ENZYME

Video 1
What are ENZYMES?
Enzymes are
biological catalysts
that speed up the rate
of the biochemical
reaction.
 Importance
Nothing works
without enzymes!
▪ Enzymes play an important role
in metabolism.
▪ All chemical reactions in living
Synthesis enzyme
organisms require enzymes to work.
• Anabolism +
- building molecules
• Catabolism Digestive enzyme

- breaking down molecules

+
 Medicinal and Clinical Uses
Therapeutics
• Enzymes can be used for aiding digestion.
Example: Amylases, Proteases and Lipase.
• They can also be used as Deworming agents.
Example: Papain.
• Enzymes can be used as surface disinfectants.
Example: Trypsin.
 Medicinal and Clinical Uses
Therapeutics
• Enzymes act as anti-clotting agents like
Fibrinolytic and Thrombolytic.
Examples: Urokinase and Streptokinase.
• A recent advance in treating heart attacks is
the use of tissue plasminogen activator (TPA),
which activates the enzyme plasminogen.
When so activated, this enzyme dissolves
blood clots in the heart and often provides
immediate relief.
 Medicinal and Clinical Uses
Diagnosis
• The levels of enzymes in the blood often indicates that there
is tissue damage in an organ and that cellular contents are
spilling out (leaking) into the bloodstream.
• The level of specific enzyme in the plasma frequently
correlates with the extent of tissue damage.

Necrosis – localized death of living tissue

 Medicinal and Clinical Uses
Diagnosis
 CLINICAL APPLICATIONS OF ENZYMES
Isoenzymes in Medical Diagnosis
▪ Enzymes that catalyze the same reactions but vary
slightly in structure are called ISOENZYMES.
▪ Example: There are five isoenzymes for lactate
dehydrogenase (LDH), an enzyme that converts lactic
acid to pyruvic acid.

• Each LDH consists of a quaternary structure containing four subunits.

• In heart muscle, the most prevalent subunit is designated as H.
• In skeletal muscle, the major subunit is designated as M.
Industrial Uses of Enzymes

• Enzymes can be used in the textile industry.

Example: Amylase as softening agent for starched
clothes.
• Enzymes have the importance in the paper
manufacturing. Examples: Endoxylanases for
bleaching of Wood pulp.
• They can be used in the manufacturing of organic
compounds. Example: Bacterial enzymes for the
manufacturing of acetone, butanol, lactic acid etc.
Biological washing powders and liquids contain enzymes
that help remove stains.

The enzymes are coated with a special wax that melts in the
wash, releasing the enzymes. Once the stains have been
broken down, they are easier for the detergent to remove.

⚫ Proteases break down proteins

in stains such as grass, blood
and sweat.
⚫ Lipases break down stains
containing fat and oil.
⚫ Carbohydrases break down
carbohydrate-based stains,
such as starch.
 Confectionary
▪ A type of enzyme called isomerase converts the sugar
glucose into fructose, another type of sugar.
Fructose is sweeter than glucose, so a smaller amount is
needed. This makes fructose syrup a useful ingredient in
slimming foods.

▪ Invertase is used to create soft-centered chocolates.

The center initially contains sucrose (cane sugar) and is
hard.
The invertase breaks
down the sucrose into the
simpler sugars glucose
and fructose, making the
center soft and runny.
• Meat industry. Papain which is
proteolytic in action, therefore
hydrolyses peptide bonds thus
for tenderizing meat and beef .

• Manufacturing of cheese.
Rennin (chymosin) found in
stomach, converts milk protein
casein to curd like calcium
paracaseinate.
 What are ENZYMES?
▪ Enzymes are biological
catalysts that speed up the
rate of the biochemical
reaction.
▪ Most enzymes are three-
dimensional globular
proteins (tertiary and
Proteins are quaternary structure).
molecules are
folded that are
folded into a
specific shape.
Important Terms to Understand Biochemical
Nature and Activity of Enzymes

• Active site
• Substrate
• Enzyme-substrate complex
• Holoenzymes
• Apoenzymes
• Cofactor Videos
• Coenzyme
• Prosthetic groups 2 and 3
 Enzymes
▪ Enzyme
molecules
contain a
special pocket
or cleft called
the active sites.
▪ Reactant/s in
biochemical
reactions are
called
substrate/s.
 ▪ When in its proper three-
dimensional shape, enzymes
has an indentation on one
side of the structure.
▪ This indentation is known as
the active site, and it is lined
with amino acid side chains.

Active site is involved in

catalysis, i.e., where the
substrate or substrates attach
to by noncovalent forces:
• hydrogen bonding
• electrostatic attractions
• hydrophobic interaction
 Composition of Enzyme
▪ Most enzymes are large protein
molecules with complex three-
dimensional shapes

▪ Non-protein enzymes: A few enzymes

are made of ribonucleic acids and
catalyze cellular reactions involving
nucleic acids.
• Ribozyme – catalytic RNA involved
in the removal of parts to form a
functional RNA molecule
 Composition of Enzyme
▪ Simple/Globular
• consist entirely of amino acid units
• Examples: pepsin, trypsin,
ribonuclease

▪ Conjugated/complex protein
(holoenzymes)
• Protein part – apoenzyme
• Non-protein part
– cofactor/coenzyme
Holoenzymes
Organic

Inorganic
 Co-factor
▪ Non-protein, inorganic substances (metals or
ions bound within enzyme molecule)

Metal Ion Enzyme

Fe2+ or Fe3+ Peroxidase

Cu2+ Cytochrome oxidas e
Zn2+ DNA polymerase
Mg 2+ Hexokinase
Mn2+ Arginase
K+ Pyruvate kinase
Ni 2+ Urease
Mo Nitrate reductase
Se Glutathione peroxidase
 No enzyme-substrate complex
Very very very slow reaction

Cofactor

Reaction speeds up

• The apoenzyme is unable to bind to its substrate.

