BCH 201/216

ENZYMES
Enzymes as Catalyst, Active
Sites, Substrate Specificity
 Enzymes as Catalyst

• Enzymes are protein (organic) catalysts that

increase the velocity of a chemical reaction
• Note: Some RNAs can act like enzymes, usually
catalyzing the cleavage and synthesis of
phosphodiester bonds.
• RNAs with catalytic activity are called ribozymes.
 Enzymes as Biological Catalysts/Properties of Enzymes
• Enzymes are proteins that increase the rate of reaction by
lowering the energy of activation
• Enzymes are highly specific, interacting with one or a few
substrates and catalyzing only one type of chemical
reaction
• Enzyme-catalyzed reactions are highly efficient, proceeding
from 10ᴧ3 – 10ᴧ8 times faster than uncatalyzed reactions.
• Not altered or consumed during reaction.

• Reusable
 Enzymes work by reducing the Activation Energy of a Reaction

• An enzyme speeds a reaction by lowering the activation

energy, changing the reaction pathway
– This provides a lower energy route for conversion of
substrate to product
• Every chemical reaction is characterized by an equilibrium

constant, Keq, which is a reflection of the difference in

energy between reactants, aA, and products, bB
An enzyme-controlled pathway
 Diagram of Energy Difference Between
Reactants and Products

• The uncatalyzed reaction has a large activation energy, Ea, seen

at left
• In the catalyzed reaction, the activation energy has been
lowered significantly increasing the rate of the reaction
 The Concept of the Active Site
• The part of the enzyme combining with the substrate is the
active site
• The shape and the chemical environment inside the active
site permits a chemical reaction to proceed with a greater
ease.
• Active sites characteristics include:
– Pockets or clefts in the surface of the enzyme
• R groups at active site are called catalytic groups
– Shape of active site is complimentary to the shape of the
substrate
– The enzyme attracts and holds the substrate using weak
noncovalent interactions
– Conformation of the active site determines the specificity of the
enzyme
• Enzymes are specific to their substrates and this specificity
 Lock and Key (Emil-Fischer’s) Enzyme Model
• In the lock-and-key model, the enzyme is assumed to be
the lock and the substrate the key
– The enzyme and substrate are made to fit exactly like a key fits
into a lock.
– This explains enzyme specificity
– Also explains the loss of activity when enzymes denature.
– However, it fails to take into account protein-conformational
changes to accommodate a substrate molecule.
 Induced Fit (Daniel Koshland’s) Enzyme Model
• The induced-fit model of enzyme action assumes that the
enzyme active site is more a flexible pocket.
• It however undergoes conformational changes to
accommodate the substrate.
• These changes then provide the chemical environment
suitable for the reaction.
• The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)
• This explains how enzymes can react with a range of
substrates of similar types as well as allosterism.
 Enzyme Catalyzed Reactions
• When a substrate (S) fits properly in an active site, an
enzyme-substrate (ES) complex is formed:
E + S  ES
• Within the active site of the ES complex, the reaction
occurs to convert substrate to product (P):
ES  E + P
• The products are then released, allowing another substrate
molecule to bind the enzyme
- this cycle can be repeated millions (or even more) times
per minute
• The overall reaction for the conversion of substrate to
product can be written as follows:
 Enzyme Specificity
Enzymes have varying degrees of specificity for substrates
1.Absolute Specificity: catalyzes one type of reaction for a
single substrate e.g urease catalyzes only hydrolysis of urea.
2.Group Specificity: catalyzes reaction involving any
molecules with the same functional group e.g hexokinase
adds phosphate group to hexoses
3.Linkage Specificity: enzyme catalyzes the formation or
break up of only certain category or type of bond e.g
chymotrypsin catalyzes hydrolysis of peptide bonds
4.Stereochemical Specificity: enzyme recognizes only one of
two enantiomers.
Enzyme Nomenclature and
Classification
 Nomenclature / enzyme classification

IUBMB (International Union of Biochemistry and Molecular

Biologists) has recommended a system of nomenclature
for enzymes in which each enzyme is assigned with two
names:

 Trivial name (common name, recommended name).

