Course Code: BIO 024 (Biochemistry/Biomolecules)

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Name: ____________________________________________________________ Class number: _______

BSN 1-A26 Schedule: ____________________________________
Section: ____________ 09/28/23
Date: _______________

Lesson title: ENZYMES Materials:

Lesson Objectives: by the end of this module, you Pen, SAS
should be able to
1. Define common terminologies under enzyme References:
2. Determine the different classifications of enzyme ▪ stoker, H. S. (2017).Biochemistry (3rd
and types of specificities ed.). (M. Finch, Ed.) Belmont CA, USA
3. Interpret the illustration and graphs depicting the ▪ Ferrier, D. (2017). Lippincott's Illustrated
enzyme activity, factors affecting the enzyme Biochemistry (7 ed.). Lippincott Williams &
Wilkins,.
activity and enzyme inhibition
▪ Nemire, Ruth & Kier, Karen. (2009)
4. Provide examples of the medical uses of Pharmacy Student Survival Guide, 2nd
enzymes. ed. Mc. Graw Hill
5.

Productivity Tip:
Try doing a house tour before starting this module. Take a quick look at some important things that makes
your daily activities moves faster. This is to give your brain an idea of what’s coming - it’s like watching a
trailer of a movie. Doing this for a minute will help your brain organize your thoughts before studying.

A. LESSON PREVIEW/REVIEW
1) Introduction (1 min)
Virtually all reactions in the body are mediated by enzymes (Greek word en “in” and zyme “yeast”), 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. Enzymes thus
direct all metabolic events. This module examines the nature of
these catalytic molecules, their mechanism of action and their
importance. (Stoker, 3rd ed.)

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2) Activity 1: What I Know Chart, part 1 (3 mins)

Instructions: "In this chart, reflect on what you know now. Do not worry if you are sure or not sure of
your answers. This activity simply serves to get you started on thinking about our topic. Answer only
the first column, "What I know" based on the question of the second column. Leave the third column
"What I learned" blank at this time.

What I Know Questions: What I Learned (Activity 4)

Enzyme activity can be affected by all three factors: pH, 1. What affects enzyme activity, pH, Temperature, pH, and concentration are all parameters that can
temperature, and substrate concentrations. Each enzyme has influence enzyme activity. Enzymes function best in specified
an optimal pH and temperature range, and substrate temperature or concentrations? temperature and pH ranges, and poor circumstances can lead an
enzyme to lose its ability to bind to a substrate.
concentration can also affect the rate of the reaction.

Yes, drugs can act as enzyme inhibitors. Enzyme inhibitors 2. Is it correct to say that, drugs are Yes, many medications are small molecule enzyme inhibitors that
target either disease-modifying enzymes in the patient or
are molecules that can bind to an enzyme and decrease its enzyme inhibitors? Support your pathogen enzymes essential for pathogen growth and
reproduction. Some proteins, in addition to tiny molecules,
activity. Some drugs work by inhibiting enzymes that are answer. operate as enzyme inhibitors. Some medicines disrupt this
involved in disease processes. interaction by inhibiting the binding site of the enzyme and
preventing actual substrate binding to the enzyme. Since this
True, enzymes are biochemically 3. Enzymes are also biochemically inhibits the enzyme's catalytic activity, these are known as
inhibitors.
used for diagnostic purposes such
as measuring blood glucose levels or used for diagnostic purposes, True True, enzymes are used in analytical tests, disease
diagnosis, treatment of enzyme deficiencies and wound
detecting certain diseases. or False? therapies.

B.MAIN LESSON
A. Activity 2: Content notes (60 min).
Instructions: Please make your own mind map or outline. Note, it will help if you check the skill
building activity first. However, do not answer the activity side by side with your notes so that you can
assess your learning later better.

I.NOMENCLATURE
-------------------------------------------------------------------------------------
Each enzyme is assigned two names. The first is its short, recommended name, convenient for
everyday use. The second is the more complete systematic name, which is used when an enzyme
must be identified without ambiguity.