• When the required cofactor, in this case a copper ion, Cu2+, is available, it
binds to the apoenzyme.
• Now the active site takes on the correct configuration, the enzyme-
substrate complex forms, and the reaction occurs.
 Coenzyme
▪ Small organic molecules
acting as carrier molecule
▪ many vitamins or Cosubstrate Prosthetic
provitamins group

▪ Types :
• Cosubstrate: binds transiently/loosely to
enzymes near the active site
Example: NAD+ (niacin, B3) - dissociate from the
enzyme in an altered state
• Prosthetic group: binds tightly/ permanently
Example: FAD (riboflavin, B2)
Coenzyme
Coenzyme
 Coenzyme

▪ Collagen contains hydroxylysine and hydroxylproline.

▪ Hydroxylation of lysine and proline in collagen formation are
catalyzed by enzymes and require ascorbic acid (Vit. C).
▪ In Vit. C deficiency, hydroxylation is impaired, and the triple helix
of the collagen is not assembled properly.
 Nomenclature of enzymes
1. The suffix –ase identifies a substance as an enzyme.
Ex.: urease, sucrase, lipase are all enzyme designations.

The suffix –in is still found in many of the digestive enzymes.

Ex.: trypsin, pepsin.

Enzymes are most commonly named by using a system that

attempts to provide information about the function (rather than
the structure) of the enzyme.

2. The type of reaction catalyzed (function) by an enzyme is

often noted with a prefix.
Ex.: Oxidase catalyzes oxidation reaction
Hydrolase catalyzes hydrolysis reaction
 Nomenclature of enzymes
Important aspects in naming enzymes are as follows:

3. The identity of the substrate is often noted in addition to

the type of reaction/function
Ex.: alcohol dehydrogenase oxidizes ethanol
glucose oxidase, pyruvate carboxylase, succinate
dehydrogenase

4. Infrequently, the substrate but not the reaction type is given

Ex.: urease, lactase
 Enzymes are classified into six functional Classes
(EC number Classification) by the
International Union of Biochemists (I.U.B.).
on the Basis of the Types of Reactions that they Catalyze

EC 1. Oxidoreductases
EC 2. Transferases
EC 3. Hydrolases
EC 4. Lyases
EC 5. Isomerases
EC 6. Ligases
Principle of the International Classification
▪ A series of four number serves to specify a particular
enzyme.
▪ The numbers are preceded by the letters EC (enzyme
commission).
▪ Example: EC: (2.7.1.1) Hexokinase

2: Class (transferase)
7: Subclass (transfer of phosphate)
1: Sub-sub class (characteristic of the reaction; alcohol -OH is
phosphate acceptor)
1 : order of the individual entries/entry number

Specific name
ATP,D-hexose-6-phosphotransferase
 Oxidoreductase
Reduction equivalent

A: + B → A + B:
▪ Oxidase – oxidation of a substrate
-involved in oxidizing or taking away electrons from a
substrate
▪ Reductase – reduction of a substrate
-involved in reducing or giving electrons to a substrate
▪ Enzyme-influenced
discoloration (browning)
that occurs when freshly
cut fruit (apples, pears,
etc.) and vegetables
(potatoes) are exposed to
air for a short period of
time.

▪ The enzyme involved, which is present in the food, is an

oxidoreductase enzyme called phenolase (or polyphenoloxidase),
a conjugated enzyme in which copper is present.
 Oxidoreductase

▪ Dehydrogenase – removal or addition of hydrogen atoms

 EC 1. Oxidoreductases
• Biochemical Activity:
– Catalyse Oxidation/Reduction Reactions
– Act on many chemical groupings to add or
remove hydrogen atoms.
• Examples:
– Lactate dehydrogenase
– Glucose Oxidase
– Peroxidase
– Catalase
– Phenylalanine hydroxylase
 Transferase

AB + C → A + BC
▪ Kinase – transfer of a phosphate group between substrates

ATP

ADP
 Transferase
▪ Transaminase – transfer of an amino group
between substrates

ALT: Alanine transaminase

 EC 2. Transferases
• Biochemical Activity:
– Transfer a functional groups (e.g. methyl or
phosphate) between donor and acceptor
molecules.
• Examples:
– Transaminases (ALT & AST)
– Phosphotransferases (Kinases)
– Transmethylases
– Transpeptidases
– Transacylases
 Hydrolase

AB + HOH → AH + BOH

•Catalyze hydrolysis reactions where water is

the acceptor of the transferred group
 Hydrolase
▪ Protease – hydrolysis of peptide linkages in proteins
 Hydrolase
▪ Carbohydrase – hydrolysis of glycosidic bonds in
carbohydrates: sucrase, lactase, cellulase

β-amylase

Maltase
 Hydrolase
▪ Lipase – hydrolysis of ester linkages in lipids
 Hydrolase
▪ Nuclease
– hydrolysis of sugar
phosphate ester
bonds in nucleic acid

▪ Phosphatase – hydrolysis of phosphate ester bonds

 EC 3. Hydrolases
• Biochemical Activity:
– Catalyse the hydrolysis of various bonds Add
water across a bond.
• Examples:
– Protein hydrolyzing enzymes (Peptidases)
– Carbohydrases (Amylase, Maltase, Lactase)
– Lipid hydrolyzing enzymes (Lipase)
– Nucleases
– Phosphatases
 Lyase (Synthase)