 Systematic name ( official name ).

 Trivial, Recommended or Common name

• The name of an enzyme identifies the reacting substance -

usually ends in -ase e.g sucrase catalyzes the hydrolysis of
sucrose
• The name may also describe the function of the enzyme e.g
oxidases catalyze oxidation reactions
• Sometimes common names are used, particularly for the
digestive enzymes such as pepsin and trypsin
• Some names describe both the substrate and the function
 IUBMB System of Enzyme Classification
• Enzymes are classified according to the type of reaction
they catalyze:

Class Reactions catalyzed

 EC 1 Oxidoreductases Oxidation-reduction
 EC 2 Transferases Transfer groups of atoms
 EC 3 Hydrolases Hydrolysis
 EC 4 Lyases Adds atoms/removes atoms
to/from a double bond
 EC 5 Isomerases Rearranges atoms
 EC 6 Ligases Uses ATP to combine
molecules
 Systematic/Official/IUBMB Name
Each enzyme is characterized by a code no. called

Enzyme Code no or EC number and contains four

figures (digits) separated by a dot e.g EC m. n. o. p

First digit (m) represents the class;

Second digit (n) stands for subclass;

Third digit (o) stands for the sub-sub class or subgroup;

Fourth digit (p) gives the serial number of the particular

enzyme in the list.
 Systematic/Official/IUBMB Name
• Using the IUBMB naming system, the official,
systematic name for Hexokinase is:
ATP:D-hexose 6-phosphotransferase E.C. 2.7.1.1.
– This name identifies hexokinase as a member of class 2
(transferases),
– subclass 7 (transfer of a phosphoryl group),
– sub-subclass 1 (alcohol is the phosphoryl acceptor),
– and "hexose-6" indicates that the alcohol
phosphorylated is on carbon six of a hexose.
• Despite the clarity of the IUB system, the names are
lengthy and relatively cumbersome, biochemists
continue to use the common name e.g hexokinase.
Oxidoreductases, Transferases and Hydrolases
Lyases, Isomerases and Ligases
 Factors Affecting Enzyme
Activity/Enzyme Regulation
 Factors Affecting Enzyme Activity

• Temperature

• Hydrogen ion concentration (pH)

• Substrate concentration

• Enzyme concentration

• Products of the reaction

• Presence of activator/inhibitor

• Allosteric effects

• Time
 The effect of temperature
• Q10 (the temperature coefficient) = the increase in reaction
rate with a 10°C rise in temperature.
• For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every 10°C rise
in temperature)
• Enzyme-controlled reactions follow this rule as they are
chemical reactions but at high temperatures proteins denature
• The optimum temperature for an enzyme controlled reaction
will be a balance between the Q10 and denaturation.
 The effect of temperature

Q10 Denaturat
Enzym ion
e
activity

0 10 20 30 40 50

© 2007 Paul Billiet ODWS

Temperature / °C
 Temperature and Enzyme Activity
• Enzymes are most active at an optimum temperature (usually 37°C in humans)
• They show little activity at low temperatures
• Activity is lost at high temperatures as denaturation occurs

• A few bacteria have enzymes that can withstand very high temperatures up to 100°C

