BIOCHEMISTR

Y
Enzymes
 Overview
Enzymes are secreted by living cells and are
complex organic chemical compounds with
definite structure.
They have the remarkable property of speeding
up chemical reactions without being themselves
affected in the process.
They make up the largest and most highly
specialized class of proteins.
Their catalytic action is the key to the
continuous replacement and renewal processes
characteristic of living organisms.
 Chemical Nature
They are colloidal substances.
They are proteins was because of their (+)
reactions to color tests of proteins.
Many of them posses chemical groups that are
non – protein by nature:
◦ Holoenzymes (conjugated proteins) have been found
to be dissociated in a protein compound (apoenzyme)
and a non – protein organic prosthetic portion.
Example: catalase (reddish brown) splits in acid
medium into a colorless protein and
ferriprotoporphyrin.
 Chemical Nature
◦ In some cases, the enzyme may contain only
amino acids and a metal like ascorbic oxidase.
Here the copper component is tightly bound to
the protein.
◦ In some, the metal combine with the substrate
and it is the metal substrate that is acted upon
by the enzyme.
◦ Some need the presence of metal ions
(activators) to activate them. They function in
combination with proteins. Example:
phosphorylase needs Mg2+ for their activity.
 Chemical Nature
◦ Metal ions may serve as bridging group to bind
substrate and enzyme through the formation of
coordination complex or may serve as catalytic group.
◦ Others need the presence of organic substances as co
– factors in order to function. these are called co –
enzymes and they act as intermediary carriers of
electrons, specific atoms or functional groups
removed or contributed to the substrate. These co –
factors may be regarded as easily dissociable moieties
of conjugated proteins. They contain vitamin which is
vital to the cell.
 Nomenclature
They are designated with the suffix ase
preceded by the term which indicates
either one of the following:
◦ The general nature of the substrate. For
example – lipase is an enzyme acting on lipids
in general.
◦ The actual name of the substrate. For example
– sucrase is the enzyme acting on sucrose.
 Nomenclature
◦ The type of reaction catalyzed. For example –
oxidase is an enzyme that catalyzes oxidation.
◦ Combination of several of these designations.
For example – xanthine oxidase catalyzes the
oxidation of xanthine.
In the case of hydrolytic enzymes, the
suffix adjective lytic is used. Examples
are amylolytic, lipolytic, proteolytic
enzymes acting on amylase, lipids, and
proteins, respectively.
 Nomenclature
The inactive form of enzyme is the pro –
enzyme or zymogen. The suffix ogen is
affixed to the name of the active enzyme.
Examples are pepsinogen, trypsinogen,
reninogen, etc. They exhibit the property of a
true enzyme only after they are activated by
suitable hydrogen ion concentration or by an
organic activator called kinase. For example,
pepsinogen is activated by HCl while
trypsinogen is activated by enterokinase from
the succus entericus.
 Classification
According to the International Enzyme
Commission, there are 6 major classes,
subdivided into subclass each with
identifying number. This is chemically
informative and is based on the nature of
the chemical reaction catalyzed by the
enzyme. They are used where accurate
identification is required as in
international research journal, abstracts,
and indexes.
 Oxido – reductase (causes oxidation –
reduction reactions)
Acts on – CH – OH (secondary
alcohol)
Acts on – C = O (ketone)
Acts on – CH = C – (alkene)
Acts on – CH – NH2 (primary amine)
Acts on – CH – NH – (secondary
amine)
Acts on NADP, NADPH
Transferases (transfer functional group)

One carbon group

Aldehyde or ketone group
Acyl groups
Glycerol groups
Phosphate groups
S – containing groups
Hydrolases (causes hydrolysis)
Esters
Glycosidic bonds
Peptide bonds
Other C – N bonds
Acyl anhydrides
Lyases (addition of double bonds)
– C = C – (alkene)

– C = O – (ketone)

– C = N – (cyanides)
Isomerases (isomerization reaction)
Racemases
Ligases (formation of bonds with ATP
cleavage)
– C–O

– C–S

– C–N

– C–C
 Chemical Kinetics
Chemical reactions may be classified on the
basis of the number of molecules that must
ultimately react to form the reaction products.
Thus we have monomolecular, bimolecular, etc.
They may also be classified on a kinetic basis by
reaction order. We have zero order, first order,
second order, and third order reactions
depending on how the reaction rate is influenced
by the concentration of the reactants under a
given set of conditions.
 Chemical Kinetics
1st order reactions are those which proceed at
a rate exactly proportional to the
concentration of one reactant: A  P. The
rate of reaction is exactly proportional to the
rate of disappearance of A or the appearance
of P.