▪ Catalyze the
dissociation of a
molecule without
using water.
 Lyase (Synthase)
▪ Dehydratase - removal of the components of water
from a double bond

ACP (Acyl carrier protein)

▪ Decarboxylase – removal of carbon dioxide from substrate

 Lyase (Synthase)

▪ Deaminase – removal of ammonia from substrate

 EC 4. Lyases
• Biochemical Activity:
– Cleave various bonds by means other than
hydrolysis and oxidation.
– Add Water, Ammonia or Carbon dioxide
across double bonds, or remove these
elements to produce double bonds.
• Examples:
– Deaminase
– Carbonic anhydrase
 Isomerase

• catalyzes the conversion of a substrate into another compound

that is isomeric with it.
• facilitates intramolecular rearrangements in which bonds are
broken and formed or they can catalyze conformational
changes.
 Isomerase
Maleate
isomerase

Maleate Fumarate
Cis-configuration Trans- configuration
 Isomerase
▪ Racemase – conversion of D to L isomer or vice versa

▪ Mutase – transfer of a functional group within a molecule

 Isomerase
▪ Epimerase – conversion of one sugar epimer into another
 EC 5. Isomerases
• Biochemical Activity:
– Catalyse isomerization changes within a
single molecule.
– Carry out many kinds of isomerization:
• L to D isomerizations.
• Mutase reactions (Shifts of chemical
groups).
• Examples:
– Isomerase
– Mutase
 Ligase

A + B → AB
•Catalyze ligation, or joining of two substrates
•Require chemical energy (e.g. ATP)
 Ligase
▪ Synthetase – formation of new bond between two
substrates with participation of ATP
 Ligase

▪ Carboxylase – formation of new bond between

substrate and carbon dioxide with participation of ATP
 EC 6. Ligases/Synthetases
• Biochemical Activity:
– Join two molecules with covalent bonds
– Catalyse reactions in which two
chemical groups are joined (or ligated)
with the use of energy from ATP.
• Examples:
– Acetyl~CoA Carboxylase
– Glutamine synthetase
Class Reaction Type Subclass
 Important characteristic
features of enzymes
▪ Catalytic power (ratio of enzyme catalyzed
rate of a reaction to the uncatalyzed rate)
▪ Specificity (selectivity of enzyme to their
substrate)
▪ Regulation (Control of enzymatic reaction)
 Enzyme Catalytic Power

❑Catalysts are substances that speed up the

rate of a chemical reaction.
▪ Enzymes cause cellular reactions to occur
millions of times faster than corresponding
uncatalyzed reactions.
▪ Enzymes can increase the rate of a reaction
by a factor of up to 1020 over uncatalyzed
reactions.
 Activation energy: an input
of energy before molecules
will react together

▪ Enzymes Lower a Reaction’s

Activation Energy
▪ Enzymes are not consumed
(reusable/recyclable)
 Activation Energy (EA)

▪ An enzyme ALTERS the rate of a reaction, (lowering of activation

energy) but not its free energy change or position of equilibrium.
 Specificity
The Models of Enzyme Action
▪ Lock and key theory (Fisher, 1890)
• In the lock-and-key model of enzyme action, the shape or
geometry of the substrate and conformation of the active site
are complementary to one another.
• Only substrates with a complementary geometry can be
accommodated at the active site, much as a lock accepts only
certain keys.
• In the lock-and-key
model, the active site in
the enzyme has a fixed,
rigid geometrical
conformation.

Enzyme-substrate complex interactions

 The Models of Enzyme Action
▪ Induced fit theory (Koshland , 1958)
• The lock-and-key model explains the action of
numerous enzymes. It is, however, too restrictive for
the action of many other enzymes.
• Experimental evidence indicates that many enzymes
have flexibility in their shapes. They are not rigid and
static. There is constant change in their shape.
• The induced-fit model is used for this type of
situation.
• The induced-fi t model allows for small changes in
the shape or geometry of the active site of an
enzyme to accommodate a substrate.
 Induced fit theory (Koshland , 1958)
• In induced induced-fit model of enzyme action, the enzyme
undergoes a conformational change upon binding to substrate.
• The shape of the active site becomes complementary to the
shape of the substrate only after the substrate binds to the
enzyme.
• This is a more thorough explanation for the active-site
properties of an enzyme because it includes the specificity of
the lock-and-key model coupled with the flexibility of the
enzyme protein.

Enzyme-substrate
complex interactions
 ▪ The forces that draw the
substrate into the active site are
many of the same forces that
maintain tertiary structure in
the folding of peptide chains.
• hydrogen bonding
• electrostatic attractions
• hydrophobic interaction

▪ Alternatively, cofactors
such as positively charged
metal ions often help bind
substrate molecules.
 C
H
O Strained
peptide bond R
O
glycopeptide glycopeptide C H S CH3
transpeptidase transpeptidase
H N

