Contents: Enzymes Terminologies Enzymes Properties Mechanism Of Enzyme Action Factors Affecting Enzyme Activity Inhibition Of Enzyme Regulation Of Enzyme Activity

1. Be Confident…!!!
ENZYMES
DEFINITION:
Enzymes are protein catalysts for chemical reaction in biological systems. They increase the rate
of chemical reactions taking place within living cells without changing themselves.
TYPES OF ENZYME:
Most enzymes are protein in nature. Depending on the presence and absence of a non-protein
component with the enzyme enzymes can exist as:
1. Simple Enzyme:
 It is made up of only protein molecules not bound to any non - proteins.
 Example: Pancreatic Ribonuclease.
2. Holo Enzyme:
 It is made up of protein groups and non-protein component.
 The protein component of this holo enzymes is called apoenzyme
 The non-protein component of the holo enzyme is called a cofactor.
o If this cofactor is an organic compound it is called a coenzyme
o If it is an inorganic group it is called activator. (Fe 2+, Mn 2+, or Zn 2+ions)
o If the cofactor is bound so tightly to the apoenzyme and is difficult to
remove without damaging the enzyme it is sometimes called a prosthetic
group
ENZYMES
SIMPLE
ENZYMES
HOLO
ENZYMES
APOENZYME
COFACTOR
COENZYME
ACTIVATOR
PROSTHETIC
GROUP

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PROPERTIES OF ENZYMES
ACTIVE SITE
 Enzyme molecules contain a special pocket or cleft called the active site. The active site
contains amino acid chains that create a three-dimensional surface complementary to
the substrate.
 The active site binds the substrate, forming an enzyme-substrate (ES) complex. ES is
converted to enzyme-product (EP); which subsequently dissociates to enzyme and
product.
 For the combination with substrate, each enzyme is said to possess one or more active
sites where the substrate can be taken up.
 The active site of the enzyme may contain free hydroxyl group of serine, phenolic
(hydroxyl) group of tyrosine, SH-thiol (Sulfhydryl) group of cysteine or imindazolle group
of histidine to interact with there is substrates.
 It is also possible that the active site (Catalytic site) is different from the binding site in
which case they are situated closely together in the enzyme molecule.
CATALYTIC EFFICIENCY
 Most enzyme- catalyzed reactions are highly efficient proceeding from 103 to 108 times
faster than uncatalyzed reactions.
 Typically each enzyme molecule is capable of transforming 100 to 1000 substrate
molecule in to product each second.
SPECIFICITY
 Enzymes are specific for their substrate.
 Specificity of enzymes are divided into:
a) Absolute specificity:
o This means one enzyme catalyzes or acts on only one substrate.
o For example: Urease catalyzes hydrolysis of urea but not thiourea.

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b) Stereo specificity:
o Some enzymes are specific to only one isomer even if the compound is
one type of molecule.
o For example: glucose oxidase catalyzes the oxidation of β-D-glucose but
not α-D glucose, and arginase catalyzes the hydrolysis of L-arginine but
not D-arginine.
o *Maltase catalyzes the hydrolysis of α- but not β –glycosides.
REGULATION
 Enzyme activity can be regulated- that is, enzyme can be, activated or inhibited so that
the rate of product formation responds to the needs of the cell.
ZYMOGENS (- inactive form of enzyme)
 Some enzymes are produced in nature in an inactive form which can be activated when
they are required. Such type of enzymes is called Zymogens (Proenzymes).
 Many of the digestive enzymes and enzymes concerned with blood coagulation are in
this group.
 Examples:
 Pepsinogen - This zymogen is from gastric juice - when required
 Pepsinogen - converts to Pepsin
 Trypsinogen - This zymogen is found in the pancreatic juice, and when it is required
gets converted to trypsin.
 The activation is brought about by specific ions or by other enzymes that are
proteolytic.
Pepsinogen + H+ Pepsin
Trypsinogen Enteropeptidase
Trypsin
 Zymogen forms of enzymes a protective mechanism to prevent auto digestion of tissue
producing the digestive enzymes and to prevent intravascular coagulation of blood.

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ISOENZYMES (Isozymes)
 These are enzymes having:
 Similar catalytic activity
 Act on the same substrate and produces the same product
 Originated at different site
 Exhibiting different physical and chemical characteristics such as:
o Electrophoretic Mobilities
o Amino Acid Composition
o Immunological Behavior
 Example: LDH (Lactate dehydrogenase) exists in five different forms each having four
Polypeptide chains.
TYPE POLYPEPTIC CHAIN
LDH - 1 H H H H
LDH - 2 H H H M
LDH - 3 H H M M
LDH - 4 H M M M
LDH - 5 M M M M
*H= Heart and M=Muscle
 Example. CPK (Creatine phospho kinase) exists in three different forms each having two
polypeptide chains.
TYPE POLYPEPTIC CHAIN
CPK - 1 B B
CPK - 2 B M
CPK - 3 M
*B=Brain and M= Muscle
MECHANISM OF ACTION OF ENZYMES
 Mechanism begins with the binding of the substrate (or substrates) to the active site on
the enzyme forming Enzyme-Substrate Complex.
 This binding of the substrate to the enzyme causes changes in the distribution of
electrons in the chemical bonds of the substrate and ultimately causes the reactions
that lead to the formation of products.

