INTRODUCTION

TO
ENZYMES
 Enzymes
• Enzymes are catalysts that change the rate of a reaction without being
changed themselves. Enzymes are highly specific and their activity can
be regulated. Virtually all enzymes are proteins, although some
catalytically active RNAs have been identified.
 Coenzymes and
prosthetic groups
• Some enzymes require the presence of cofactors, small
nonprotein units, to function. Cofactors may be inorganic
ions or complex organic molecules called coenzymes. A
cofactor that is covalently attached to the enzyme is called
a prosthetic group (like FAD). A Holoenzyme is the
catalytically active form of the enzyme with its cofactor

• Holoenzyme = enzyme + cofactor

• , whereas an apoenzyme is the protein part on its own.
• apoenzyme+= protein part
• Many coenzymes are derived from dietary vitamin
precursors, and deficiencies in them lead to certain
diseases.
• Nicotinamide adenine dinucleotide (NAD+),
nicotinamide adenine dinucleotide phosphate (NADP+),

• flavin adenine dinucleotide (FAD) and flavin mononucleotide

(FMN) are widely occurring coenzymes involved in oxidation–
reduction reactions.
• Some enzymes require molecules other than proteins for enzymic
activity. Holoenzyme refers to the active enzyme with its nonprotein
component, whereas the enzyme without its nonprotein moiety is
termed an apoenzyme and is inactive. If the nonprotein moiety is a
metal ion such as Zn2+ or Fe2+, it is called a cofactor. If it is a small
organic molecule, it is termed a coenzyme.

• Coenzymes that only transiently associate with the enzyme are

called cosubstrates. Cosubstrates dissociate from the enzyme in an
altered state (NAD+ and coenzyme A are examples. If the coenzyme
is permanently associated with the enzyme and returned to its
original form, it is called a prosthetic group (FAD is an example).
Coenzymes frequently are derived from vitamins. For example,
NAD+ contains niacin, coenzyme A contains pantothenic acid, and
FAD contains riboflavin
 The active site
• The active site is the region of the enzyme that binds the
substrate, to form an enzyme–substrate complex, and
transforms it into product.
• The active site is a three-dimensional entity, often a cleft
or crevice on the surface of the protein, in which the
substrate is bound by multiple weak interactions.
• Two models have been proposed to explain how an enzyme
binds its substrate:
• the lock-and-key model and the induced-fit model.
Nomenclature and classification of enzymes
A. Recommended name
• 1- Enzyme are generally named after adding the
suffix (ase) to the name os the substrate.
Urea + H2O Urease 2NH3 +CO2
Lactose + H2O Lactase Glucose +galactose
Dipeptide +H2O dipeptidase ( A.A1+A.A2)
Note: Some enzymes retain their original trivial
names,, for example, trypsin and pepsin.]
B. Systematic name
• The (IUBMB) developed a system of nomenclature
in which enzymes are divided into six major classes
(Figure )

• The suffix -ase is attached to a fairly complete

description of the chemical reaction catalyzed,
including the names of all the substrates; for
example D- glyceraldehyde 3-phosphate:NAD+
oxidoreductase.
 Enzyme classification
• Enzymes are classified into six major groups on the
basis of the type of reaction that they catalyze.
Each enzyme has a unique four-digit classification
number.
Enzymes with similar reaction specify
are grouped into each of the six major
classes:
 The substrate specificity
• The substrate specificity of an enzyme is
determined by the properties and spatial
arrangement of the amino acid residues forming
the active site.
• The serine proteases trypsin, chymotrypsin and
elastase cleave peptide bonds in protein substrates on
the carboxyl side of positively charged, aromatic and
small side-chain amino acid residues, respectively, due
to complementary residues in their active sites.
 Properties of Enzymes
• A. Active sites
• B. 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 1,000
substrate molecules into product each second. The
number of molecules of substrate converted to
product per enzyme molecule per second is called
the turnover
 C- Specificity of Enzymes
• One of the properties of enzymes that makes them
so important as diagnostic and research tools is the
specificity they exhibit relative to the reactions
they catalyze.
• absolute specificity A few enzymes exhibit
absolute specificity; that is, they will catalyze only
one particular reaction.
• Other enzymes will be specific for a particular
type of chemical bond or functional group.
• In general, there are four distinct types of
specificity:
• 1. Absolute specificity - the enzyme will catalyze
only one reaction.
• 2. Group specificity - the enzyme will act only on
molecules that have specific functional groups,
such as amino, phosphate and methyl groups.
• 3. Linkage specificity - the enzyme will act on a
particular type of chemical bond regardless of the
rest of the molecular structure.
• 4. Stereochemical specificity - the enzyme will
act on a particular steric or optical isomer.
E- Regulation
• Enzyme activity can be regulated, that is, enzymes can be
activated or inhibited, so that the rate of product formation
responds to the needs of the cell.