• Most enzymes however

are fully denatured at 70°C

 pH and Enzyme Activity
• Enzymes are most active at optimum pH
• Amino acids with acidic or basic side-chains have the proper
charges when the pH is optimum
• Activity is lost at low or high pH as tertiary structure is disrupted
 Optimum pH for Selected Enzymes
• Most enzymes of the body have an optimum pH of about 7.4
• However, in certain organs, enzymes operate at lower and higher
optimum pH values
 Enzyme Concentration and Reaction Rate
• The rate of reaction increases as enzyme concentration
increases (at constant substrate concentration)
• At higher enzyme concentrations, more enzymes are available
to catalyze the reaction (more reactions at once)
• There is a linear relationship

between reaction rate and

enzyme concentration
(at constant substrate
concentration)
 Substrate Concentration and Reaction Rate
• The rate of reaction increases as substrate concentration
increases (at constant enzyme concentration)
• Maximum activity occurs when the enzyme is saturated
(when all enzymes are binding substrate)
• The relationship between reaction rate and substrate
concentration is exponential,
and asymptotes (levels off)
when the enzyme is saturated
 Enzyme Inhibitors
• Inhibitors are chemicals that reduce the rate of enzymic
reactions.
• The are usually specific and they work at low
concentrations.
• They block the enzyme but they do not usually destroy it.

• Many drugs and poisons are inhibitors of enzymes in the

nervous system.
Enzyme Inhibitors
 Enzyme Inhibitors
• Enzyme inhibitors are substances that bind to the enzyme
reversibly or irreversibly, decreases the activity of enzyme
in a process is known as enzyme inhibition.
• Enzyme inhibitors are used to gain information about the
shape of the active site of the enzyme and amino acids
residues in the active site.
• They are used to gain information about regulation or
control of metabolic pathway.
 Enzyme Inhibitors
• They can be used for drug designing.

• They are important for correcting metabolic imbalance.

• They are used for designing herbicides, pesticides and for

killing pathogens e.g. antibiotics, antivirals, antifungals.
 Enzyme Inhibitors - Classification
I. On the basis of specificity:

1. Co-enzyme inhibitor:
•Inhibits co-enzymes only. E.g. cyanide hydrazine, hydroxyl amine inhibits co-enzyme
pyridoxal phosphate.

2. Ion-cofactor inhibitor:
•E.g. fluoride chelate Mg2+ ion of enolase enzyme.

3. Prosthetic group inhibitor:

•E.g. cyanide inhibit Heme of cytochrome oxidase.

4. Apoenzyme inhibitor:
•E.g. antibiotics

5. Physiological modulator:
 Enzyme Inhibitors - Classification
II. On the basis of origin:

1. Natural enzyme inhibitor:

•E.g. Aflatoxin, – amanitin

2. Artificial enzyme inhibitor (synthetic):

•E.g. drugs
 Enzyme Inhibitors - Classification
III. On the basis of reversibility or otherwise

1. Reversible inhibition: The enzyme inhibition in which the

enzymatic activity can be regained after removal of inhibitors.
•- Competitive

•- Non-competitive

•- Uncompetitive

•- Mixed

2. Irreversible inhibition: The enzyme inhibition in which the

enzymatic activity cannot be regained after removal of inhibitors.
 Enzyme Inhibitors - Competitive inhibition

Competitive inhibition
•Competitive inhibitors are substrate analogues that bind to
substrate binding site of enzyme i.e. active site so competition
occurs between inhibitor and substrate for binding to enzyme.
•This type of inhibitor is overcome by increasing the
concentration of substrate.
•The kinetics of reaction is Vmax remains same and Km
increases.
Enzyme Inhibitors - Competitive inhibition
 Enzyme Inhibitors - Competitive inhibition

• In this reaction, initially inhibitor binds to enzyme but with

increase in concentration of substrate causes release of
inhibitor.
• Then, substrate bind enzymes so that the Vmax remains same
while Km increases.

Example:
• Succinate dehydrogenase convert succinate to fumarate.

Succinate —succinate dehydrogenase————–> Fumarate +

NADH +H+
 Enzyme Inhibitors - Competitive inhibition

• Malate is competitive inhibitor of succinate due to structural analogy.

Malate + NAD+ —–succinate dehydrogenase———> Oxaloacetate

• Sulphonamide is competitive inhibitor of PABA during
tetrahydrofolate synthesis.

