ENZYMES

PART 2
Objectives:
1. Discuss the factors that affect enzyme and
principal of mechanisms of enzyme catalysis
2. Differentiate the 2 Models used in Enzymes.
3. Relate enzyme inhibition to the mechanism of
action of some drugs.
SPECIFICITY OF ENZYMES
Three types:
a) Stereochemical specificity
b) Reaction specificity
c) Substrate specificity
1. Stereospecificity
- ● Optical specificity – only one of the isomers which
acts as a substrate for an enzyme action (e.g. succinic
dehydrogenase while acting on succinic acid will give
only fumaric acid and not malic acid which is its
isomer)
Stereospecificity is due to apoenzyme part of
holoenzyme, while the reaction specificity is due to
the coenzyme or prosthetic group.
2. Reaction Specificity

A substrate can undergo many reactions, but in a

reaction specificity, one enzyme can catalyze only
one of the various reactions. (e.g. oxaloacetic acid
undergoes several reactions, but each reaction is
catalyzed by its own separate enzyme which
catalyzes only that reaction and none of the others)
3. Substrate Specificity
2 types:
a) Absolute specificity – comparatively rare such as urease which
catalyzes hydrolysis of urea
b) Relative specificity – divided as
b.1) Group specificity – Examples of group specificity are
trypsin, chymotrypsin. Trypsin hydrolyzes the residues of only lysine
and arginine, while chymotrypsin hydrolyzes residues of only
aromatic amino acids.
b.2) Bond specificity – bond specificity is observed in case to
proteolytic enzymes, glycosidases, and lipases which act on peptide
bonds, glycosidic bonds, and ester bonds.
FACTORS AFFECTING ENZYME ACTION
1. Effect of Temperature
- enzyme is most active on its optimum temperature
- temperature increases the total energy of the chemical
system with the result the activation energy is increased
- enzyme activity decreases when the temperature of
reaction is below or above the optimum temperature
- increase in temperature also causes denaturation of
enzymes
2. Effect of pH
- enzymatic reaction depends on the pH of the medium
- optimum pH is 4-9
- hydrogen ions in the medium may alter ionization of
active site or substrates (ionization is a requirement for
ES complex)
- pH may influence separation of coenzyme from
holoenzyme complex
3. Effect of enzyme concentration
- enzymatic reaction is directly proportional to the enzyme
concentration
4. Effect of product concentration
- products formed as a result of enzymatic reaction may
accumulate, and this excess of product may lower the
enzymatic reaction by occupying the active site of the enzyme
- it is also possible that under certain conditions of high
concentration of products, a reverse reaction may be favored
forming back the substrate
5. Effect of substrate concentration
- reaction is directly proportional to the substrate
concentration, but only true up to a certain
concentration after which the increasing
concentration of substrate does not further increase
the velocity of the recation
6. Effect of activators and coenzyme
- certain enzymes is dependent of metal ion activators and
coenzymes
7. Effect of modulators and inhibitors
- Whenever the active site is not available for binding of
the substrate, the enzyme activity may be reduced.
- Substances which stop or modify enzymatic reaction are
inhibitors and modulators which can adversely affect the
rate of enzymatic reaction
8. Effect of time
- The time required for completion of an enzyme
reaction increases with decreases temperature from
its optimum.
- Under the optimum conditions of pH and
temperature, time required for enzymatic reaction is
less
The effect of the Enzymes on the
Activation Energy of a Rection
• To speed up chemical
reaction, it changes the path
by which the reaction occur,
providing a lower energy
route for the conversion of
the substrate into the
product, the substance that
result from the enzyme-
catalyzed reaction.
The effect of a Substrate
concentration on Enzyme-Catalyzed
Reaction
• The rates of uncatalyzed chemical reactions often double
every time the substrate concentration is doubled.
• 2 stage of enzyme-catalyzed reaction
A. Formation of the enzyme substrate complex
B. Conversion of the substrate to product and the release
of the product and enzymes from the resulting
enzyme-product complex. (rate-limiting steps)
Enzyme Substrate Complex
Step 1 Step 2 Step 3 Step 4
Enzyme Enzyme- Transition Enzyme- Enzyme-
+ Substrate State Product +
Substrate Complex Complex Product

Active Site – The part of the enzyme that binds with the substrate.
Characteristics of the Active Site
- Enzyme active sites are pocket or cleft in the surface
of the enzyme.
- The shape of the active site is complementary to the
shape of the substrate. (or Fits nearly into the active
site of the enzyme)
- An enzyme attracts and holds its substrate by weak,
non-covalent interaction.
- The conformation of the active site determines the
specificity of the enzyme because only the substrate
that fits into the active site will be used in a reaction
Models Used in Enzyme

• LOCK-AND-KEY MODEL
• INDUCED FIT MODEL

Anyone who can differentiate the 2 Models.