A. Recommended name
Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the reaction (for
example, glucosidase and urease), or to a description of the action performed (for example, lactate
dehydrogenase and adenylyl cyclase). [Note: Some enzymes retain their original trivial names, which
give no hint of the associated enzymic reaction, for example, trypsin and pepsin.]

B. Systematic name
In the systematic naming system, enzymes are divided into six major classes (Figure 2.1), each with
numerous subgroups. For a given enzyme, the suffix -ase is attached to a fairly complete description of
the chemical reaction catalyzed, including the names of all the substrates; for example, lactate: NAD+
oxidoreductase. [Note: Each enzyme is also assigned a classification number.] The systematic names
are unambiguous and informative, but are frequently too cumbersome to be of general use. (Lippincott,
7th ed.)

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(Stoker, 3rd ed.)

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CLASSIFICATION OF ENZYME BASED ON THE REACTION BEING CATALYZED

Main Class Selected Type of Reaction Catalyzed

subclasses
• oxidases • oxidation of a substrate
OXIDOREDUCTAS • reductases • reduction of a substrate
ES • dehydrogenases • introduction of double bond (oxidation) by formal removal of two H atoms
from substrate, the H being accepted by a coenzyme
• Transaminases • transfer of an amino group between substrates
TRANSFERASES
• Kinases • transfer of a phosphate group between substrates
• Lipases • hydrolysis of ester linkages in lipids
• Proteases • hydrolysis of amide linkages in proteins
HYDROLASES • Nucleases • hydrolysis of sugar–phosphate ester bonds in nucleic acids
• Carbohydrases • hydrolysis of glycosidic bonds in carbohydrates
• phosphatases • hydrolysis of phosphate–ester bonds
• dehydratases • removal of H2O from a substrate
• decarboxylases • removal of CO2 from a substrate
LYASES
• deaminases • removal of NH3 from a substrate
• hydratases • addition of H2O to a substrate
• Racemases • conversion of D isomer to L isomer,or vice versa
ISOMERASES • Mutases • transfer of a functional group from one position to another in the same
molecule
• Synthetases • formation of new bond between two substrates, with participation of ATP
LIGASES • Carboxylases • formation of new bond bet. a substrate and CO2, with participation of ATP

II.PROPERTIES OF ENZYME (Lippincott, 7th ed)

-----------------------------------------------------------------------------------------------------------------------------------------
Enzymes are protein catalysts that increase the velocity of a chemical reaction, and are NOT
consumed during the reaction. [Note: Some RNAs can act like enzymes, usually catalyzing the
cleavage and synthesis of phosphodiester bonds. RNAs with catalytic activity are called ribozymes,
and are much less commonly encountered than protein catalysts.]

A. Active site
Enzyme molecules contain a special pocket or cleft called the active site. The active site (10-
20% of the entire protein structure) contains amino acid side chains that participate in substrate
binding and catalysis (Figure 2.2). The substrate binds the enzyme,
forming an enzyme–substrate (ES) complex. Binding is thought to cause
a conformational change in the enzyme (induced fit) that allows
catalysis. ES is converted to an enzyme–product (EP) complex that
subsequently dissociates to
enzyme and product.

2.2
Substrate - is the reactant in an
enzyme-catalysed reaction

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▪ the active site is the region of an enzyme where substrate molecules

bind and undergo a chemical reaction.
▪ The active site consists of residues that form temporary bonds with the
substrate (binding site) and residues that catalyse a reaction of that
substrate (catalytic site).

Enzyme–substrate complex is the intermediate reaction species

that is formed when a substrate binds to the active site of an
enzyme.

2 types of ESC or Models of enzyme activity

1. Lock and key model - the active site in the enzyme has a fixed, rigid geometrical
conformation. Only substrates with a complementary geometry
can be accommodated at such a site, much as a lock accepts
only certain keys. This is the simplest type and a product of
enzyme specificity.

2. Induced fit model- allows for small changes in the shape or

geometry of the active site of an enzyme to accommodate a
substrate. The induced fit is a result of the enzyme’s flexibility; it
adapts to accept the incoming substrate.