2nd order reactions are those in which the rate
is proportional to the product of the
concentration of two reactants or the 2nd
power of a single reactant: A + B  P
 Chemical Kinetics
3rd order reactions are relatively rare and
are those whose velocity is proportional to
the product of three concentration terms.
Reactions are zero order with respect to
the reactants. The rate of reaction depends
on the concentration of the catalyst or on
some factor other than the concentration
of the molecular species undergoing
reaction.
 Nature of Catalysis
Chemical reactions are governed by the
flow of energy. This is the relationship
between Chemistry and Thermodynamics.
Energy transformations are described as
follows:
◦ Enthalpy (H) = heat content of the system
◦ Entropy (S) = portion of enthalpy used for
increasing disorder or useless energy
◦ Free energy (F) = energy available for work
 Nature of Catalysis
The 1st Law of Thermodynamics is the
Law of Conservation of Energy. It is an
expression of an ideal reversible system in
which the energy utilized to alter the
system is released in exactly equal
amount when the system is returned to its
original state
 Nature of Catalysis
The 2nd Law of Thermodynamics states
that the total energy in an isolated system
does not increase nor decrease, the
transformation of energy from one form to
another proceeds in such a way that the
capacity for performing work decreases.
As potential energy changes to kinetic
energy, disorder increases and part of the
energy is unavailable for work.
 Nature of Catalysis
At maximum entropy, equilibrium is
established, available free energy is zero
and no further change occurs unless
additional energy is supplied from the
outside.
The formation of entropy is not totally
useless. Its increase during biological
processes which is irreversible provides
the driving force and gives direction to all
biological activities.
 Concept of Free Energy
If the free energy content of P is less than
that of A, the change is a negative
quantity (exergonic, releases energy) and
the reaction occurs spontaneously.
If deltaF involves an increase in energy,
as when P is reconverted to A and it is
positive (endergonic, takes up energy) and
the reaction will occur only when energy
is supplied from the outside.
 Concept of Free Energy
Chemical reactions such as A  P takes
place because some molecules of A
contain more energy than the rest of the
molecules, enough to attain an “activated
state” in which chemical bonds may be
made or broken to form products of a
substance at a given temperature.
 Concept of Free Energy
This is the transition state which is the energy
rich state of the interacting molecules at the top
of the activation barrier.
◦ A Transition State P
The rate of reaction is directly proportional to
the concentration of the transitional state
species.
A rise in temperature increases thermal motion
and energy resulting in the increase in number
of molecules capable of entering the transition
state.
 Concept of Free Energy
Living organisms do not influence the rate of
metabolic reactions by invoking changes in
temperature so catalyzed reaction is needed.
Catalysts accelerate chemical reactions by
lowering the free energy of activation.
They combine with reactants to produce the
transition state having less energy than the
transition state of an uncatalyzed reaction.
When the reaction products are produced, the
free catalyst is regenerated.
Kinetics of Enzyme Catalyzed Reactions
The study of the rate of enzyme catalyzed
reactions is very important because the reaction
must proceed rapidly enough to supply the
needs of the organism.
Toxic products should also be eliminated with
sufficient speed to prevent their accumulation so
as not to produce an injurious effect.
When a needed substance is not supplied or a
toxic product is not removed at sufficient speed,
disease may occur.
 Factors Influencing Rate of Enzyme
Reactions
 Concentration of enzyme and its substrate.
◦ Other conditions being constant, the rate of reaction
depends on the concentration of enzyme and
substrate.
 With substrate concentration at saturating point,
increasing the enzyme increases substrate catalysis.
The limiting factor is substrate concentration
because of its capacity of lowering the energy of
activation thereby increasing the number of
molecules activated.
 With the concentration of enzyme fixed, increasing
substrate concentration, a typical hyperbolic curve
is produced.
 Factors Affecting Rates of Enzyme
Reactions
Dependence of Enzyme Reaction Rates
on Co – Factors:
◦ Effect of temperature: The effect of
temperature is twofold. Increase in rate with
increasing temperature until a maximal rate is
achieved.
◦ A region of high temperature in which the rate
decreases with increased temperature due to
thermal inactivation of the enzyme
(denaturation).