Example:
• Treatment of methanol poisoning:

Methanol —–alcohol dehydrogenase————-> Formaldehyde (toxic)

Ethanol ——alcohol dehydrogenase———> Acetaldehyde

 Enzyme Inhibitors - Non-competitive inhibition

Non-competitive inhibition:
•In this inhibition, there is no competition between substrate
and inhibitor because the inhibitor binds to enzyme other than
substrate binding site.
•Since the binding site of substrate and inhibitor to enzyme is
different, inhibitor doesn’t affect the affinity of enzyme to
substrate.
•In this case, the inhibition cannot be overcome by increasing
substrate concentration.
 Enzyme Inhibitors - Non-competitive inhibition

• The kinetic reaction is that Vmax decreases and Km

remains same.
• This means that substrate concentration has no effect on
inhibition.
• Binding of substrate and inhibitor are equal.

• The inhibitor changes the conformation of enzyme after

binding so that substrate cannot bind to enzyme.
• This results in decrease of Vmax.
Enzyme Inhibitors - Non-competitive inhibition
 Enzyme Inhibitors - Non-competitive inhibition

Example:
•Heavy metal poisoning. Hg, Pb etc. distort the -SH group
containing enzyme at allosteric site.
•Doxycycline is non-competitive inhibitor of proteinase
enzyme of bacteria.
•The non-competitive inhibitor can be removed by pH
treatment or by hydrolysis.
•In case of metal poisoning, chelator is used.
 Enzyme Inhibitors - Uncompetitive inhibition
Uncompetitive inhibitor:
•This type of inhibition is seen in multi-substrate reaction.

•It is rare type of inhibition.

•The process of inhibition is same as non-competitive but it

only binds to ES-complex.
•At first substrate binds to enzyme to form ES-complex.

•After binding of substrate to active site of enzyme, the

binding site for inhibitor forms at allosteric site so that
inhibitor bind.
 Enzyme Inhibitors - Uncompetitive inhibition
• The binding of inhibitor distorts the active as well as
allosteric site of enzyme, inhibiting catalysis.
• In this inhibition, Vmax as well as Km both decreases.

Examples:
• Inhibition of lactate dehydrogenase by oxalate.

• Inhibition of alkaline phosphatase by L-phenylalanine.

 Enzyme Inhibitors - Mixed inhibition
Mixed inhibition:
•This type of inhibition is commonly seen in multi-substrate
reaction.
•It is the combination of competitive as well as non-
competitive inhibition.
•The mixed inhibitor can bind to both active site and allosteric
site.
•The kinetics of reaction is that Vmax decreases and Km
increases.
 Enzyme Inhibitors - Mixed inhibition
• The Vmax decreases because inhibitor non-competitively binds
to allosteric site and distort enzyme.
• Similarly, Km increases because inhibitor can also bind to active
site competition with substrate.
• This type of inhibition cannot be removed by increasing
substrate concentration.

Examples:
• Ketoconazole is mixed inhibitor bind to 5–α reductase enzyme.

• Pallidium ion is mixed inhibitor of oxidoreductase enzyme.

Regulation of Enzyme Activities
 Regulation of Enzyme Activities
• Enzyme activity must be regulated so that the proper levels of
products are produced at all times and places
• This control occurs in several ways:
– biosynthesis at the genetic level
– covalent modification after biosynthesis
– feedback inhibition
– Storage as inactive forms called zymogens
– regulatory enzymes
– allosteric modification
• A common covalent enzyme modification is the addition or removal
of a phosphate group
 Under high-energy conditions (high ATP), phosphorylation is favored
 Under low-energy conditions (low ATP), dephosphorylation is favored
 This regulates the balance between biosynthesis and catabolism
 Zymogens
• Zymogens (proenzymes) are inactive forms of enzymes
• They are activated by removal of peptide sections
• For example, proinsulin is converted to insulin by removing
a 33-amino acid peptide chain.
 Digestive Enzymes
• Digestive enzymes are produced as zymogens, and are then
activated when needed.
• Most of them are synthesized and stored in the pancreas,
and then secreted into the small intestine, where they are
activated by removal of small peptide sections.
• The digestive enzymes must be stored as zymogens
otherwise they would damage the pancreas.
 Allosteric Enzymes