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LOWERING OF ACTIVATION ENERGY
 Enzymes carry out their function of lowering Activation Energy by temporarily
combining with the chemicals involved in the reaction.
 Activation energy is the energy required to convert one mole of reacting substance from
ground state to the transition state.
 Enzyme are said to reduce the magnitude of this activation energy.
 Enzyme reacts with the Substrate to form Enzyme-Substrate Complex. This is the
Transition state.
 In Transition state, the probability of making or breaking a chemical bond to form the
product is very high.
 This results in acceleration of the reaction forming products.
 During the formation of an ES complex, the substrate attaches itself to the specific
active sites on the enzyme molecule by Reversible interactions formed by Electrostatic
bonds, Hydrogen bonds, Vanderwaals forces, hydrophobic interactions.

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THEORY OF ENZYME ACTION
 Two theories about action are:
1. Lock and Key Model:
 Postulated in 1890 by Emil Fischer.
 The lock is the enzyme and the key is the substrate.
 Specific shape of substrate allows it to fit into the Enzyme.
 Only the correctly sized key (substrate) fits into the key hole (active site)
of the lock (enzyme).
 Smaller keys, larger keys (incorrectly shaped or sized substrate
molecules) do not fit into the lock (enzyme).
2. Induced Fit Model:
 Formulated by Daniel Koshland in 1958
 The substrate plays a role in determining the final shape of the enzyme.
 The enzyme is partially flexible.
 Only the proper substrate is capable of inducing the proper alignment pf
the active site.

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FACTORS AFFECTING ENZYME ACTIVITY
 Physical and chemical factors are affecting the enzyme activity.
 These include:
1. Temperature
2. Effect of pH
3. Substrate/enzyme concentration
4. Relationship of Velocity to Enzyme Concentration
1. TEMPERATURE:
 Starting from low temperature as the temperature increases to certain degree the
activity of the enzyme increases because the temperature increase the total energy of
the chemical system.
 There is an optimal temperature at which the reaction is most rapid (maximum). Above
this the reaction rate decreases sharply, mainly due to denaturation of the enzyme by
heat.
 The temperature at which an enzyme shows maximum activity is known as the optimum
temperature for the enzyme. For most body enzymes the optimum temperature is
around 370c, which is body temperature.

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2. EFFECT OF PH:
 pH is a measure of the concentration of hydrogen ions in a solution.
 The higher the hydrogen ion concentration, the lower the pH
 Most enzymes function efficiently over a narrow pH range.
 A change in pH above or below this range reduces the rate of enzyme reaction
considerably.
 Changes in pH lead to the breaking of the ionic bonds that hold the tertiary structure of
the enzyme in place. The enzyme begins to lose its functional shape, particularly the
shape of the active site, such that the substrate will no longer fit into it, the enzyme Is
said to be denatured.
 Also changes in pH affect the charges on the amino acids within the active site such that
the enzyme will not be able to form an enzyme-substrate complex.
 The pH at which an enzyme catalyses a reaction at the maximum rate is called the
optimum pH. This can vary considerably from pH 2 for pepsin to pH 9 for pancreatic
lipase

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3. CONCENTRATION OF SUBSTRATE:
 At fixed enzyme concentration pH and temperature the activity of enzymes is influenced
by increase in substrate concentration.
 An increase in the substrate concentration increases the enzyme activity till a maximum
is reached. Further increase in substrate concentration does not increase rate of
reaction.
 This condition shows that as concentration of substrate is increased, the substrate
molecule combine with all available enzyme molecules at their active site till not more
active sites are available (The active Sites become saturated). At this state the enzyme is
obtained it maximum rate (V max).
 The characteristic shape of the substrate saturation curve for an enzyme can be
expressed mathematically by the Michaelis Menten equation:
Equation Missing
Where: V= Velocity at a given concentration of substrate (initial reaction velocity)
Vmax = Maximal velocity possible with excess of substrate
[S] = concentration of the substrate at velocity V
Km = michaelis-constant of the enzyme for particular substrate.
 Relationship between [S] and Km:
o Km shows the relationship between the substrate concentration and the
velocity of the enzyme catalyzed reaction.
o Take the point, in which 50% of the active site of the enzyme will be
saturated by substrate, Assume that at ½ Vmax-50% of the active site of
enzyme becomes saturated. Therefore:
Equation Missing
 Characteristics of Km:
o Km- can define as the concentration of the substrate at which a given
enzyme yields one-half its max. Velocity (i.e Km is numerically equal to the
substrate concentration of which the reaction velocity equal to ½ Vmax)