 Factors Affecting Rates of Enzyme
Reactions
Co – Factors (continuation)
◦ Effect of pH
 Most enzymes have a
characteristic pH at which their
activity is maximal. Above and
below this pH, the activity
declines
 Factors Affecting Rates of Enzyme
Reactions
◦ The pH activity relationship depends on:
 pK of ionizable groups of active site on the
enzyme which participates in binding the
substrate.
 pK of functional groups of the substrate which
participates in binding to the enzyme.
 pK of the functional groups of the enzyme
molecules responsible for the catalytic act.
 pK of other groups of the enzyme molecule
whose state of ionization may determine
specific catalytically active conformation of the
molecule.
 Factors Affecting Rates of Enzyme
Reactions
Co – Factors (continuation)
◦ The influence of pH may be due to
 Enzyme denaturation
 Effects on charged state of enzyme and substrate
◦ The pH activity relationship of an enzyme
may be a factor in intracellular control of
enzyme activity since cells contain hundreds
of enzymes, all differently responsive to pH,
the intracellular pH may represent an
important element in the complex network of
controls over cell metabolism.
 Factors Affecting Rates of Enzyme
Reactions
Co – Factors (continuation)
◦ The effect of time: Time is an important
element in defining other conditions which
regulate the rate of enzyme action. Accdg to
Gotner, there cannot be an optimum H+
concentration nor optimum temperature
independent of time.
◦ Products of Reaction: The rate of enzyme
action decreases as products of the reaction
increases because of the reversibility of
enzyme action.
 Factors Affecting Rates of Enzyme
Reactions
Co – Factors (continuation)
◦ Light and other physical agents: Light rays affect the
activity of enzymes. Blue and red lights increase
activity of salivary amylase, while ultraviolet rays and
radium exert inhibitory effect. Violent shaking
destroys the enzyme by causing denaturation.
◦ Chemical Agents: Some chemical agents accelerate
enzyme action. If salivary amylase is dialyzed it
becomes inactive but the activity is regenerated by
the addition of sodium, potassium, and other
chlorides. The chlorides here are coenzymes which
are specific for the activity of salivary amylase.
 Reversibility and Synthetic Action
 The reversibility of the enzyme action is the basis
for the various synthetic phenomenon occurring in
the body. For example, ingested proteins are
hydrolyzed into amino acids by the proteolytic
enzymes of the intestinal tract before they are
absorbed. In the tissues, they are resynthesized into
proteins through the action of proteases found in the
tissue cells,
Proteolytic enzymes, intestines
Proteins Amino Acids
Proteases, tissue cells
 Enzyme Inhibitors
Two types of inhibition
◦ Reversible: The substrate and the inhibitor
compete for the same site.
 Competitive inhibitor competes with the substrate
for binding to the active site of the enzyme. Because
they usually resemble the normal substrate in the 3
dimensional structure & cheats the enzyme into
binding to it.
 Non – competitive inhibitor binds at a different site
from that of the substrate so it has no effect to
normal binding. It affects enzyme activity by
altering the conformation of the enzyme molecule
so that reversible inactivation of the catalytic site
 Enzyme Inhibitors
◦ Irreversible: The inhibitor does not combine
with the free enzyme nor affect the reaction
with its normal substrate. It combines with the
enzyme – substrate complex forming an
inactive enzyme – substrate – inhibitor
complex:
ES + I  ESI
◦ It is not reversed by increasing substrate
concentration. Rather the degree of inhibition
may increase upon increasing the substrate
concentration.
 Metabolic Inhibition
The enzyme may be inhibited by
compounds having a structure related to
the natural substrate. Utilization of the
required metabolites by the
microorganisms is dependent on enzymes
and these enzymes may be inhibited by
substances whose structures are related to
those of the metabolites. These type of
inhibitors are called antimetabolites.
 Metabolic Inhibition
They may also block the utilization of
metabolites that are synthesized by the organism
itself.
It is an important aid in the study of metabolic
pathways because by providing the required
metabolite, the inhibition of growth can be
overcome.
There is evidence to show that
pharmacologically active substances alter the
metabolic processes by virtue of their action on
enzymes or enzyme system.
 Enzyme Specificity
Two distinct features to determine the
specificity of an enzyme for a substrate:
◦ Substrate must possess the specific chemical
bond capable of being attacked by the enzyme
◦ Substrate must possess functional groups
which binds and positions the substrate
molecule on the catalytic site.
 Types of Enzyme Specificity
Absolute specificity: The enzyme
catalyzes a single reaction in a single
substrate. The substrate molecule fits the
active site in a lock and key relationship.
It will not attack even the closely related
molecules.
Group specificity: This is a lower degree
of specificity. The enzyme acts on a
particular chemical group.
 Type of Enzyme Specificity
Relative specificity (Reaction specificity):
This is the lowest degree of specificity.
The enzyme reacts with a variety of
similar compounds at different rates.