2.3

B. Catalytic efficiency
Enzyme-catalyzed reactions are highly efficient, proceeding from 103–108 times faster than
uncatalyzed reactions. The number of molecules of substrate converted to product per enzyme
molecule per second is called the turnover number, or kcat and typically is 102–104s-1.

C. Specificity
Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one
type of chemical reaction. [Note: The set of enzymes made in a cell determines which metabolic
pathways occur in that cell.]

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4 TYPES OF SPECIFICITY

ABSOLUTE GROUP
• enzyme will catalyze only one reaction. • the enzyme will act only on molecules that
• Ex. Catalase enzyme catalyzes only have a specific functional group, such as
Hydrogen peroxide (H2O2) to H2O and hydroxyl, amino, or phosphate groups.
O2 • Ex. Carboxypeptidase is group-specific; it
cleaves amino acids, one at a time, from the
carboxyl end of a peptide chain.

LINKAGE STEREOCHEMICAL
• the enzyme will act on a particular type • the enzyme will act on a particular
of chemical bond, irrespective of the rest stereoisomer. Chirality is inherent in an enzyme
of the molecular structure. active site because amino acids are chiral
• Ex. Phosphatases compounds. An L-amino acid oxidase will
•hydrolyze phosphate-ester bonds in all catalyze the oxidation of the L-form of an amino
types of phosphate esters acid but not the D-form of the same amino acid

D. Structural class, Holoenzymes, apoenzymes, cofactors, and coenzymes

Enzymes can be divided into two general structural classes: simple enzymes and conjugated
enzymes. A simple enzyme is an enzyme composed only of protein (amino acid chains). A
conjugated enzyme is an enzyme that has a nonprotein part in addition to a protein part. By
itself, neither the protein part nor the nonprotein portion of a conjugated enzyme has catalytic
properties.

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 or the protein part of the conjugated enzyme 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+ is an example). 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 (see vitamin module).
For example, NAD+ contains niacin and FAD contains riboflavin.

Apoenzyme + nonprotein moiety (cofactor) = holoenzyme

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 (Figure 2.3). Such
compartmentalization serves to isolate the reaction substrate or product from other competing
reactions. This provides a favorable environment for the reaction, and organizes the thousands
of enzymes present in the cell into purposeful pathways.

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III.HOW ENZYMES WORK (MECHANISM OF ACTION) (Lippincott, 7th ed)

------------------------------------------------------------------------------------------------------------------------------
Two different perspectives: First, catalysis in terms of energy changes that occur during the
reaction, that is, enzymes provide an alternate, energetically favorable reaction pathway different from
the uncatalyzed reaction. Second, describes how the active site
chemically facilitates catalysis.

A. Energy changes occurring during the reaction

Virtually all chemical reactions have an energy barrier separating
the reactants and the products. This barrier, called the free energy of
activation, is the energy difference between that of the reactants and a
high-energy intermediate that occurs during the formation of product. For
example, Figure 2.4 shows the changes in energy during the conversion
of a molecule of reactant A to product B as it proceeds through the
transition state (high-energy intermediate), T*:

A↔T*↔B

1.Free energy of activation: The peak of energy in Figure 2.4 is the

difference in free energy between the reactant and T*, where the high-
energy intermediate is formed during the conversion of reactant to
product. Because of the high (ΔG) free energy of activation, the rates of
uncatalyzed chemical reactions are often slow.
2.4

2.Rate of reaction: For molecules to react, they must contain sufficient

energy to overcome the energy barrier of the transition state. In the
absence of an enzyme, only a small proportion of a population of molecules may possess enough
energy to achieve the transition state between reactant and product. The rate of reaction is determined
by the number of such energized molecules. In general, the lower the free energy of activation, the
more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate
of the reaction.

3.Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions
prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation
(Figure 2.4). The enzyme does not change the free energies of the reactants or products and,
therefore, does not change the equilibrium of the reaction (see illustration). It does, however, accelerate
the rate with which equilibrium is reached.