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o Km- is characteristic of n enzyme and a particular substrate, and reflects the
affinity of the enzyme for that substrate.
o Km- values vary from enzyme to enzyme and used to characterize different
enzymes.
o Km- values of an enzyme help to understand the nature and speed of the
enzyme catalysis.
o Small Km - A numerically small (Low) km reflects a high affinity of the enzyme
for substrate because a low conc. of substrate is needed to half saturate the
enzyme- that is reach a velocity of ½ Vmax.
o High Km - A numerically large (high) Km reflects a low affinity of enzyme for
substrate b/c a high conc. of substrate is needed to half saturate the enzyme.
o High Km Value f an enzyme means the catalysis of that enzyme is slow
compared to low Km.
o Km does not vary with the concentration of enzyme.
4. RELATIONSHIP OF VELOCITY TO ENZYME CONCENTRATION:
 The rate of the reaction is directly proportional to enzyme concentration at all
substrate concentration.
 For example, if the enzyme concentration halved, the initial rate of the reaction (Vo)
is reduced to one half that of the original.
 Order of Reaction
o When [S] is much less than Km, the velocity of the reaction is roughly
proportional to the substrate concentration.
o The rate of reaction is then said to be first order configuration with respect
to substrate.
o When [S] is much greater than Km, the velocity is constant and equal to V
max. The rate of reaction is then independent of substrate concentration and
said to be zero order with respect to substrate concentration.

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ENZYME INHIBITION
Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an
inhibitor and the process is known as inhibition.
TYPES OF INHIBITION
 There are two major types of enzyme inhibition:
1. Irreversible Inhibition:
 The type of inhibition that cannot be reversed by increasing substrate
concentration or removing the remaining free inhibitor is called Irreversible
inhibition.
 Bind to enzymes through non covalent bonds.
 Example: Inhibition of triose phosphate dehydrogenate by iodo acetate
which block the activity of the enzyme.
2. Reversible Inhibition:
 This type of inhibition can be:
a) Competitive Inhibition
b) Non-competitive Inhibition
c) Uncompetitive Inhibition
A. Competitive Inhibition:
 This type of inhibition occurs when the inhibitor binds reversibly to the same site
that the substrate would normally occupy, therefore, competes with the substrate
for that site.
 In competitive inhibition the inhibitor and substrate compete for the same active
site on the enzyme as a result of similarity in structure.
 A classicalexample is Malonate that competes with succinate and inhibits the action
of succinate dehydrogenase to produce fumarate in the Krebs cycle.

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 Effect of Competitive inhibitors:
o Effect on Vmax: The effect of a competitive inhibitor is reversed by
increasing at a sufficiently high substrate concentration; the reaction velocity
reaches the Vmax observed in the absence of inhibitor.This means that the
effect remains same.
o Effect on Km: A competitive inhibitor increases the apparent Km for a given
substrate. This means that in the presence of a competitive inhibitor more
substrate is needed to achieve ½ Vmax.
 Examples of Competivie Inhibitors - Statin Drugs:
o This group of antihyperlipidemic agents competitively inhibits the first
committed step in cholesterol synthesis.
o This reaction is catalyzed by hydroxymethylglutaryl–CoA reductase (HMG-
CoA reductase)
o Statin drugs, such as atorvastatin (Lipitor) and pravastatin (Pravachol),1 are
structural analogs of the natural substrate for this enzyme, and compete
effectively to inhibit HMG-CoA reductase.

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o By doing so, they inhibit de novo cholesterol synthesis, thereby lowering
plasma cholesterol levels.
B. Non-Competitive Inhibition:
 In non-competitive inhibition the inhibitor binds at different site rather than the
substrate-binding site.
 When the inhibitor binds at this site there will be a change in conformation of the
enzyme molecules, which leads to the reversible inactivation of the catalytic site.
 Non-competitive inhibitors bind reversibly either to the free-enzyme or the ES
complex to form the inactive complexes
 An Example: The inhibition of L-threonine dehydratase by L-isoleucine. Such type of
Enzyme is called Allosteric Enzyme, which has a specific sites or allosteric site other
than the substrate-binding site.
 Effect of Non - Competitive inhibitors
o Effect on Vmax: Non-Competitive inhibition cannot be overcome by
increasing the concentration of substrate. Thus, non-competitive inhibitors
decrease the Vmas of the reaction.
o Effect on Km: Non-competitive inhibitors do not interfere with the binding of
substrate to enzyme. Thus, the enzyme shows the same Km in the presence
or absence of the noncompetitive inhibitor.