• An allosteric enzyme binds a regulator molecule at a site

other than the active site (an allosteric site)
• Regulators can be positive or negative:

- a positive regulator enhances the binding of substrate

and accelerates the rate of reaction.
- a negative regulator prevents the binding of the substrate
to the active site and slows down the rate of reaction (non-
competitive inhibition)
 Feedback Control
• In feedback control, a product acts as a negative regulator
• When product concentration is high, it binds to an allosteric
site on the first enzyme (E1) in the sequence, and production
is stopped
• When product concentration is low, it dissociates from E1
and production is resumed
• Feedback control allows products to be formed only when
needed
Reaction/Enzyme Kinetics
 Reaction/Enzyme Kinetics
• When an enzyme is added to a substrate, the reaction that
follows occurs in three stages with distinct kinetics:
Phase Concentration of ES Rate of product formation

Rapid burst of ES Initially slow, waiting for

Pre-steady state
complexes form ES to form, then speeds up

ES concentration
Constant rate of formation,
remains constant as it is
Steady-state (equilibrium) faster than the pre-steady
being formed as quickly as
state
it breaks down

Slow as there are fewer ES

Substrate depletes so fewer
Post-steady state complexes; slows down as
ES complexes form
substrate runs out
 Reaction/Enzyme Kinetics
• The pre-steady state phase is very short, as equilibrium is
reached within microseconds.
• Therefore, if you measure the rate in the first few seconds of
a reaction, you will be measuring the reaction rate in the
steady state.
• This is the rate used in Michaelis-Menten Kinetics.
 Michaelis-Menten Kinetics
• Michaelis-Menten kinetics is a model of enzyme kinetics which
explains how the rate of an enzyme-catalysed reaction
depends on the concentration of the enzyme and its substrate.
• Let’s consider a reaction in which a substrate (S) binds
reversibly to an enzyme (E) to form an enzyme-substrate
complex (ES), which then reacts irreversibly to form a product
(P) and release the enzyme again.
• S + E ⇌ ES → P + E

• Two important terms within Michaelis-Menten kinetics are:

 Michaelis-Menten Kinetics
Two important terms within Michaelis-Menten kinetics are:
•Vmax – the maximum rate of the reaction, when all the enzyme’s
active sites are saturated with substrate.
•Km (also known as the Michaelis constant) – the substrate
concentration at which the reaction rate is 50% of the Vmax.
•Km is a measure of the affinity an enzyme has for its substrate, as
the lower the value of Km, the more efficient the enzyme is at
carrying out its function at a lower substrate concentration.
 Michaelis-Menten Kinetics

• The Michaelis-Menten equation for the reaction above is:

• This equation describes how the initial rate of reaction (V0)

is affected by the initial substrate concentration ([S]).
• It assumes that the reaction is in the steady state, where
the ES concentration remains constant.
 Michaelis-Menten Kinetics

• When a graph of substrate concentration against the rate of

the reaction is plotted, we can see how the rate of reaction
initially increases rapidly in a linear fashion as substrate
concentration increases (1st order kinetics).
• The rate then plateaus, and increasing the substrate
concentration has no effect on the reaction velocity, as all
enzyme active sites are already saturated with the
substrate (0 order kinetics).
Michaelis-Menten Kinetics - Graph of the rate of
reaction against substrate concentration,
demonstrating Michaelis – Menten kinetics, with
Vmax and Km highlighted.
 Michaelis-Menten Kinetics

• This plot of the rate of reaction against substrate

concentration has the shape of a rectangular hyperbola.
• However, a more useful representation of Michaelis–
Menten kinetics is a graph called a Lineweaver–Burk plot,
which plots the inverse of the reaction rate (1/r) against the
inverse of the substrate concentration (1/[S]).
 Michaelis-Menten Kinetics

