Dr Nazia khan
Assistant professor
College of medicine
 Virtually all reactions in the body are mediated by
enzymes, which are protein catalysts that increase the
rate of reactions without being changed in the overall
process. Among the many biologic reactions that are
energetically possible, enzymes selectively channel
reactants (called substrates) into useful pathways
Definition
 Enzymes are biocatalysts synthesized by living cells.
 They are protein in nature.
 (Except Ribozyme which is RNA).
 They are colloidal
(A system in which finely divided particles, which are approxi
mately 10 to 10,000 angstroms in size, are dispersed within aco
ntinuous medium in a manner that prevents them from being
filtered easily or settled rapidly.The particulate matter so dispe
rsed
 Thermolabile in character and
 Specific in their action.
Cofactor
 The protein part of the enzyme on its own is not
always adequate to bring about the catalytic activity.
Many enzymes requires certain non protein small
additional factors collectively referred to as cofactor for
catalysis.
 The cofactor may be organic or inorganic in nature.
Enzymes may be
1. Simple: composed of two proteins
2. Complex: composed of protein and a small organic
molecule
Holoenzyme= apoenzyme+ prosthetic group/coenzyme
Coenzyme: The non protein organic low molecular
weight and dialysable substance associated with enzyme
function is known as coenzyme.
 The binding between apoenzyme and non protein
component is non covalent
Prosthetic group:
 The functional enzyme is referred to as holoenzyme
which is made up of a protein part(apoenzyme) and a
non protein part(coenzyme).
 The term prosthetic group is used when a non protein
moiety is tightly bound(covalent bond) to the enzyme
which is not easily separated by dialysis
Activator:
 The term activator is referred to the inorganic cofactor
(Mg, Ca, Mn etc) necessary to enhance enzyme
activity.
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PROPERTIES OF ENZYMES
 Enzymes are protein catalysts that increase the velocity of a
chemical reaction, and are not consumed during the reaction.
A. Active sites Enzyme molecules contain a special pocket or
cleft called the active site.
B. Catalytic efficiency :Enzyme-catalyzed reactions are highly
efficient, proceeding from 103–108 times faster than
uncatalyzed reactions.
C. Specificity Enzymes are highly
specific, interacting with one or
a few substrates and catalyzing
only one type of chemical reaction
D. Holoenzymes: Some enzymes require molecules other
than proteins for enzymic activity. The term holoenzyme
refers to the active enzyme with its nonprotein
component, whereas the enzyme without its nonprotein
moiety is termed an apoenzyme and is inactive
E. Regulation: Enzyme activity can be regulated, that is,
increased or decreased, so that the rate of product
formation responds to cellular need.
F. Location within the cell: Many enzymes are localized in
specific organelles within the cell .
IUBMB
 IUBMB - International Union of Biochemistry and
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Molecular Biology.
Used for nomenclature of enzymes.
A four digit enzyme commission (EC) number is assigned
to each enzyme representing the class (first digit), sub class
(second digit), sub sub class (third digit) and the individual
enzyme (fourth digit)
The nomenclature was determined by the Enzyme
Commission in 1961 (with the latest update having
occurred in 1992)
All enzymes are assigned an “EC” number
NOMENCLATURE
 Each enzyme is assigned two names.
A. Recommended name : Most commonly used enzyme names have the
suffix “- ase ” attached to the substrate of the reaction (for example,
glucosi- dase and urease ), or to a description of the action performed
(for example, lactate dehydrogenase and adenylyl cyclase
B.
Systematic name: In the systematic naming system, enzymes are
divided into six major classes, each with numerous subgroups. for
example, lactate:NAD+ oxi- doreductase .The systematic names are
unambiguous and informative, but are frequently too cumbersome
to be of general use.
Classification of enzymes
 EC 1. Oxidoreductases
 EC 2. Transferases
 EC 3. Hydrolases
 EC 4. Lyases
 EC 5. Isomerases
 EC 6. Ligases
Class 1: Oxidoreductases
 Catalyze the transfer of hydrogen or oxygen atoms or
electrons from one substrate to another.
 Also called oxidases, dehydrogenases, or reductases
 Catalyzes redox reactions, therefore an electron
donor/acceptor is also required to complete the reaction.
 Ex: Alcohol dehydrogenase.
Class 2: Transferases
 Transfer chemical groups from one molecule to another or
to another part of the same molecule
 Catalyze group transfer reactions.