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 Examples of Non – Competitive Inhibitors:
 Some inhibitors act by forming covalent bonds with specific groups of
enzymes.
 Example:
o Lead forms covalent bonds with the sulfhydryl side chains of cysteine in
proteins. The binding of the heavy metal shows noncompetitive
inhibition.
o Ferrochelatase, an enzyme that catalyzes the insertion of Fe2+ into
protoporphyrin (a precursor of heme), is an example of an enzyme
sensitive to inhibition by lead
o Other examples of noncompetitive inhibition are certain insecticides,
whose neurotoxic effects are a result of their covalent binding at the
catalytic site of the enzyme acetylcholinesterase (an enzyme that cleaves
the neurotransmitter, acetylcholine).
C. Uncompetitive Inhibition:
 Uncompetitive Inhibitor binds only to ES complex at locations other than the
catalytic site.
 Substrate binding modifies enzyme structure, making inhibitor-binding site available.

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 Inhibition cannot be reversed by substrate. In this case apparent Vmax and Km
decreased.
ENZYME INHIBITORS AS DRUGS
 At least half of the ten most commonly dispensed drugs in the United States act as
enzyme inhibitors.
 For example:
o The widely prescribed â-lactam antibiotics, such as penicillin and amoxicillin2
act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may
also act by inhibiting extracellular reactions.
o This is illustrated by angiotensin-converting enzyme (ACE) inhibitors. They
lower blood pressure by blocking the enzyme that cleaves angiotensin I to
form the potent vasoconstrictor, angiotensin II.
o These drugs, which include captopril, enalapril, and lisinopril, cause
vasodilation and a resultant reduction in blood pressure
REGULATION OF ENZYME ACTIVITY
IMPORTANCE OF REGULATION
 Regulation of enzyme activity is important to coordinate the different metabolic
processes.

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 It is also important for homeostasis i.e. to maintain the internal environment of the
organism constant.
MECHANISMS FOR REGULATION OF ENZYME ACTIVITY
 Regulation of enzyme activity can be achieved by two general mechanisms:
1. Control of enzyme quantity:
a) Alternating the rate of enzyme synthesis and degradation:
 As enzymes are protein in nature, they are synthesized from
amino acids under gene control and degraded again to amino
acids after doing its work.
 Enzyme quantity depends on the rate of enzyme synthesis and
the rate of its degradation.
 Increased enzyme quantity may be due to an increase in the rate
of synthesis, a decrease in the rate of degradation or both.
 Decreased enzyme quantity may be due to a decrease in the rate
of synthesis, an increase in the rate of degradation or both.
b) Induction:
 Induction means an increase in the rate of enzyme synthesis by
substances called inducers.
 According to the response to inducers, enzymes are classified
into:
i. Constitutive enzymes, the concentration of these enzymes
does not depend on inducers.
ii. Inducible enzymes the concentration of these enzymes
depends on the presence of inducers
c) Respiration:
 Repression means a decrease in the rate of enzyme synthesis by
substances called repressors.
 Repressors are low molecular weight substances that decrease
the rate of enzyme synthesis at the level of gene expression.

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 Repressors are usually end products of biosynthetic reaction, so
repression is sometimes called feedback regulation.
d) Derespression:
 Removal of the repressor or its exhaustion, enzyme synthesis
retains its normal rate.
2. Control of catalytic efficiency of enzymes:
a) Allosteric Regulation:
 Allosteric means another site.
 Allosteric enzyme is formed of more than one protein subunit.
 It has two sites:
i. Catalytic site for substrate binding and
ii. Allosteric site that is the regulatory site, to which an
effector binds.
 If binding of the effector to the enzyme increases it activity, it is
called positive effector or allosteric activator e.g. ADP is allosteric
activator for phosphofructokinase enzyme.
 If binding of the effector to the enzyme causes a decrease in its
activity, it is called negative effector or allosteric inhibitor e.g.
ATP and citrate are allosteric inhibitors for phosphofructokinase
enzyme.
b) Feedback Inhibition:
 It means that an end product directly inhibits an enzyme early in
biosynthetic pathways.
 It does not affect enzyme quantity.
 It decreases enzyme activity.

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 It is a direct and rapid process that occurs in seconds to -
minutes.
 For example, CTP inhibits aspartate- transcarbamylase enzyme in
pyrimidine synthesis
c) Proenzyme ( Zymogen):
 Some enzymes are secreted in inactive forms called proenzymes
or zymogens.
 Examples for zymogens include:
o Pepsinogen
o Trypsinogen
o Chymotrypsinogen
o Prothrombin
o Clotting factors
 Zymogen is inactive because it contains an additional polypeptide
chain that masks (blocks) the active site of the enzyme.
 Activation of zymogens can occur by one of the following methods:
Pepsinogen → Pepsin
Trypsinogen → Trypsin
Plasminogen → Plasmin
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