• This produces a straight line, allowing for the easier

interpretation of various quantities and values from the
graph.
• For example, the y-intercept of the graph is equivalent to
the Vmax.
• The Lineweaver-Burk plot is also useful when determining
the type of enzyme inhibition present by, comparing its
effect on Km and Vmax.
Michaelis-Menten Kinetics - Different types of enzyme
inhibition as shown on a Lineweaver-Burk plot
Cofactors, Coenzymes and
Prosthetic Groups
 Cofactors, Coenzymes and Prosthetic Groups
• These are small nonprotein organic, inorganic molecules and metal
ions that participate directly in substrate binding or catalysis.
• These extend the repertoire of catalytic capabilities beyond those
afforded by the limited number of functional groups present on the
aminoacyl side chains of peptides.
• Enzymes that require a cofactor, coenzyme or prosthetic group but
do not have one bound are called apoenzymes or apoproteins.
• An enzyme together with the cofactor(s) required for activity is called
a holoenzyme (or haloenzyme).
 Cofactors, Coenzymes and Prosthetic Groups

• The term holoenzyme can also be applied to enzymes that

contain multiple protein subunits, such as the DNA
polymerases;
• Here the holoenzyme is the complete complex containing
all the subunits needed for activity
• There are ambiguities in the use of the terms cofactors,
coenzymes and prosthetic groups as different authorities
define these terms differently.
 Prosthetic Groups
• Constant in their definition is that prosthetic groups are tightly
or stably incorporated into the enzyme’s structure by covalent
or noncovalent forces.
• Examples include pyridoxal phosphate, flavin mononucleotide
(FMN), flavin adenine dinucleotide (FAD), thiamin
pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn,
and Zn.
• Metals are the most common prosthetic groups.

• The roughly one-third of all enzymes that contain tightly bound

metal ions are termed metalloenzymes.
 Prosthetic Groups

• Metal ions that participate in redox reactions generally are

complexed to prosthetic groups such as heme or iron-sulfur
clusters.
• Metals also may facilitate the binding and orientation of
substrates,
• the formation of covalent bonds with reaction
intermediates (Co2+ in coenzyme B12),
• or interact with substrates to render them more
 Cofactors
• Cofactors serve functions similar to those of prosthetic groups but
bind in a transient, dissociable manner either to the enzyme or to
a substrate such as ATP.
• Unlike the stably associated prosthetic groups, cofactors therefore
must be present in the medium surrounding the enzyme for
catalysis to occur.
• The most common cofactors also are metal ions.

• Enzymes that require a metal ion cofactor are termed metal-

activated enzymes to distinguish them from the metalloenzymes
for which metal ions serve as prosthetic groups.
 Coenzymes
• Coenzymes are usually organic molecules that are loosely bound
to enzymes and serve as recyclable shuttles-or group transfer
agents-that transport many substrates from their point of
generation to their point of utilization.
• Association with the coenzyme also stabilizes substrates such as
hydrogen atoms or hydride ions that are unstable in the aqueous
environment of the cell.
• Coenzymes transport chemical groups from one enzyme to
another.
• Examples include NADH, NADPH and ATP.
 Coenzymes
• Some coenzymes, such as riboflavin, thiamine and folic acid, are
vitamins, or compounds that cannot be synthesized by the body
and must be acquired from the diet.

The chemical groups carried include

• the hydride ion (H−) carried by NAD or NADP+,

• the phosphate group carried by adenosine triphosphate,

• the acetyl group carried by coenzyme A,

• formyl, methenyl or methyl groups carried by folic acid and

• the methyl group carried by S-adenosylmethionine.