 Ex: Hexokinase
 These are of the general form:
A-X + B ↔ BX + A
Classs 3: Hydrolases
 Catalyze hydrolytic reactions
 Cleave a bond using water to produce two
molecules from one
 Ex: Lipase
A-X + H2O ↔ X-OH + HA
Class 4: Lyases
 Catalyze non-hydrolytic removal of functional groups from
substrates
 Remove a group from or add a group to double bonds
 Often creating a double bond in the product
 Or the reverse reaction, ie, addition of function groups
across a double bond.
Ex:Aldolases.
Class 5: Isomerases
 Catalyzes isomerization reactions.
 Interconvert isomeric structures by molecular
rearrangements
 Ex: Triose phosphate isomerase
Class 6: Ligases
 Catalyzes the synthesis of various (mostly C-X) bonds
 Coupled with the breakdown of energy containing
substrates, usually ATP
 Join two separate molecules by the formation of a new
chemical bond usually with energy supplied by the
cleavage of an ATP
 Ex:Glutamine synthetase
How enzymes work
 The mechanism of enzyme action can be viewed from
two different perspectives.
 First: Enzymes provide an alternate, energetically
favorable reaction pathway different from the
uncatalyzed reaction.
 The second perspective: describes how the active site
chemically facilitates catalysis
Mechanism of enzyme action
Mechanism of enzyme action
 Fischer’s Lock and key model
 Koshland’s Induced Fit Model
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The Lock and Key Hypothesis
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Fit between the substrate and the active site of the
enzyme is exact
Like a key fits into a lock very precisely
The key is analogous to the enzyme and the substrate
analogous to the lock.
Temporary structure called the enzyme-substrate
complex formed
Products have a different shape from the substrate
Once formed, they are released from the active site
Leaving it free to become attached to another substrate
The Induced Fit Hypothesis
 Some proteins can change their shape
(conformation)
 When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
 The active site is then moulded into a precise
conformation
 Making the chemical environment suitable for the
reaction
 The bonds of the substrate are stretched to make
the reaction easier (lowers activation energy)
MODE OF ACTION OF ENZYMES
 After binding takes place, catalysis generates transition
state complexes leading to the formation of reaction
products.
 Enzymes accelerate reaction rates, but do not
alter the equilibrium point of the reaction.
 Enzymes increase reaction rates by decreasing the
amount of energy required to form the transition
state.
 Activation energy is the energy required to reach
the transition state
 The activation energy it is the minimum, necessary,
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amount of energy required for a reaction to proceed.
This barrier is the reason why the rate of many
chemical reactions is very slow without the presence of
enzymes, heat, or other catalytic forces.
There are two common ways to overcome this barrier
and thereby accelerate a chemical reaction.
First, the reactants could be exposed to a large amount
of heat
A second strategy is to lower the activation energy
barrier. Enzymes lower the activation energy to a point
where a small amount of available heat can push the
reactants to a transition state.
 The lower the energy of activation, more molecules have sufficient
energy to pass through the transition state at a given temperature.
 Enzymes lower the energy of activation and thus allow a reaction
to proceed rapidly.
Enzyme Kinetics
Enzyme Kinetics
Michaelis and Menten, in 1913, developed a simple
model for examining the kinetics of enzyme catalyzed
reactions.
• The model assumes that the enzyme [E] reversibly
combines with its substrate [S] to form an
intermediate enzyme-substrate complex [ES] that
subsequently breaks down to product. ES is
relatively stable.
• The series of events can be shown thus:
k1
k2
E + S ↔
ES ↔ E + P
k-1
k-2
• The Michaelis-Menten equation describes how reaction
velocity varies with substrate concentration
Where:
V1 = initial reaction velocity
Vmax = maximal velocity
Km = Michaelis constant = (k-1 + k2)/k1
[S] = substrate concentration
Michaelis Constant (Km)
 The Km is a constant and is characteristic of an enzyme
and its particular substrate
 It is a measure of the affinity of the enzyme for that
substrate. (Low values indicate high affinity.)
 It is also numerically equal to substrate concentration at
which the reaction velocity is equal to ½ Vmax.
A small or low Km:
• Reflects a high affinity of
enzyme for substrate.
• A very low concentration
of substrate is needed to
reach a velocity that is, say,
half maximal.
A large or high Km:
 Reflects low affinity of
enzyme for the substrate
 A high concentration of
substrate is needed to
saturate one half the enzyme.
 At [S] near point A the rate is
directly proportional to
substrate concentration and the
reaction rate is said to first
order (i.e., dependent upon
[S]).
 At [S] near point C and at very
high substrate concentrations,
the rate is nearly independent of
substrate concentration and the
reaction rate is said to be zero
order (i.e., not dependent upon
[S]).
 At [S] near point B, the rate is ½
Vmax. The substrate
concentration at point B is by
definition equal to Km.