HC CH3

N COO-
Ser OH Ser O C
H
H
O
 Specificity of Enzymes
▪ Enzyme specificity is the extent to which an enzyme’s activity is
restricted to a specific substrate, a specific group of substrates, a
specific type of chemical bond, or a specific type of chemical
reaction.
▪ Enzymes exhibit different degrees of selectivity for substrates, as
determined by the active site.
▪ Some active sites accommodate only one particular compound,
whereas others can accommodate a “family” of closely related
compounds.
▪ A molecular recognition
mechanism which operates
through the structural and
conformational
complementary between
enzyme and substrate.
 Geometric specificity
▪ Single enzyme can act on different substrates having
similar molecular geometry
▪ Specificity is less.
▪ Alcohol dehydrogenase can oxidize both ethanol and
methanol to yield corresponding aldehydes since
both alcohols have similar molecular geometry.
Ethanol
Methanol
 Bond Specificity
▪ Relative or linkage specificity
▪ The enzyme will act on a particular type of chemical bond,
irrespective of the rest of the molecular structure.
▪ Enzymes are specific to substrates having similar bonds/structure.
• Phosphatases hydrolyze phosphate-ester bonds in all types of
phosphate esters.
• -amylases hydrolyze -1-4 glycosidic bond linkage in starch and
glycogen ( the enzyme is specific only to the -1-4 glycosidic bond
and not to the substrate
• Peptide bonds are
hydrolyzed by
proteinases
(peptidases member
of the group of
proteinases)
 Group Specificity
▪ The enzyme will act only on molecules that have a specific
functional group, such as hydroxyl, amino, or phosphate groups.
▪ The enzyme is specific to a bond and groups surrounding the bonds.
▪ Moderate specificity (More than that of bond specificity)
▪ Carboxypeptidase (carboxylase) is group-specific because it cleaves
amino acids, one at a time, from the carboxyl end of a peptide
chain.

▪ Aminopeptidase cleaves amino acids, one at a time, from the amino

end of a peptide chain.
 Group Specificity
▪ Pepsin, a digestive enzyme, can hydrolyze a peptide bond in which
the amino group is contributed by an aromatic amino acid such as
phenyl alanine, tyrosine and tryptophan.

▪ Trypsin, a serine protease of digestive system, can hydrolyze a

peptide bond in which the amino group is contributed by any basic
amino acid such as lysine, arginine and histidine.
 Substrate Specificity
▪ Absolute specificity
▪ Specificity is very high.
▪ The enzyme will catalyze only one reaction.
▪ Enzymes showing substrate specificity are specific
only to one substrate and one reaction.
▪ Catalase catalyzes the conversion of hydrogen
peroxide (H2O2) to O2 and H2O. Hydrogen peroxide
is the only substrate it will accept.
▪ Lactase can only hydrolyze the β-1-4 glycosidic bond
of lactose to yield galactose and glucose.
▪ Maltase can only act on the -1-4 glycosidic linkage
of two glucose molecules in maltose.
 Co-factor Specificity
▪ Co-factors are nonprotein part of enzyme required for the
functioning of some enzymes.
▪ Enzymes which requires co-factors for their activity shows co-
factor specificity.
▪ Only correct combination of substrates and co-factor allows
enzymatic reactions.
▪ In the absence of specific co-factor, the enzymes will be
inactive even if there are plenty of substrates.
 Stereo-specificity
▪ Stereochemical or Optical specificity
▪ The enzyme will act on a particular stereoisomer.
▪ Chirality is inherent in an enzyme active site because amino acids
are chiral compounds.
▪ The highest specificity shown by any class of enzymes
▪ An L-amino acid oxidase will catalyze the oxidation of the L-form of
an amino acid but not the D-form of the same amino acid.The
enzyme is specific not only to substrate but also to its optical
configuration.
 Stereo-specificity
▪ -amylase hydrolyzes only the -glycosidic bonds but not the β-
glycosidic bonds
 Enzyme Specificity
▪ (1st, highest) Stereo (Stereochemical) specificity – specific to substrate
and its optical conformation
▪ (2nd) Co-factor specificity – specific to exact substrate, co-factor
combination
▪ (3rd) Substrate (Absolute) specificity – specific to only one substrate and
reaction
▪ (4th) Group specificity – specific to bonds and groups surrounding the
bonds
▪ (5th) Bond (Relative) specificity – specific to bonds
▪ (6th, least) Geometrical specificity – substrate having similar molecular
geometry

Geometrical < Bond < Group < Substrate < Co-factor < Stereo
 Factors Affecting Enzyme Activity

▪ Enzyme activity is a measure of the rate at which an

enzyme converts substrate to products in a biochemical
reaction.
▪ Four factors affect enzyme activity:
• Temperature
• pH
• Concentration of Enzyme
• Concentration of Substrate
 Factors Affecting Enzyme Activity
1) Temperature
• Higher temperatures mean molecules are moving faster and
colliding more frequently.
• As the temperature of an enzymatically catalyzed reaction
increases, there are more collisions between substrate molecules
and enzymes, so the rate (velocity) of the reaction also increases.
▪ The temperature that produces maximum activity for an enzyme
is known as the optimum temperature for that enzyme.
▪ Further increase in temperature results in a decrease in reaction
velocity as a result of temperature-induced denaturation of the
enzyme leading to derangement in the native (tertiary) structure
of the protein and active site.
▪ Enzymes become inactive.
▪ For human enzymes, the optimum temperature is around 37C,
normal body temperature. The optimum temperature for most
human enzymes is between 35 and 40°C.
▪ A person who has a fever where body core temperature exceeds
40C can be in a life-threatening situation because such a
temperature is sufficient to initiate enzyme denaturation.
▪ The loss of function of critical enzymes, particularly those of the
central nervous system, can result in dysfunction sufficient to
cause death.
Optimum temperature for enzymes is variable:
• Thermophilic bacteria found in the hot springs have
optimum temperatures of 70°C.
 Extremozymes
▪ An extremophile is a microorganism that thrives in extreme
environments, environments in which humans and most other
forms of life could not survive.
▪ Extremophile environments include the hydrothermal areas such
as hydrothermal vents on the ocean floor where temperatures and
pressures can be extremely high.
▪ The ability of extremophiles to survive under such harsh conditions
is related to the amino acid sequences present in the enzymes
(proteins) of extremophiles. These sequences are stable under the
extraordinary conditions present.
The enzymes present in extremophiles are called extremozymes.
An extremozyme is a microbial enzyme active at conditions that would
inactivate human enzymes as well as enzymes present in other types of
higher organisms.
▪ The enzymes present in some detergent formulations, which must
function in hot water, are the result of research associated with
high-temperature microbial enzymes.
▪ The cold-water-wash laundry detergents contain enzymes
originally characterized in cold-environment microbial organisms.
 Factors Affecting Enzyme Activity
2) pH
▪ The pH of an enzyme’s
environment can affect its
activity.
▪ This is not surprising because
the charge on acidic and basic
amino acids located at the
active site depends on pH.
▪ Small changes in pH (less than
one unit) can result in enzyme
denaturation and subsequent
loss of catalytic activity.
 Factors Affecting Enzyme Activity
pH
▪ Most enzymes exhibit
maximum activity over a very
narrow pH range.
▪ Only within this narrow pH
range do the enzyme’s amino
acids exist in properly charged
forms.
▪ Optimum pH is the pH at
which an enzyme exhibits
maximum activity.
▪ Biochemical buffers help
maintain the optimum pH for
an enzyme.
• Optimum pH for enzymes is variable.
• Most body enzymes usually falls within the physiological pH range
of 7.0–7.5.
• Notable exceptions are the digestive enzymes pepsin and trypsin