Models Used in Enzyme
• LOCK-AND-KEY MODEL
- By Emil Fischer in 1894.
- The specific action of an enzyme with a
single substrate can be explained using
a Lock and Key analogy first postulated in
1894 by Emil Fischer. In this analogy, the
lock is the enzyme and the key is the
substrate. Only the correctly sized key
(substrate) fits into the key hole (active
site) of the lock (enzyme
• INDUCED FIT MODEL
By Daniel E. Koshland, Jr. in 1958
The induced-fit theory assumes that the substrate plays a role in
determining the final shape of the enzyme and that the enzyme
is partially flexible. This explains why certain compounds can
bind to the enzyme but do not react because the enzyme has
been distorted too much. Other molecules may be too small to
induce the proper alignment and therefore cannot react. Only
the proper substrate is capable of inducing the proper alignment
of the active site.
MECHANISM OF ENZYMES CATALYSIS
• Enzyme catalyze reactions by interacting with
the substrate and the subsequent transition
state. These interaction release energy and
therefore stabilize the transition state, which
ultimately lower the activation energy of the
reaction.
Types of catalytic mechanisms that enzymes
employ;
1. Covalent Catalysis
2. Catalysis by Proximity and orientation
3. Acid-base catalysis
4. Metal ion catalysis
ENZYME INHIBITION

 rates of enzyme-catalyzed reactions can be

decrease by a group of substances called
inhibitors
 enzyme inhibitor is a substance that slows or
stops the normal catalytic function of an enzyme
by binding to it
Reversible Competitive Inhibition
 Competitive enzyme inhibitor is a molecule that
sufficiently resembles an enzyme substrate in shape and
charge distribution that it can compete with the
substrate for occupancy of the enzyme’s active site
• When a competitive inhibitor binds to the enzyme active
site, inhibitor remains unchanged (no reaction occurs),
but its physical presence at the site prevents a normal
substrate molecule from occupying the site – result is
decrease in enzyme activity.
• Formation of enzyme-competitive inhibitor is a
reversible process because it is maintained by weak
interactions (H bonds). With time (fractions of seconds),
the complex breaks up. The empty active site is then
available for a new occupant. Substrate and inhibitor
again compete for the empty active site. Thus, the active
site of an enzyme binds either inhibitor or normal
substrate on a random basis. If inhibitor concentration is
greater than substrate concentration, the inhibitor
dominates.
 Competitive inhibition can be reduced by increasing
the concentration of the substrate.
 Numerous drugs act by means of competitive
inhibition. Example, antihistamines are competitive
inhibitors of histidine decarboxylation, the
enzymatic reaction that converts histidine to
histamine which causes allergy and cold symptoms:
watery eyes and runny nose
2) Reversible Noncompetitive
Inhibition
noncompetitive enzyme inhibitor is a molecule
that decreases enzyme activity by binding to a site
on an enzyme other than the active site
 substrate can still occupy the active site, but the
presence of the inhibitor causes a change in the
structure of the enzyme sufficient to prevent the
catalytic groups at the active site from properly
effecting their catalyzing action

• Binding o these inhibitors modifies the shape of
the active site in much the same way that the
binding of an allosteric effector does.
• Since this binding is nonspecific, these inhibitors
inactivate a broad range of enzyme
unlike in competitive inhibition, increasing the concentration of
the substrate does not completely overcome the inhibitory effect
in this case. However, lowering the concentration of a
noncompetitive inhibitor does free up many enzymes, which
then return to normal activity