D- Location within the cell

• Many enzymes are localized in specific organelles
within the cell. Such compartmentalization serves to
isolate the reaction substrate or product from other
competing reactions.
 Isoenzymes
• Isoenzymes are different forms of an
enzyme which catalyze the same reaction,
but which exhibit different physical or
kinetic properties.

• The isoenzymes of lactate dehydrogenase (LDH)

can be separated electrophoretically and can be
used clinically to diagnose a myocardial
infarction.
How Enzymes
Work
• Figure: Effect of
an enzyme on the
activation energy
of a reaction
Factors affects the Enzyme's reaction rate:-
1- pH and temperature dependency of
enzyme activity
• The effect of enzymes is strongly dependent on the pH
value. When the activity is plotted against pH, a bell-
shaped curve is usually obtained .
• With animal enzymes,
the pH optimum—i. e., the pH value at which enzyme
activity is at its maximum—is often close to the pH value
of the cells (i. e., pH 7).However, there are also exceptions
to this. For example, the proteinase pepsin
The temperature dependency of
enzymatic activity
The temperature dependency of enzymatic activity is usually
asymmetric. With increasing temperature, the increased
thermal movement of the molecules initially leads to a rate
acceleration.
At a certain temperature, the enzyme then becomes unstable,
and its activity is lost within a narrow temperature
difference as a result of denaturation.
The optimal temperatures of the enzymes in higher organisms
rarely exceed 50 °C, while enzymes from thermophilic
bacteria found in hot springs, for instance, may still be
active at 100 °C.
• Effect of pH on the ionization of the active site: The
concentration of H+ affects reaction velocity in several ways.
First, the catalytic process usually requires that the enzyme
and substrate have specific chemical groups in either an
ionized or un-ionized state in order to interact. For example,
catalytic activity may require that an amino group of the
enzyme be in the protonated form (–NH3+). At alkaline pH,
this group is deprotonated, and the rate of the reaction,
therefore, declines.
The pH optimum varies for different
enzymes:

• The pH at which maximal enzyme activity is achieved is

different for different enzymes, and often reflects the
[H+] at which the enzyme functions in the body. For
example, pepsin, a digestive enzyme in the stomach, is
maximally active at pH 2.
Enzyme Concentration
 4- Effect of Substrates

• Effect of substrate concentration on reaction velocities

for two enzymes: enzyme 1 with a small Km, and
enzyme 2 with a large Km.
Michaelis-Menten Equation
 Important conclusions about
Michaelis-Menten kinetics
• Characteristics of Km: Km—the Michaelis constant—is
characteristic of an enzyme and its particular substrate,
and reflects the affinity of the enzyme for that substrate.
Km is numerically equal to the substrate concentration at
which the reaction velocity is equal to ½Vmax. Km does
not vary with the concentration of enzyme.
• Effect of substrate concentration on reaction velocities
for two enzymes: enzyme 1 with a small Km, and
enzyme 2 with a large Km.
– Small Km: A numerically small (low) Km reflects a
high affinity of the enzyme for substrate, because a low
concentration of substrate is needed to half-saturate the
enzyme—that is, to reach a velocity that is
½Vmax (Figure 5.9).