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B.Chemistry of the active site

The active site is not a passive receptacle for binding the substrate, but rather is a complex molecular
machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product. A
number of factors are responsible for the catalytic efficiency of enzymes, including the following:

1. Transition-state stabilization: The active site

often acts as a flexible molecular template that binds the substrate and
initiates its conversion to the transition state, a structure in which the
bonds are not like those in the substrate or the product (see T* at the
top of the curve in Figure 2.4). By stabilizing the transition state, the
enzyme greatly increases the concentration of the reactive intermediate
that can be converted to product and, thus, accelerates the reaction.
2.
2. Catalysis: The active site can provide catalytic
groups that enhance the probability that the transition state is formed. In
some enzymes, these groups can participate in general acid-base
catalysis in which amino acid residues provide or accept protons. In
other enzymes, catalysis may involve the transient formation of a
covalent ES complex. [Note: Chymotrypsin , an enzyme of protein
digestion in the intestine, includes general base, general acid, and
covalent catalysis]
1.
3. Visualization of the transition state: The
enzyme-catalyzed conversion of substrate to product can be visualized
as being similar to removing a sweater from an uncooperative infant
(see figure at the side). The process has a high energy of activation
because the only reasonable strategy for removing the garment (short of
ripping it off) requires that the random flailing (wave or swing or cause to
wave or swing wildly) of the baby results in both arms being fully extended
over the head—an unlikely posture. However, we can envision a parent
acting as an enzyme, first coming in contact with the baby (forming ES),
then guiding the baby’s arms into an extended, vertical position,
analogous to the ES transition state. This posture (conformation) of the
baby facilitates the removal of the sweater, forming the disrobed baby,
which here represents product. [Note: The substrate bound to the
enzyme (ES) is at a slightly lower energy than unbound substrate (S)
and explains the small “dip” in the curve at ES.]

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IV. FACTORS AFFECTING ENZYMATIC ACTIVITY (Lippincott, 7th ed.)

------------------------------------------------------------------------------------------------
This section describes factors that influence the reaction velocity of
enzymes. Enzymic responses to these factors give us valuable clues as
to how enzymes function in living cells (that is, in vivo).

A. Substrate concentration
1. Maximal velocity: The rate or velocity of a reaction (v) is the
number of substrate molecules converted to product per unit
time; velocity is usually expressed as μmol of product formed
per minute. The rate of an enzyme-catalyzed reaction increases
with substrate concentration until a maximal velocity (Vmax)
Figure 2.5: effect of substrate
is reached (Figure 2.5). The leveling off of the reaction rate at concentration on rxn velocity
high substrate concentrations reflects the saturation with
substrate of all available binding sites on the enzyme molecules
present.

Saturation
means that all
enzyme active
sites were fully
occupied and
so the reaction
rate remains
constant

2. Hyperbolic shape of the enzyme kinetics curve: Most

enzymes show Michaelis-Menten kinetics (see next pages), in which the plot of initial reaction
velocity (vo) against substrate concentration ([S]), is hyperbolic (similar in shape to that of the
oxygen-dissociation curve of myoglobin, see illustration). In contrast, allosteric enzymes do not
follow Michaelis-Menten kinetics and show a sigmoidal curve
(see illustation) that is similar in shape to the oxygen
dissociation curve of hemoglobin (see illustration).

B. Temperature
1. Increase of velocity with temperature: The reaction
velocity increases with temperature until a peak/maximum
velocity is reached (Figure 2.6). This increase is the result of
the increased number of molecules having sufficient energy to
pass over the energy barrier and form the products of the
reaction.
Note: Optimum temperature is the temperature at which an
enzyme exhibits maximum activity (that is substrate is Figure 2.6: effect of temperature
converted to product). on an enzyme reaction

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2. Decrease of velocity with higher temperature:

Further elevation of the temperature results in a decrease
in reaction velocity as a result of temperature-induced
denaturation of the enzyme (see Figure 2.6). The
optimum temperature for most human enzymes is
between 35 and 40°C. Human enzymes start to denature
at temperatures above 40°C, but thermophilic bacteria
found in the hot springs have optimum temperatures of
70°C.