• Examples of noncompetitive inhibitors include heavy metal

ions lead ion, silver ion, and mercury ion. The binding sites
for these ions are sulfhydryl (SH groups) located away from
the active site. Metal sulfide linkages are formed, an effect
that disrupts secondary and tertiary structure.
Uncompetitive Inhibition
• Occurs when (II) binds only to the enzyme-
substrate complex (ESES) and not free EE. One
can hypothesize that on binding S, a
conformational change in EE occurs which
presents a binding site for II. Inhibition occurs
since ESIESI can not form product. It is a dead
end complex which has only one fate, to return
to ESES.
3) Irreversible Inhibition
irreversible enzyme inhibitor is a molecule that
inactivates enzymes by forming a strong covalent
bond to an amino acid side-chain group at the
enzyme’s active site
 inhibitors do not have structures similar to that of
enzyme’s normal substrate
 inhibitor-active site bond is sufficiently strong that
addition of excess substrate does not reverse the
inhibition process
Objectives:
1. Explain Allosteric Enzymes and Feedback
control of enzyme.
2. Discuss the biomedical implication of enzyme in
terms of diagnosis and treatment.
Regulation of the Enzyme Activity
It is often regulated by the cell. Often the reason
for this is to conserve energy because if the cell
runs out of chemical energy, it will die; therefore
many mechanism exist to converse energy.
A. Allosteric Enzymes – Enzymes that have more
than one a single site. It has an active sites that
can be altered by binding of a small molecules
called effector molecules or regulators.
- Negative allosterism – inihibit enzyme action
- Positive Allosterism- stimulate enzyme action
B. Proenzyme
The production of the
enzyme in an active
form. The proenzyme is
converted by proteolysis
to the active from when
it has reached the site of
its activity,
C. Protein Modification
This is a process in which a chemical group is
covalently added to or removed from the protein.
This covalent modification either activates the
enzyme or turn it off
D. Feedback Control
- Enzymes are often regulated by environmental
conditions.
- It is an enzyme regulation process in which
formation of a product inhibits an earlier
reaction in the sequence.
- The reaction product of one enzymes may
control the activity of another, especially in a
complex system in which enzymes work
cooperatively.
• The final product in the chain may
inhibit the activity of the first
enzyme. When the concentration of
the final product is low, all of the
reactions proceed rapidly.
• As the concentration increases,
however the enzyme 1 becomes
inhibited and eventually stops.
• The accumulation of the final
product serves as a message that
tells enzyme 1 to shut down because
the cell has enough final product for
its present needs
Enzyme in Medical Diagnosis and
Treatment
• Assay of enzymes present in blood plasma or
serum have been routinely carried out in clinical
chemistry laboratories
• Diagnostic enzymes refers to the enzymes
that are used directly or as components of the
assay system for the determination of number of
substances
• Changes in the concentrations of various
biomolecules are indications of abnormal
metabolic activities,infections,infectious and
non-infectious diseases and inflammatory
conditions.
• Use to detect and quantify certain substances
• Serum
- is the component that is neither a blood cell
(serum does not contain white or red blood cells)
nor a clotting factor; it is the blood plasma with the
fibrinogens removed. Serum includes all proteins
not used in blood clotting (coagulation) and all the
electrolytes, antibodies, antigens and hormones.
Enzyme Normal Body Fluid Disease Diagnosed
Activity
Alanine amino- 3-17 U/L Serum Hepatitis
transferase (ALT)
Acid phosphatase 2.5-12 U/L Serum Prostate Cancer

Alkaline Phosphatase 13-38 U/L Serum Liver or Bone Disease

(ALP)
Amylase 19-80 U/L Serum Pancreatic Disease or
Mumps
Aspartate amino- 7-19 U/L Serum Heart Attack or Hepatitis
Transferase
7-49 U/L Cerebrospinal Heart Attack or Hepatitis
Fluid
U/L- International units per liter; WU/mL= Wroblewski units per milliliter
Enzyme Normal Body Fluid Disease
Activity Diagnosed
Lactate Dehydrogenase 100-350 Serum Heart Attack
(LDH) WU/mL
Creatine 7-60 U/L Serum Heart Attack
Phosphokinase (CPK)
Phosphohexose 15-76 U/L Serum Heart Attack
isomerase (PHI)

U/L- International units per liter; WU/mL= Wroblewski units per milliliter
Medical importance of Non-functional
enzymes
Measurement of these enzymes is important for:
•Diagnosis of diseases- as disease of different
organs cause elevation of different plasma
enzymes.
•Prognosis of the disease- Follow up treatment pre
and post measurement of enzymes
Sources of non-functional enzymes
•Cell damage
•Obstruction of normal pathways: ex:
obstruction of bile duct increases
alkaline phosphatase
•Increase of the enzyme synthesis: ex
Bilirubin increases the rate of synthesis
of alkaline phosphatase in obstructive
liver disease.
•Increased permeability: eg hypoxia.
How it is measure?
- It is measure through blood samples.
- Then blood samples where undergo
Electrophoresis, chromatographic techniques
and isoelectric focusing.