– Large Km: A numerically large (high) Km reflects a

low affinity of enzyme for substrate because a high
concentration of substrate is needed to half-saturate the
enzyme.
 Effect of substrate
concentration on
reaction velocity for
an enzyme-catalyzed
reaction.
• 1- When [S] is much
less than Km

• 2- When [S] is much

greater than Km

• When [S] = Km
 Relationship of velocity to enzyme
concentration:
• The rate of the reaction is directly proportional to the enzyme
concentration at all substrate concentrations. For example, if
the enzyme concentration is halved, the initial rate of the
reaction (Vo), as well as that of Vmax, are reduced to half
that of the original.
• Order of reaction: When [S] is much less than Km, the
velocity of the reaction is approximately proportional to
the substrate concentration (seeFigure).The rate of
reaction is then said to be first order with respect to
substrate.

• When [S] is much greater than Km, the velocity is

constant and equal to Vmax. The rateof reaction is then
independent of substrate concentration, and is said to be
zero order with respect to substrate concentration (see
Figure )
 Lineweaver-Burk plot
• it is impossible to obtain a definitive value
from a typical Michaelis-Menten plot.
Because KM is the concentration of substrate
at V max/2,
• it is likewise impossible to determine an
accurate value of KM. However, V max.
• It must transforming to a straight-line plot by
Taking the reciprocal of both sides of
equation gives.
• This called a Lineweaver-Burk or double-reciprocal plot,
• A plot of 1/V 0 versus 1/[S], yields a straight line with an
intercept of 1/V max and a slope of K M/V max (Figure).
The intercept on the x-axis is -1/K M.
 4- Inhibition of Enzyme Activity
• 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 bind to enzymes through
noncovalent bonds,
(thus dilution of the enzyme–inhibitor complex
results in dissociation of the reversibly bound
inhibitor, and recovery of enzyme activity. )
• Mechanism-based inhibitors. The
effectiveness of many drugs and toxins
depends on their ability to inhibit an enzyme.
The strongest inhibitors are covalent
• inhibitors, compounds that form covalent
bonds with a reactive group in the enzyme
active site, or transition state analogues that
mimic the transition state complex.
A. Competitive inhibition
• the inhibitor binds reversibly to the same site that
the substrate would normally occupy and, therefore,
competes with the substrate for that site.
• Vmax is unchanged
• Km is increased
 A- Competitive inhibition
• Effect on Vmax: The effect of a competitive inhibitor is
reversed by increasing [S]. At a sufficiently high substrate
concentration, the reaction velocity reaches the Vmax
observed in the absence of inhibitor.
• 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
½Vmax.
• Effect on the Lineweaver-Burk plot: in which the plots of the
inhibited and uninhibited reactions intersect on the y-axis at
1/Vmax (Vmax is unchanged). The inhibited and uninhibited
reactions show different x-axis intercepts, indicating that the
apparent Km is increased in the presence of the competitive
inhibitor because it moves closer to zero from a negative value
• 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
simvastatin (Zocor),1 are structural analogs of the
natural substrate for this enzyme, and compete
effectively to inhibit HMG-CoA reductase. By
doing so, they inhibit cholesterol synthesis,
thereby lowering plasma cholesterol levels
 B. Noncompetitive inhibition
• Its characteristic effect on Vmax. Noncompetitive inhibition occurs
when the inhibitor and substrate bind at different sites on the enzyme.
The noncompetitive inhibitor can bind either free enzyme or the ES
complex, thereby preventing the reaction from occurring
 B. Noncompetitive inhibition
• Effect on Vmax: Noncompetitive inhibition cannot be
overcome by increasing the concentration of substrate.
Thus, noncompetitive inhibitors decrease the apparent
Vmax of the reaction.
• Effect on Km: Noncompetitive 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.
• Effect on Lineweaver-Burk plot: Noncompetitive
inhibition is readily differentiated from competitive
inhibition by plotting 1/vo versus 1/[S] and noting that
the apparent Vmax decreases in the presence of a