• A variation from normal pH can also affect substrates, causing

either protonation or deprotonation of groups on the substrate.
• The interaction between the altered substrate and the enzyme
active site may be less efficient than normal—or even impossible.
 Factors Affecting Enzyme Activity
3) Concentration of the enzyme

• Because enzymes are not consumed in the reactions

they catalyze, the cell usually keeps the number of
enzymes low compared with the number of substrate
molecules.
• This is efficient because the cell avoids paying the
energy costs of synthesizing and maintaining a large
work force of enzyme molecules.
• Thus, in general, the concentration of substrate in a
reaction is much higher than that of the enzyme.
Concentration of the enzyme
• If the amount of substrate present is kept constant and the
enzyme concentration is increased, the reaction rate
proportionately increases because more substrate
molecules can be accommodated in a given amount of time
• The greater the enzyme concentration, the greater the
reaction rate. .

Initially there is a reaction

(although very slow)
 Factors Affecting Enzyme Activity
4) Concentration of substrate

• The rate of reaction

increases as substrate
concentration increases (at
constant enzyme
concentration) and
thereafter remains constant
– this activity pattern is
called a saturation curve

• Maximum activity occurs

when the enzyme is
saturated (when all enzymes
are binding substrate)
Concentration of substrate
• As substrate concentration
increases, the point is eventually
reached where enzyme
capabilities are used to their
maximum extent.
• The rate remains constant from
this point on.

• Each substrate must occupy an enzyme active site for a finite

amount of time, and the products must leave the site before the
cycle can be repeated.
• When each enzyme molecule is working at full capacity, the
incoming substrate molecules must “wait their turn” for an empty
active site.
• At this point, the enzyme is said to be under saturation conditions.
Turnover Number
• The rate at which an enzyme accepts substrate molecules and
releases product molecules at substrate saturation is given by its
turnover number.
• An enzyme’s turnover number is the number of substrate
molecules transformed per minute by one molecule of enzyme
under optimum conditions of temperature, pH, and saturation.
• Some enzymes have a much faster mode of operation than others.
 Thermodynamics tells if reaction is
spontaneous (-G) or not (+G)!!!

▪ An enzyme alters the rate of a reaction, (lowering of activation

energy) but not its free energy change or position of equilibrium
 Enzyme Kinetics
(How fast is fast??? How slow is slow???
▪ It is the analysis of the velocity (or rate) of a chemical
reaction catalyzed by an enzyme, and how the velocities
can change on the basis of environmental parameters
modifications.

WHAT DO YOU HAVE TO KNOW?

▪ Velocities
▪ The rate of an enzyme-catalysed reaction as defined in
mathematical way
▪ The order of a reaction (zero order/ first-order reaction)
 Enzyme Kinetics
Steady-state
▪ Under experimental conditions [S]>>>[E].
▪ The [ES] quickly reaches a constant value in such dynamic system,
and remains constant until complete P formation: Steady State
assumption

E = Enzyme S = Substrate P = Product

ES = Enzyme-Substrate complex
k1 = rate constant for the forward
reaction
k-1 = rate constant for the breakdown of
the ES to substrate
0 Time
k2 = rate constant for the formation of
Early stage Steady state
ES formation the products
[ES] is constant
Substrate Concentration and Reaction Rate

Steady
state
▪ Each substrate must occupy an enzyme active site for a finite
amount of time, and the products must leave the site before the
cycle can be repeated.
▪ When each enzyme molecule is working at full capacity, the incoming
substrate molecules must “wait their turn” for an empty active site.
At this point, the enzyme is said to be under saturating conditions.
 Michaelis Menten Equation
The Michaelis-Menten equation describes how reaction velocity
varies with substrate concentration:

where:
vo = initial reaction velocity
Vmax = maximal velocity
[S] = substrate concentration
Km = Michaelis constant = (k-1 + k2)/k1

The rate or velocity of a reaction (v) is the number

of substrate molecules converted to product per unit time.
Important conclusions about
Michaelis-Menten kinetics
k −1 + k 2
KM =
Km
k1
1. Km—the Michaelis constant—
▪ reflects the affinity of the
enzyme for that substrate
▪  (low) Km reflects a  (high)
affinity of the enzyme for
substrate, because a low
concentration of substrate is
needed to reach a velocity
that is ½ Vmax
▪  large (high) Km reflects a
 low affinity
If Km = [S], → Vo = ½ Vmax.
[S] + [S]
Important conclusions about
Michaelis-Menten kinetics

2. Order of reaction:
▪ When Km >>>[S], the
velocity of the reaction
is approximately [S] <<< Km
proportional to the Km >>>[S]

substrate
concentration.

vo  [S]
Michaelis-Menten

The rate of a enzymatic reactions depends on the

substrate concentration
▪ The rate of
reaction is
first order
with respect
vo  [S] to substrate.