Stereochemical specificity (Spatial
specificity): this occurs when enzymes act
only on one of the optical isomers.
 Relation of Conformation of Enzyme
Molecule to Catalytic Activity
The catalytic activity is dependent on the
appropriate conformation of each enzyme.
However, this conformation is not absolute.
The active site (both binding forces and
participating groups) may not pre – exist in
the form required until the approach of the
substrate.
Koshland suggested that surface
conformation may then be altered resulting
in an “induced fit”.
Relation of Conformation of Enzyme
Molecule to Catalytic Activity
The catalytic site of an enzyme contains
specific functional groups which can bind
the substrate and bring about catalysis.
Enzyme activity is dependent in a general
way on the specific three – dimensional
conformation of the molecule and that
denaturation of the enzyme means a loss
of its activity.
 Relation of Conformation of Enzyme
Molecule to Catalytic Activity
Some enzymes do not need other parts of the
structure so that folding of the peptide chain
results to bring together the essential groups.
The advantages of this arrangement are:
◦ It makes possible for large enzyme molecules to exert
stress on the substrate by means of conformational
change in the enzyme structure.
◦ Conformational change also makes possible the
unloading of the products of the reaction from the
active site after the conformation reverts back to its
original state.
 Acid – Base Catalysis of Organic
Reactions
Acids and bases are the most versatile and
universal catalysts of organic reactions
and they can be divided into 3 types:
◦ Specific acid and base catalysis
◦ General acid- general base catalysis
◦ Lewis acids or Lewis base catalysis
 Mechanism of Enzyme Action
Adsorption Theory: It is suggested that
the big surface area of the enzyme serves
to gather in favorable proximity, the
different reactant, substrates, and
cofactors producing marked local increase
in concentration, so collisions occur,
leading to specific reactions.
 Mechanism of Enzyme Action
Activation by Strain or Bond Distortion
Theory: It maintains that the specific
attachment to the enzyme surface induces
stratin or deformation of the bonds which
are to be broken.
 Mechanism of Enzyme Action
The Functional Groups at the Catalytic Site
Theory: It is believed that at the active site
there is present amino acid residues or other
groupings such as metal ions which can serve
as acid or base or as nucleophilic or
electrophilic residues and these participate in
the reactions. The metal can bind with
groupings in the substrate and act as strain
producing agent forming a chelated
intermediary compound.
 Multiple Enzyme System
Enzymes work together in sequential chains
in which the products of the first becomes
the substrate of the next and so on.
Three Types
◦ Simple: Enzymes are in solution in the cytoplasm
and the independent molecular entities are not
physically associated with each other at any time
during the action. The small substrate molecules
which have high rate of diffusion finds their way
from one enzyme to another.
 Multiple Enzyme System
◦ Highly Organized: The individual enzymes are
physically associated and function together as
enzyme complexes.
◦ Most highly organized: This is true of the
enzymes associated with large supra molecular
structures such as membranes or
mitochondria.
 Self Regulating Properties of Enzyme
Systems
Regulatory enzymes which make possible
the control and integration of the activity
of multienzyme systems have been
identified.
The end product of the sequence of
reactions can inhibit the 1st enzyme with
the result that the rate of the entire
sequence catalyzing the conversion of the
substrate to its product can be regulated.
 Self Regulating Properties of Enzyme
Systems
In most self – regulating enzyme system, it is
the first enzyme of the sequence which is
inhibited by the end product of the sequence.
This enzyme is called the regulatory or
allosteric enzyme and the inhibitory metabolite
(isoleucine) is the effector or modulator. If the
later is inhibitory, it is a negative effector or
modulator. In self – regulating systems, the
regulatory enzyme is polyvalent coz it
responds to more than one specific modulator.
 Self Regulating Properties of Enzyme
Systems
The regulatory enzyme may also possess
a specific positive modulator. Most often
the positive modulator is the substrate of
the regulatory enzyme or some other
metabolic intermediates.
Regulatory enzymes also have a common
property. They catalyze reactions which
are essentially irreversible under
intracellular conditions.
 Self Regulating Properties of Enzyme
Systems
Regulatory enzymes are of two types: allosteric
or non – covalently regulated enzymes & the
covalently regulated enzymes or those
modulated by covalent modification of some
specific functional groups necessary for activity.
The 1st step in multi – enzyme reaction sequence
is often called the committed step. Once it takes
place, all the ensuing reactions take place. The
inhibition of the 1st step in the sequence by an
allosteric modulator yields maximum economy
of metabolite.
