• Amylase which acts on α-1-4 glycosidic ,bond in starch dextrin and
glycogen, shows bond specificity.
• In this type of specificity, the enzyme is specific not only to the type of
bond but also to the structure surrounding it.
Pepsin is an endopeptidase enzyme, that hydrolyzes central peptide bonds
in which the amino group belongs to aromatic amino acids e. g phenyl
alanine, tyrosine and tryptophan.
• In this type of specificity ,the enzymes acts only on one substrate
Uricase ,which acts only on uric acid, shows substrate specificity.
Maltase , which acts only on maltose, shows substrate specificity.
OPTICAL / STEREO-SPECIFICITY
• In this type of specificity , the enzyme is not specific to substrate
but also to its optical configuration
D amino acid oxidase acts only on D amino acids.
L amino acid oxidase acts only on L amino acids.
• There are two types of dual specificity.
The enzyme may act on one substrate by two different reaction types.
• Isocitrate dehydrogenase enzyme acts on isocitrate (one substrate) by
oxidation followed by decarboxylation(two different reaction types) .
The enzyme may act on two substrates by one reaction type
• Xanthine oxidase enzyme acts on xanthine and hypoxanthine(two
substrates) by oxidation (one reaction type)
• Regulation of enzyme occurs in following ways
– Allosteric regulation
– Activation of Latent Enzyme
– Control of enzyme synthesis
– Enzyme Degradation
• Additional sites other than active sites –
• Types of allosteric enzyme:
– K-class: effectors changes the Km
– V-class: effectors changes the Vmax
• Most of allosteric enzymes are oligomeric in
• Non-reversible binding of effector molecule at
the allosteric sites – conformational change in
the active site of enzyme
Activation of Latent Enzyme
• Some enzymes remain inactive,
• It gets activated at the site of action by the
breakdown of one or more peptide bonds
• Ex: chymotrypsin, pepsinogen and
• Certain enzymes keeps interconverting from
active to inactive and vice-versa depending on
the need of body
• Ex: Glycogen phosphorylase, Phosphorylase b
• The enzyme remains confined to particular area
of cell/body which makes it exclusive
• For instance: fatty acid synthesis takes place in
cytosol whereas fatty acid oxidation takes in
Organelle Enzyme/metabolic pathway
Cytoplasm Aminotransferase, peptidases, glycolysis, HMP shunt
Mitochondria Fatty acid oxidation, Kreb’s Cycle, Urea Cycle, ETC
Nucleus Biosynthesis of DNA and RNA
Endoplasmic Reticulum Protein Biosynthesis, Triacylglycrol and phospholipid synthesis
Lysosomes Lysozyme, phosphatases, phospholipases, hydrolases, proteases
Golgi Appartus Glucos-6 phosphatease, glucosyl and galactosyl transferase
Peroxisomes Catalases, Urea oxidase, D-amino acid oxidase
Control of enzyme synthesis
• Most of the enzyme particularly the rate limiting
ones are present in very low concentration
• Based on the amount of enzyme present in the
body, enzymes are
– Constitutive enzymes: its levels are not controlled
and it remain almost constant
– Adaptive enzymes: their level increases or decreases
as per body needs
• Synthesis of enzyme are regulated by gene.
• Regulation by induction / repression
• Enzymes have their self-destructing
• But it is highly variable and in general
– The key and regulatory enzyme are most rapidly
– Not so important enzyme have longer half life
• Ex: LDH4 – 5-6 days, LDH1 – 8-12 hrs, amylase
– 3-5 hrs
• When same reaction is catalyzed by two or more
different molecular forms of an enzyme, it is
• It may occur in the same species, in the same
tissue, or even in the same cell.
• The different forms of the enzyme generally differ
in kinetic or regulatory properties
• Ex: hexokinase - 4, lactate dehydrogenase (LDH) –
5, creatinine phosphate (CPK) - 3 , creatinine
kinase (CK) - 3, Alkaline phosphate (ALP) – 6,
Alcohol dehydrogenase (ADH) - 2
Diagnostic Importance of Enzyme
• Estimation of enzyme activities in biological
fluid is of great clinical importance.
• The enzyme can be divided in 2 groups
– Plasma Specific or plasma functional enzyme
– Non-plasma specific or plasma non-functional
• Present in the plasma normally and have specific
• Their value is higher in plasma than tissue
• They are mainly synthesized in liver and enter the
• Ex: Lipoprotein lipase, plasmin, thrombin, choline
• Impairment of liver function or genetic disorder –
leads to enzyme deficiency
• Wilson disease – deficiency of ceruloplasmin
• These enzymes are present in the low level in
plasma compared to the tissue
• Estimation of activities of these enzymes serves for the
diagnosis and prognosis of several disease - markers of
• The raised enzyme level may indicate
– Cellular damage
– Increased rate of cell turnover
– Proliferation of cells
– Increased synthesis of enzymes
• Activity increased in acute pancreatitis
• Normal level – 0.2-1.5 IU/l
• Peak value in 8-12 hrs – onset of disease and
returns to normal in 3-4 days
• Urine analysis
• Serum analysis – chronic pancreatitis, acute
parotitis (mumps) and obstruction of
Serum glutamate pyruvate
• Also known as Alanine transaminase (ALT)
• Normal level – 3-4.0 IU/l
• Acute hepatitis of viral or toxic origin
• Jaundice and cirrohosis of liver
Serum glutamate oxaloacetate
• Also known as Aspartate transaminase
• Normal 4-4.5 IU/l
• Increase in myocardial infarction and also in
• SGPT is more specific for liver disease and
SGOT for MI – SGPT more cytosomal enzyme
while SGOT is cytosol and mitochondria
• Elevated in bone and liver disease
• Normal : 25-90 IU/l
• Diagnosis for
– Carcinoma of bone
– Obstructive jaundice
– Paget’s Disease
• Normal : 0.5 -4 KA units/dl
• Increased in cancer of prostate gland and
• Good tumor marker
Lactate dehydrogenase (LDH)
• At least five different isozymes
• Assess the timing and extent of heart damage
due to myocardial infarction MI (heart attack)
– 12 hrs of MI: blood level of total LDH increases, and
there is more LDH2 than LDH1
– 24 hrs of MI: more LDH1 than LDH2
Type Composition Location
LDH1 HHHH Heart and erythrocyte
LDH2 HHHM Heart and erythrocyte
LDH3 HHMM Brain and kidney
LDH4 HMMM Skeletal muscle and liver
LDH5 MMMM Skeletal muscle and liver
Creatinine phosphokinase (CPK)
• Normal : 10-50 IU/l
• Diagnosis of
– MI - Very early detection
– Muscular dystrophy
• Biochemistry – U. Satyanarayan, U. Chakerpeni
• Lehninger Principles of Biochemistry, Fourth
Edition - David L. Nelson, Michael M. Cox.