 Isoenzyme
• Isozymes (also known as isoenzymes) are enzymes that
differ in amino acid sequence but catalyze the same
chemical reaction.
• These enzymes usually display different kinetic parameters
(i.e. different KM values), or different regulatory properties.
• The existence of isozymes permits the fine-tuning of
metabolism to meet the particular needs of a given tissue
or developmental stage (for example lactate
dehydrogenase (LDH)).
 Isoenzyme
• In biochemistry, isozymes (or isoenzymes) are isoforms
(closely related variants) of enzymes.
• In many cases, they are coded for by homologous genes
that have diverged over time.
• Although, strictly speaking, allozymes represent different
alleles of the same gene, and isozymes represent different
genes whose products catalyse the same reaction, the two
words are usually used interchangeably.
 Isoenzyme
• Isozymes were first described by hunter and Markert
(1957) who defined them as different variants of the same
enzyme having identical functions and present in the same
individual.
• This definition encompasses

• (1) enzyme variants that are the product of different genes

and thus represent different loci (described as isozymes)
• (2) enzymes that are the product of different alleles of the
same gene (described as allozymes).
 Isoenzyme
• Isozymes are usually the result of gene duplication, but can
also arise from polyploidisation or hybridization.
• Over evolutionary time, if the function of the new variant
remains identical to the original, then it is likely that one or
the other will be lost as mutations accumulate, resulting in
a pseudogene.
 Isoenzyme
• However, if the mutations do not immediately prevent the
enzyme from functioning, but instead modify either its
function, or its pattern of gene Expression, then the two
variants may both be favoured by natural selection and
become specialised to different functions.
• For example, they may be expressed at different stages of
development or in different tissues.
 Isoenzyme
• Allozymes may result from point mutations or from
insertion-deletion (indel) events that affect the dna coding
sequence of the gene.
• As with any other new mutation, there are three things
that may happen to a new allozyme:
• It is most likely that the new allele will be non-functional —
in which case it will probably result in low fitness and be
removed from the population by natural selection.
 Isoenzyme
• Alternatively, if the amino acid residue that is changed is in
a relatively unimportant part of the enzyme, for example a
long way from the active site then the mutation may be
selectively neutral and subject to genetic drift.
• In rare cases the mutation may result in an enzyme that is
more efficient, or one that can catalyse a slightly different
chemical reaction, in which case the mutation may cause
an increase in fitness, and be favoured by natural selection.
 Isoenzyme
• An example of an isozyme is glucokinase, a variant of
hexokinase which is not inhibited by glucose 6-phosphate.
• Its different regulatory features and lower affinity for
glucose (compared to other hexokinases), allows it to serve
different functions in cells of specific organs, such as
control of insulin release by the beta cells of the pancreas,
or initiation of glycogen synthesis by liver cells.
• Both of these processes must only occur when glucose is
abundant, or problems occur.
 Plasma enzymes In Clinical Diagnosis
• Plasma enzyme assays can detect abnormal levels of
enzymes in the blood. The assay measures units of activity
in a sample and so will only measure functional enzyme.
• If levels of an enzyme are abnormally raised, it may
indicate leakage from damaged tissue that the enzyme is
normally found in.
• Abnormally low enzymes may indicate either a non-
functional enzyme, produced more slowly than usual or
being broken down quickly, owing to maybe genetic defect.
 Plasma enzymes In Clinical Diagnosis
Enzyme Location Causes of raised Causes of
levels low levels

Expressed everywhere
Acute/chronic tissue
Specific isoenzymes:
damage, e.g.
•LDH1 – heart,
myocardial infarction.
erythrocytes
Degree of elevation can
1. Lactate •LDH2 – white blood cells
indicate the extent of
dehydrogenase •LDH3 – lung
damage
•LDH4 – white blood cells,
Isoenzymes may help
kidney, pancreas
localise the site of
•LDH5 – liver, skeletal
injury
muscle
 Plasma enzymes In Clinical Diagnosis