Factors affecting enzyme activity
 Important factors that affect enzyme activity are
1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. pH
5. Product concentration
6. Presence of activators or inhibitors
7. Availability of coenzymes
Effect of Enzyme Concentration
 At a given substrate
concentration, the initial
velocity of an enzyme
catalyzed reaction is
proportional to the enzyme
concentration
 Property made use of in
determining the level of
particular enzyme in
plasma, serum or tissues
Effect of Substrate Concentration
 The rate of an enzyme
catalyzed reaction increases
with substrate concentration
in a rectangular hyperbolic
curve.
 This increase in rate occurs
until maximum velocity
(Vmax) is reached
 Allosteric enzymes: are
enzymes that change their
conformational ensemble
upon binding of an effector,
which results in an apparent
change in binding affinity
Vmax reflects the saturation of all the available
binding sites on the enzyme with substrate
Effect of Temperature
 Reaction velocity increases with
an increase in temperature till a
peak is reached, often at 40-60oC.
 This is the result of the increased
number of molecules having
sufficient energy to pass the
energy barrier & form products.
 A further increase in
temperature causes denaturation
of enzyme & decreases the
reaction velocity.
 An important exception is the
Taq polymerase from
thermophilic bacteria that is
active at very high temperatures
and is used for PCR (polymerase
chain reaction).
Effect of pH
• Most enzymes in the
human body function
optimally in the
physiological pH range
(around pH 7.4).
• Some exceptional
enzymes include pepsin
with a pH optimum of
1.5-2.0, secreted in gastric
juice.
• Changes in pH affect
ionic charge of amino
acid side chains of
enzymes (histidine,
glutamate, cysteine) and
dramatically affect
catalytic function.
Effect of product concentration
 The accumulation of reaction products generally
decreases the enzyme velocity.
 For certain enzymes the product combines with the
active site of enzyme and forms a loose complex and
thus inhibits the enzyme activity.
Effect of activators: ↑ enzyme activity.
 Metal activated enzymes: metal not held tightly. Ex:
ATPase (Mg and Ca) and Enolase (Mg)
 Metalloenzymes: metals are held tightly. Ex: Alcohol
dehydrogenase (Zn)
Availability of Coenzymes: ↑ enzyme activity.
Clinical application:
 The catalytic activity of enzymes facilitates their detection :
ELISA
 The analysis of certain enzymes aids diagnosis
 Principal serum enzymes used in clinical diagnosis. Many
of the enzymes are not specific for the disease listed.
Serum Enzyme
Major Diagnostic Use
1. Aminotransferases
Myocardial infarction , viral hepatitis
2. Amylase
Acute pancreatitis
3. Creatine kinase
Muscle disorders and myocardial infarction
4. Lactate dehydrogenase
Myocardial infarction
5. Lipase
Acute pancreatitis
6. Phosphatase, acid
Metastatic carcinoma of the prostate
7. Phosphatase, alkaline
Various bone disorders
Enzyme Inhibition
 Any substance that can diminish the velocity of an enzyme-
catalyzed reaction is called an inhibitor.
 Irreversible inhibitors bind to enzymes through covalent
bonds. Reversible inhibitors typically bind to enzymes
through noncovalent bonds
 The inhibitor may be organic or inorganic in nature.
 There are three broad categories of enzyme inhibition:
 1. Reversible inhibition.
 2.irreversible inhibition.
Inhibitors
 Inhibitors are chemicals that reduce the rate of
enzymic reactions
 They 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
Types of enzyme inhibition
 Reversible inhibitors:
There are two categories:
Competitive and non competitive.
Reversible Competitive Inhibition
A competitive inhibitor:
 Has a structure similar to substrate
 Occupies active site
 Competes with substrate for active site
 Inhibition is reversed by increasing substrate
concentration
Vmax is unchanged.
Km is increased
Malonate as an Example of a Competitive Inhibitor
 Succinate Dehydrogenase is an
important enzyme of the TCA cycle
that converts succinate to fumarate
 Malonate is structurally very
similar to succinate and acts as a
competitive inhibitor of the
enzyme
Malonate cannot form a C=C
 Inhibition by malonate can be
reversed by increasing the
concentration of succinate
 Statin drugs as examples of competitive inhibitors:
 This group of antihyperlipidemic agents competitively
inhibits the first committed step in cholesterol
synthesis.
 This reaction is catalyzed by hydroxymethylglutaryl–
CoA reductase ( HMG-CoA reductase).