1st order
 Important conclusions about
Michaelis-Menten kinetics [S] >>> Km

2. Order of reaction:
▪ When [S] >>> Km, the velocity
is constant and equal to Vmax.

vo = Vmax
▪ The rate of reaction is then
zero order
independent of substrate
concentration, and is said to be
zero order with respect to
substrate concentration
[S] = Low → High
 Enzyme Efficiency

▪ Enzyme efficiency = Kcat/ Km unit:

1/(Molarity*second)= 1/[(mol/L)*s)] = L/ (mol*s)
▪ It provides information about two combined facts:
• substrate binding (Km)
• catalysis (Kcat, substrate conversion into product).
▪ The enzyme efficiency can be increased with
• Larger Kcat (rapid turnover)
• Smaller Km (high affinity for substrate) .
Michaelis-Menten Experimental determination
Equation
of KM and Vmax
Several rearrangements of the
Michaelis-Menten equation transform
it into a straight-line equation.
Lineweaver-Burk
double-reciprocal plot:

1 KM 1 1
= +
v Vmax [ S ] Vmax

▪ In Vo vs [S] plot, it is Y = mx + b
not always possible to m – slope
b= Y-intercept
measure Vmax
because of the gradual
upward slope.
 Inhibition of Enzymes
Inhibition: Velocity of an enzymatic reaction is
decreased or inhibited by some agent (inhibitors)

▪ Enzyme inhibitor
• Any substance that acts directly on an enzyme to
slow or stop the normal catalytic function of an
enzyme by binding to it
• It can be cellular metabolites, or foreign substances
such as drugs or toxins that have either a
therapeutic or toxic (can be lethal) effect.
• Usually specific and work at low concentrations
 Types of Inhibition
❑ Irreversible inhibition
▪ Inhibitor causes stable, covalent alterations in
the enzyme.

❑ Reversible inhibition
▪ Inhibitor interact with the enzyme through
noncovalent association/dissociation reactions.
a) Competitive
b) Un-competitive
c) Non-competitive/Mixed
 Irreversible Inhibition
▪ An irreversible enzyme
inhibitor is a molecule Enzyme
that inactivates enzymes
by forming a strong
S
covalent bond to an O I
amino acid side-chain
group at the enzyme’s
active site (Inhibitor can
not dissociate from the
enzyme.)
▪ Inhibitor cause conformational change at the active site
of the enzyme, thus, destroying the capacity of enzyme
to function as catalyst (The enzyme is permanently
deactivated. )
 Irreversible Inhibition - Reaction
Mechanism
E+S ES E+P
+ ▪ The inhibitor–active site bond is sufficiently
I strong that addition of excess substrate
does not reverse the inhibition process.
▪ The inhibitor permanently inactivates the
enzyme.
▪ The net effect is removal of enzyme from
EI the reaction.
Vmax decreases
No effect on Km
▪ Enzyme activity is not regained by increasing
the concentration of substrate or dialysis.
 Irreversible Inhibition
Examples:
▪ The actions of chemical warfare agents (nerve
gases) and organophosphate insecticides are
based on irreversible inhibition.
▪ Aspirin: causes covalent modification in a
cyclooxygenase involved in inflammation

Cyclooxigenase inhibition by aspirin

cyclooxygenase
 Irreversible (Suicide) Inhibitor
▪ Suicide inhibitors or mechanism-based inactivators hijack
the normal enzyme reaction mechanism to inactivate the
enzyme.
▪ These inhibitors are relatively unreactive until they bind to
the active site of a specific enzyme.
- The inhibitors are converted to a more effective
inhibitor with the help of the enzyme to be inhibited.
- The enzyme literally commits suicide.
▪ Examples: Penicillin or ampicillin cause covalent
modification of a transpeptidase which catalyzes the
synthesis of the bacterial cellular wall.
 Penicillin is a suicide inhibitor
R
O Penicillin
C
S CH3
H
H N

HC CH3

N COO-
C
H
O Strained
peptide bond R
O
glycopeptide C
glycopeptide H S CH3
transpeptidase transpeptidase
H N

HC CH3

N COO-
Ser OH Ser O C
H
H
O

▪ Glycopeptide transpeptidase catalyzes the formation of cross-links

between D-amino acids in the cell walls of bacteria.
▪ The enzyme also catalyzes the hydrolysis of peptide bonds such as
the strained peptide bond in penicillin through covalent bonding
with active site serine. This results to active site modification, thus,
the enzyme a sort of “commits suicide”.
 Reversible Inhibitor
▪ Inhibitor binds non-covalently (weak interaction)
with enzyme
▪ If inhibitor is removed, then the action of enzyme
is fully restored (Reversible)
▪ The activity of enzyme is fully restored on
removing the inhibitor by dialysis
 Competitive Inhibitor
▪ A competitive enzyme inhibitor is a molecule that sufficiently
resembles an enzyme substrate in shape and charge distribution
and they bind to active site of the enzyme, thus, preventing
binding of the substrate to enzyme.
▪ When a competitive inhibitor binds to an enzyme active site, the
inhibitor remains unchanged (no reaction occurs), but its
physical presence at the site prevents a normal substrate
molecule from occupying the site. The result is a decrease in
enzyme activity.
 Competitive Inhibitor