Hepatocellular
injury
Widely distributed (acute/chronic liver
but predominantly disease), gall Pregnancy,
bladder disease,
found in the liver, diabetes,
2. Aspartate kidney failure,
heart, skeletal beriberi
transaminase (AST) rhabdomyolysis,
muscle, kidneys, (vitamin B1
MI (myocardial
brain and infarction) deficiency)

erythrocytes Degree of elevation

can indicate the
extent of damage
 Plasma enzymes In Clinical Diagnosis

Hepatocellular
injury
(acute/chronic
3. Alanine Widely distributed
liver disease), bile
transamiase but predominantly
duct problems
(ALT) in liver
More specific
marker of hepatic
injury than AST
 Plasma enzymes In Clinical Diagnosis

Greatest
Prostate
concentration
carcinoma, biliary
4. Alkaline in prostate
obstruction, high
phosphatase Specific isoenzymes
bone turnover
(ALP) are also found in the
(physiological or
liver, bone, kidney,
pathological)
intestine and placenta
 Plasma enzymes In Clinical Diagnosis

Expressed in various
tissues
MM – skeletal
Specific isoenzymes:
muscle dystrophy
•MM – skeletal musc
5. Creatine MB – myocardial
le, heart
kinase infarction in last 2-
•MB – heart
3 days
•BB – brain, neurons,
BB – brain tumour
thyroid, kidney,
intestine
 Plasma enzymes In Clinical Diagnosis

Pancreatitis,
Exocrine pancreas, infections, DKA,
6. Amylase
saliva perforated ulcer,
renal failure
 Plasma enzymes In Clinical Diagnosis

Pancreatitis
7. Lipase Exocrine pancreas (more specific
than amylase)
 Plasma enzymes In Clinical Diagnosis

Widely expressed
Specific isoenzymes Diagnosis and
8. Acid
in the liver, treatment of
phosphatase
erythrocytes, platelets prostate cancer
and bone
 Blood Clotting Enzymes
Introduction:
•Blood clotting is also called coagulation.

•The process of coagulation starts when some tissue is

injured and bleeding is started.
•Injured tissue releases a clotting factor that stimulates
extrinsic and intrinsic pathways.
•The clotting factor released from damaged tissue also
initiates the conversion of inactive zymogen prothrombin into
an active enzyme known as thrombin.
 Blood Clotting Enzymes
• Enzyme thrombin catalyzes the conversion of soluble
plasma proteins fibrinogen into insoluble fibrous protein
fibrin.
• Fibrin makes a mesh-like structure around the platelet plug
made initially and the blood cells are trapped in this mesh
to form a clot.
• After the complete repair of the damaged region, the clot is
dissolved by an enzyme plasmin.
 Blood Clotting Enzymes
• Thrombin plays a major role in converting fibrinogen (a
glycoprotein complex) to fibrin which functions primarily to
occlude blood vessels to stop bleeding.
• Thrombin is a naturally occurring enzyme, which is
responsible for blood clotting.
 Blood Clotting Enzymes

• Serine protease proteins are important enzymes involved in

the process of blood coagulation.
• Blood coagulation is an importance defense mechanism that
prevents the host mammal organism from losing excess
blood or from forming unwanted blood clot.
• The process of coagulation can be initiated by both intrinsic
factors and extrinsic factors.
 Blood Clotting Enzymes

• A cascade of event is followed which activate these

enzymes; normally the enzymes are inactive state a
condition called zymogens.
• Zymogens by their virtual condition of being inactive
prevent unwanted blood clotting which may have a far
reaching consequence such as thrombosis.
 Blood Clotting Enzymes
• Blood clotting in a series of processes, in which the
zymogens’ need to be activated by reacting with its
glycoprotein co-factors.
• Among the serine protease is the thrombin enzyme factor
five (v) responsible for clearing clot in the blood.
• The enzyme is usually present circulating in plasma which
is made up of a single monomer chain, it life span can range
from 12 to 36 hours.
ThankYou