noncompetitive inhibitor, whereas Km is unchanged
• Examples of noncompetitive inhibitors: Some inhibitors act
by forming covalent bonds with specific groups of enzymes.
• example, lead forms covalent bonds with the sulfhydryl side
chains of cysteine in proteins. The binding of the heavy
metal shows noncompetitive inhibition. 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.
• Other examples of noncompetitive inhibition are certain
insecticides, whose neurotoxic effects are a result of their
irreversible binding at the catalytic site of the enzyme
acetylcholinesterase (an enzyme that cleaves the
neurotransmitter, acetylcholine).
C- Uncompetitive Inhibitor
• An inhibitor that is uncompetitive with respect to
a substrate will bind only to enzyme containing
that substrate. Suppose, for example,
• inhibitor that is a structural analog of B and binds
to the B site could only bind to an enzyme that
contains A. That inhibitor would be called
uncompetitive with respect to A.
• It would decrease both the Vmax of the enzyme
and its apparent Km for A.
Mixed inhibition.
 5- Covalent modification of enzyme
• The rates of most enzymes are responsive to
changes in substrate concentration, because the
intracellular level of many substrates is in the range
of the Km
• an increase in substrate concentration prompts an
increase in reaction rate, which tends to return the
concentration of substrate toward normal.
• In addition, some enzymes with specialized
regulatory functions respond to allosteric effectors
or covalent modification, or they show altered rates
of enzyme synthesis (or degradation) when
physiologic conditions are changed
 A. Allosteric binding sites
• Allosteric enzymes. Allosteric activators or inhibitors
are compounds that bind at sites other than the active
catalytic site and regulate the enzyme through
conformational changes affecting the catalytic site.
• The presence of an allosteric effector can alter the
affinity of the enzyme for its substrate, or modify the
maximal catalytic activity of the enzyme, or both.
• Effectors that inhibit enzyme activity are termed
negative effectors, whereas those that increase
enzyme activity are called positive effectors.
• Figure: Effects of negative - or positive + effectors on an
allosteric enzyme. A. Vmax is altered. B. The substrate
concentration that gives half-maximal velocity (K0.5) is
altered.
• 1-Homotropic effectors: When the substrate
itself serves as an effector, the effect is said to
be homotropic. Most often, an allosteric
substrate functions as a positive effector.
• . 2-Heterotropic effectors: The effector may be
different from the substrate, is said to be
heterotropic. For example, consider the
feedback inhibition shown in Figure. The
enzyme that converts D to E has an allosteric
site that binds the end product, G
• Feedback inhibition provides the cell with a product it
needs by regulating the flow of substrate molecules
through the pathway that synthesizes that product.

• example, the glycolytic enzyme phosphofructokinase-1 is

allosterically inhibited by citrate, which is not a substrate
for the enzyme
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 .
• Protein phosphorylation is recognized as one of the
primary ways in which cellular processes are
regulated.
• 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
• One of the fuels used by skeletal muscles for
jogging is glucose, which is converted to glucose 6-
phosphate (glucose 6-P) by the enzymes hexokinase
(HK) and glucokinase (GK). Glucose 6-P is
metabolized in the pathway of glycolysis to
generate ATP.
• This pathway is feedback regulated, so that as the
muscles use ATP, the rate of glycolysis will
increase to generate more ATP.
• At the resting, the muscles
and liver will convert
glucose 6-phosphate to
glycogen (a fuel storage
pathway, shown in blue).
Glycogen
• synthesis is feed-forward
regulated by the supply of
glucose and by insulin and
other hormones that signal
glucose availability.
 C. Induction and repression of enzyme
synthesis
• Cells can also regulate the amount of enzyme
present—usually by altering the rate of enzyme
synthesis.
• The concentration of an enzyme can be regulated
by changes in the rate of enzyme synthesis (e.g.,
induction of gene transcription) or the rate of
degradation.