▪ Effective concentration of
enzyme is reduced with the
formation of the EI complex.
Vmax = Vmax’
▪ This in effect shifts the reaction
to the left.
▪ The affinity of enzyme towards Km < Km’

substrate is reduced in the

presence of inhibitor
▪ Km, normal < Km, inhibitor
 The Lineweaver-Burk plot is diagnostic
for competitive inhibition
1 KM 1 1
= +
v Vmax [ S ] Vmax
Km(normal) < Km’
y = mx + b

Vmax(normal) = Vmax’
Malonate is an inhibitor of succinate dehydrogenase.
 Competitive Inhibitors
▪ Antihistamines are competitive inhibitors of histidine
decarboxylation, the enzymatic reaction that converts
histidine to histamine. Histamine causes the usual allergy and
cold symptoms: watery eyes and runny nose.
▪ The treatment for methanol poisoning involves giving a
patient intravenous ethanol.
• The same enzyme, alcohol dehydrogenase, detoxifies both
methanol and ethanol.
• Ethanol has 10 times the affinity for the enzyme than
methanol has.
• Keeping the enzyme busy with ethanol as the substrate
gives the body time to excrete the methanol before it is
oxidized to the potentially deadly formaldehyde.
▪ Bacteria require folic acid for replication of genetic material
▪ Sulfa drugs binds to the enzyme that makes folic acid preventing
other substrates from binding
▪ As a result, folic acid is not longer made and the bacterial cell dies
▪ Since animal cells don’t make folic acid themselves, they do not
have this enzyme and so sulfa drugs has no effect on them
 Uncompetitive Inhibitor
▪ An uncompetitive inhibitor binds only to the ES complex
and not to the free enzymes

E+S ES E+P
+
I

ESI ESI is an active complex.

(cannot react)

Km lower. The affinity of enzyme towards substrate is

increased in the presence of inhibitor.
(More formation of ES complex)
 Km lower. The affinity of enzyme towards substrate is
increased in the presence of inhibitor. (More formation of ES
complex)
Vmax lower due to the inactive ESI (enzyme-substrate-
inhibitor) complex, lesser substrate converted to product

E+S ES E+P
+
I
Vmax’ < Vmax
ESI

Example of uncompetitive
inhibition: alkaline
Km ’ < Km
phosphatase inhibition by
phenylalanine
 The Lineweaver-Burk plot is diagnostic
for uncompetitive inhibition
1 KM 1 1
= +
v Vmax [ S ] Vmax
y = mx + b

Vmax’ < Vmax(normal)

Km ’ < Km(normal)
 Noncompetitive (Mixed) Inhibitor
▪ A noncompetitive enzyme inhibitor is a molecule
that decreases enzyme activity by binding to a site
other than the active site and causes changes in
the overall three-dimensional shape of the enzyme.
 Noncompetitive (Mixed) Inhibitor
▪ Combination of competitive and uncompetitive
▪ A noncompetitive inhibitor binds enzyme regardless of
whether the substrate is bound.
▪ Adding more substrate will not affect the reaction because
the active site is unavailable.
▪ Lowering the concentration of a noncompetitive inhibitor
sufficiently does free up many enzymes, which then return
to normal activity.
E+S ES E+P
+ +
I I

EI + S ESI
Inactive IE Complex Inactive ESI Complex
(cannot react) (cannot react)
▪ Vmax decreases.
▪ Km is unchanged.
The Lineweaver-Burk plot is diagnostic for
noncompetitive inhibition
1 KM 1 1
= +
v Vmax [ S ] Vmax
y = mx + b

Vmax’ < Vmax (normal)

Km ’ = Km(normal)
 Noncompetitive Inhibitors
• Heavy metal ions Pb2+, Ag+, and Hg2+.
• 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.
Fructose 1,6-bisphosphatase inhibition by AMP
 Non-competitive
Competitive Uncompetitive
Or Mixed
 Regulation of Enzyme Activity
Living systems must regulate the enzymatic catalytic activity to:
▪ Coordinate metabolic processes
▪ Promote adaptations to environmental changes
▪ Growth and complete the living cycle in the correct way

Metabolic Process:
1. A cell that continually produces large amounts of an enzyme
for which substrate concentration is always very low is
wasting energy. The production of the enzyme needs to be
“turned off.”
2. A product of an enzyme-catalyzed reaction that is present in
plentiful (more than needed) amounts in a cell is a waste of
energy if the enzyme continues to catalyze the reaction that
produces the product. The enzyme needs to be “turned off.”
 Regulation of Enzyme Activity
Mechanisms of regulation:

1. Control of the enzyme availability

• Feedback control associated with allosteric enzymes
2. Control of the enzymatic activity
• Covalent modifications of the conformation or
structure
3. Proteolytic enzymes and zymogens
 Control of the enzyme availability
Feedback control associated with allosteric enzymes
Characteristics of allosteric enzymes are as follows:
1. All allosteric enzymes have quaternary structure, that is, they
are composed of two or more protein chains.
2. All allosteric enzymes have two kinds of binding sites: those
for substrate and those for regulators.
3. Active and regulatory binding sites are distinct from each
other in both location and shape. Often the regulatory site is
on one protein chain and the active site is on another.
4. Binding of a molecule at the regulatory site causes changes in
the overall three-dimensional structure of the enzyme,
including structural changes at the active site.
▪ Allosteric enzyme is an enzyme with two or more protein
chains (quaternary structure) and two kinds of binding sites
(substrate and regulator).
 Allosteric Regulation
▪ Substances that bind at regulatory sites of allosteric enzymes
are called regulators.
• The binding of a positive
regulator (activator) increases
enzyme activity. The shape of
the active site is changed such
that it can more readily accept
substrate.
• The binding of a negative
regulator (a noncompetitive
inhibitor) decreases enzyme
activity. Changes to the active
site are such that substrate is
less readily accepted.
 KINETIC PERSPECTIVE