 Statin drugs, such as atorvastatin (Lipitor) and
pravastatin (Pravachol) are structural analogs of the
natural substrate for this enzyme, and compete
effectively to inhibit HMG-CoA reductase . By doing so,
they inhibit de novo cholesterol synthesis, thereby
lowering plasma cholesterol levels
Reversible Noncompetitive Inhibition
A noncompetitive inhibitor
 Not a structural analogue
 Binds to the enzyme but not at active site
 Changes the shape of enzyme
 Substrate binds but catalysis does not occur in the
presence of inhibitor
 Effect is not reversed by adding substrate
 Km is unaltered.
 Vmax is decreased proportionately to inhibitor
concentration.
Lead Poisoning as an Example of a
Noncompetitive Inhibitor
 Lead binds with the sulfhydryl group of cysteine
residues in enzymes. The cysteine residues are not
part of the active site.
 Lead causes noncompetitive inhibition of enzymes
like the Ferrochelatase (required in the synthesis
of heme for incorporation into heme proteins such
as hemoglobin).
Examples of Enzyme Inhibition –
Drug Therapy: Enzymes Used for Therapeutic Purpose
 Methotrexate, an inhibitor of dihydrofolate reductase, is used
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in cancer chemotherapy to inhibit DNA synthesis in rapidly
growing cells (i.e., tumors).
Aspirin is used to inhibit the synthesis of prostaglandins (by
cyclooxygenases 1 and 2) which are at least partly responsible
for the aches and pains of arthritis.
Sulfonamides (folate synthase) are used to inhibit folic acid
synthesis, essential for the metabolism and growth of diseasecausing bacteria.
Allopurinol is used to inhibit xanthine oxidase in treatment of
hyperuricemia and gout.
Statins are used to inhibit HMG-CoA reductase and lower
blood cholesterol levels.
Dicumarol is a structural analog of vitamin K and is used as an
anticoagulant.
Regulation of Enzyme Activity
 The regulation of the reaction velocity of enzymes is
essential if an organism is to coordinate its numerous
metabolic processes.
 The rates of most enzymes are responsive to changes
in substrate concentration
 Thus, an increase in substrate concentration prompts
an increase in reaction rate.
1. Regulation of allosteric enzymes
2. Regulation of enzymes by covalent modification
3. Induction and repression of enzyme synthesis
A. Allosteric Regulation of Enzymes
 Some enzymes bind small, physiologically important
molecules that modulate their activity
 These are known as allosteric enzymes and the small
regulatory molecules to which they bind are known as
allosteric effectors
 Allosteric effectors bind to the enzyme at a site
different from the catalytic site
 Binding of effector causes conformational changes to
the catalytically active site(s)
 The hallmark of effectors is that when they bind to
enzymes, they alter the catalytic properties of an
enzyme's active site.
 Those that increase catalytic activity are known as positive
effectors.
 Effectors that reduce or inhibit catalytic activity are negative
effectors
1. Homotropic effectors: When the substrate itself serves as
an effector, the effect is said to be homotropic..
2. Heterotropic effectors: The effector may be different from
the substrate, in which case the effect is said to be
heterotropic.
B. Regulation of enzymes by covalent modification
 Many enzymes may be regulated by covalent modification, most
frequently by the addition or removal of phosphate groups from
specific serine, threonine, or tyrosine residues of the enzyme. Protein
phosphorylation is recognized as one of the primary ways in which
cellular processes are regulated.
1. Phosphorylation and dephosphorylation: Phosphorylation reactions
are catalyzed by a family of enzymes called protein kinases that use
adenosine triphosphate (ATP) as a phosphate donor. Phosphate groups
are cleaved from phosphorylated enzymes by the action of
phosphoprotein phosphatases
2. Response of enzyme to phosphorylation: Depending on the specific
enzyme, the phosphorylated form may be more or less active than the
unphosphorylated enzyme. For example, phosphorylation of glycogen
phosphorylase (an enzyme that degrades glycogen) increases activity,
whereas the addition of phosphate to glycogen synthase (an enzyme
that synthesizes glycogen) decreases activity
C. Induction and repression of enzyme synthesis
 Cells can also regulate the amount of enzyme present by altering the
rate of enzyme degradation or, more typically, the rate of enzyme
synthesis.
 The increase (induction) or decrease (repression) of enzyme
synthesis leads to an alteration in the total population of active sites.
 Enzymes subject to regulation of synthesis are often those that are
needed at only one stage of development or under selected
physiologic conditions. For example, elevated levels of insulin as a
result of high blood glucose levels cause an increase in the synthesis
of key enzymes involved in glucose metabolism .In contrast, enzymes
that are in constant use are usually not regulated by altering the rate
of enzyme synthesis.
 Alterations in enzyme levels as a result of induction or repression of
protein synthesis are slow (hours to days), compared with
allosterically or covalently regulated changes in enzyme activity,
which occur in seconds to minutes.