 Properties of Regulatory Enzymes
 They are much larger, more complex, and more
difficult to purify than enzymes not endowed with
regulatory properties.
 They show anomalous properties.
 Some are unstable 0oC but stable at room
temperature.
 All have more than one polypeptide chain.
 They have atypical dependence of reaction velocity
on substrate concentration not easily treated by
simple Michaelis Menten relationship.
 They show atypical inhibition by modulating
substance.
Major Classes of Regulatory Enzymes
Homotropic enzyme: The substrate molecule is
not only a substrate but also a positive
modulator which accelerates the activity of the
enzyme depending on its concentration.
Heterotropic enzyme: They are stimulated or
inhibited by specific naturally occurring effector
or modulator other than the substrate, such as
the end product isoleucine. They possess not
only a binding or catalytic site for the substrate
but also a second site at which modulator
molecule binds.
Kinetics of Regulatory Enzymes
Homotropic enzymes show a sigmoid rather that
hyperbolic curve of Michaelis Menten. This
implies that the binding of the first substrate
molecule to the enzyme enhances the binding of
a second substrate molecule and thus enhance
activity.
Some respond to rising substrate concentration
to yield an increase in affinity of the enzyme for
its substrate without a change in maximum
velocity.
Kinetics of Regulatory Enzymes
In heterotropic enzymes the effector
molecule may inhibit or stimulate the
reaction at any given substrate
concentration.
Each enzyme has its own characteristic
kinetic behavior.
Their specificity for the modulator
molecule whether (+)/(-) may be just as
great as their specificity for the normal
substrate.
Kinetics of Regulatory Enzymes
The capacity of regulatory enzymes to be
activated or inhibited by its specific
modulator can be abolished without
damaging its catalytic activity, by treating
with an agent which produces chemical
modification of the modulator site.
The enzyme is said to be desensitized.
This is irreversible.
 Mechanism of Regulation
It is postulated that the binding of the
modulator to the specific site causes a
conformational change in the three
dimensional structure of the regulatory
enzyme molecule. The form produced
results in, intrinsically, either more active
or less active catalytically than the form
with the modulator site unoccupied.
 Isozymes
A number of enzymes have been found
exist in multimolecular forms within a
single specie or even a single cell. They
are distinct molecular species differing in
net electrical charge but performing
identical function. They are called
“isozymes.”
 Lysozyme
This is an enzyme found in human tears
and egg white. It catalyzes the hydrolytic
cleavage of complex polysaccharides in
the protective cell walls of some bacteria.
It is called lysozyme because it can lyze
or dissolve bacterial cells producing a
bactericidal effect.
Enzyme Defect Due to Genetic Mutation
There are genetic diseases in man in which an
enzyme is inactive or defective in its catalytic or
regulatory function.
This enzyme has been found to contain one or
more wrong amino acids in the polypeptide
chain due to mutation in the DNA coding.
Catalytic activity depends not only on the
presence of specific amino acid in the catalytic
and regulatory sites but also on the overall three
dimensional conformation.
Enzyme Defect Due to Genetic Mutation
Any substitution of a single amino acid in
some critical position of the chain may
alter or destroy its catalytic activity
Worst when the altered enzyme is a
member of enzyme system catalyzing a
central metabolic pathway the result may
be alarming or even fatal disturbance of
metabolism.
 Antienzymes
They are substances formed by the body as a
reaction against an enzyme introduced into the
circulation.
There are some anti – enzymes in nature.
◦ Blood serum contains anti – trypsin which inhibits the
action of trypsin.
◦ The mucosa of the stomach and intestines contains
also anti – pepsin and anti – trypsin which inhibits the
action of pepsin and trypsin, respectively
◦ In the blood of pregnant women about 4 weeks after
inception of pregnancy there appear certain enzymes
which are capable of splitting the protein of placenta.
 Clinical Applications of Enzyme
Chemistry
Enzyme action finds practical application
in the study of diseases. Examples are:
◦ Examination of gastric juice for the diagnosis
of gastric pathologies.
◦ Analysis of duodenal contents for pancreatic
and other enzymes.
◦ Determination of the activity of blood plasma
phosphatase in cases of rickets, carcinoma,
arthritis, and tuberculosis.
 Clinical Applications of Enzyme
Chemistry
Other Examples:
◦ Determination of serum amylase and serum
lipase in cases of pancreatic pathology.
◦ Determination of serum glutamic pyruvic acid
transaminase (SGPT) which is elevated in
cases of liver pathologies.
◦ Determination of serum glutamic oxaloacetic
acid transaminase (SGOT) which is increased
in myocardial infarction.