Activators (+)
Inhibitors(-)
▪  Vmax lower for there are
lesser substrate converted
to product.
▪  Km higher for there is
lesser affinity of enzyme
with substrate.
 Allosteric Inhibition
❑ Enzyme: Cytochrome C Oxidase

❑ Regular function:
▪ Speeds up the reduction of oxygen to water in cellular
respiration
▪ Without it, the reaction will not occur fast enough and the
organism will die because not enough energy is released.

❑ Inhibition
▪ CN- attaches to the –SH groups in the enzyme.
▪ This destroys the disulfide bridges and thus changing the
tertiary structure of the enzyme.
▪ Change in the shape results in the change in the active site
thus the substrate cannot bind and cytochrome c oxidase is
nonfunctional.
 Feedback Control/Inhibition
▪ Feedback inhibition is operative in devices that
maintain a constant temperature (thermostat).
 When it is cold, the thermostat turns on the heater to
produce heat.
 When it is too warm, the heat will cause the
thermostat to turn off the heater. Heat has a negative
effect on the thermostat.
Cold Thermostat Heater Heat

turning off
 Feedback Control/Inhibition
▪ Feedback control is a process in which activation or inhibition of
the first reaction in a reaction sequence is controlled by a product
of the reaction sequence.
▪ The product of each step is the substrate for the next enzyme.

▪ What will happen in this reaction series if the final product (D) is a
negative regulator of the first enzyme (enzyme 1)?
• At low concentrations of D, the reaction sequence proceeds rapidly.
• At higher concentrations of D, the activity of enzyme 1 becomes
inhibited (by feedback), and eventually the activity stops.
• At the stopping point, there is sufficient D present in the cell to meet
its needs.
• Later, when the concentration of D decreases through use in other cell
reactions, the activity of enzyme 1 increases and more D is produced.
 Feedback Control/
End Product Inhibition

▪ As the enzyme’s
end product
accumulates, it
inhibits the
enzyme by binding
to the first enzyme
in the pathway.
▪ This shuts down
the entire
sequence.
Feedback
Inhibition
 Applications of inhibitors

• Negative feedback: end point or end product

inhibition
• Medicine antibiotics, sulphonamides, sedatives and
stimulants.
• Poisons snake bite, plant alkaloids and nerve gases
 Covalent Modification of Enzymes
▪ Covalent modification is a process in which enzyme activity is
altered by covalently modifying the structure of the enzyme
through attachment of a chemical group to or removal of a
chemical group from a particular amino acid within the
enzyme’s structure.
▪ Example: Phosphate group addition (phosphorylationor)
removal (dephosphorylation) of an enzyme.
• This process is the off/on or on/off switch for the enzyme.
• Glycogen phosphorylase, an enzyme involved in the
breakdown of glycogen to glucose, is activated by the
addition of a phosphate group.
• Glycogen synthase, an enzyme involved in the synthesis of
glycogen is deactivated by phosphorylation.
• ATP molecule as source of the added phosphate group
Proteolytic Enzymes and Zymogens
• A second mechanism for regulating cellular enzyme activity is
based on the production of enzymes in an inactive form.
• These inactive enzyme precursors are then “turned on” at the
appropriate time.
• Example is zymogen. A zymogen (proenzyme) is the inactive
precursor of a proteolytic enzyme.
• A proteolytic enzyme (most digestive and blood-clotting
enzymes) is an enzyme that catalyzes the breaking of peptide
bonds that maintain the primary structure of a protein.
Because proteolytic enzymes would destroy the tissues that
produce them, proteolytic enzymes are generated in an
inactive form (zymogen) and then later, when they are
needed, are converted to their active form.
 Proteolytic Enzymes and Zymogens
• Activation of a zymogen requires
an enzyme-controlled reaction
that removes some part of the
zymogen structure.
• Such modification changes the
three-dimensional structure
(secondary and tertiary
structure) of the zymogen, which
affects active site conformation.
• For example, the zymogen pepsinogen is converted to the active
enzyme pepsin in the stomach, where it then functions as a digestive
enzyme.
• Pepsin would digest the tissues of the stomach wall if it were
prematurely generated in active form.
• Pepsinogen activation involves removal of a peptide fragment from its
structure
 Recap
Enzyme-substrate
complex
Class Reaction Type Subclass
 Enzyme specificity
❑ (1st, highest) Stereo (Stereochemical) specificity – specific to substrate
and its optical conformation
❑ (2nd) Co-factor specificity – specific to exact substrate, co-factor
combination
❑ (3rd) Substrate (Absolute) specificity – specific to only one substrate and
reaction
❑ (4th) Group specificity – specific to bonds and groups surrounding the
bonds
❑ (5th) Bond (Relative) specificity – specific to bonds
❑ (6th, least) Geometrical specificity – substrate having similar molecular
geometry

Geometrical < Bond < Group < Substrate < Co-factor < Stereo
Enzyme inhibition
Enzyme regulation