C. pH
1. Effect of pH on the ionization of the active site: The
concentration of H+ affects reaction velocity in several Figure 2.7: effect of pH on an enzyme
ways. First, the catalytic process usually requires that the reaction
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.

2. Effect of pH on enzyme denaturation: Extremes of pH can also lead to denaturation of the

enzyme, because the structure of the catalytically active protein molecule depends on the ionic
character of the amino acid side chains.

3. The pH optimum varies for different enzymes: The pH at which maximal enzyme activity is
achieved is different for different enzymes, and often reflects optimum pH. For example,
pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other
enzymes, designed to work at neutral pH, are denatured by such an acidic environment (Figure
2.7). (You may check for extremophiles or enzymes that can live in more harsh environment.)

D. Enzyme concentration

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; 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. If the amount of substrate present is kept
constant and the enzyme concentration is increased, the
reaction rate increases because more substrate
molecules can be accommodated in a given amount of
time (see Figure 2.). The greater the enzyme
concentration, the greater the reaction rate.

Figure 2.8: effect of eznyme

concentration on rxn velocity

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V. MICHAELIS-MENTEN EQUATION (Don’t worry there’ll be no computation yet)

---------------------------------------------------------------------------------------------------------------------------------------
A. Reaction model
Leonor Michaelis and Maude Menten proposed a simple model that accounts for most of the
features of enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its
substrate to form an ES complex that subsequently yields product, regenerating the free
enzyme. The model, involving one substrate molecule, is represented below:

where
S is the substrate
E is the enzyme
ES is the enzyme–substrate complex
P is the product
k1, k-1, and k2 are rate constants

B.Michaelis-Menten equation
The Michaelis-Menten equation describes how reaction velocity varies
with substrate concentration:

The following assumptions are made in deriving the Michaelis-

Menten rate equation:

1. Relative concentrations of E and S:

The concentration of substrate ([S]) is much greater than the
concentration of enzyme ([E]), so that the percentage of total
substrate bound by the enzyme at any one time is small.

2. Steady-state assumption:
2.9 [ES] does not change with time (the steady-state assumption), that is,
the rate of formation of ES is equal to that of the breakdown of ES (to
E + S and to E + P). In general, an intermediate in a series of
reactions is said to be in steadystate when its rate of synthesis is
equal to its rate of degradation.

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3. Initial velocity: Initial reaction velocities (vo) are used in the analysis of enzyme reactions.
This means that the rate of the reaction is measured as soon as enzyme and substrate are
mixed. At that time, the concentration of product is very small and, therefore, the rate of the
back reaction from P to S can be ignored.

C.Important conclusions about Michaelis-Menten kinetics

1. 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
1⁄2Vmax. Km does not vary with the concentration of enzyme.
a. 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
1⁄2Vmax (Figure 2.9).
b. 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.

2. 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
2.10 reduced to half that of the original.

3. Order of reaction: When [S] is much less than Km, the velocity of

the reaction is approximately proportional to the substrate

concentration (Figure 2.10). 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 rate of reaction
is then independent of substrate concentration, and is said to be
zero order with respect to substrate concentration (see Figure 2.10).

D. Lineweaver-Burk plot
2.11 When vo is plotted against [S], it is not always possible to determine
when Vmax has been achieved, because of the gradual upward
slope of the hyperbolic curve at high substrate concentrations.
However, if 1/vo is plotted versus 1/[S], a straight line is obtained (Figure 2.11). This plot, the
Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and
Vmax, as well as to determine the mechanism of action of enzyme inhibitors.
1. The equation describing the Lineweaver-Burk plot is:

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VI. INHIBITION OF ENZYME ACTIVITY

---------------------------------------------------------------------------------------------------------------------------------------
Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. In
general, irreversible inhibitors bind to enzymes through covalent bonds. Reversible inhibitors typically 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. The two most commonly encountered types
of reversible inhibition are competitive and noncompetitive.

A. Competitive inhibition
This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate
would normally occupy and, therefore, competes with the substrate for that site.
1. 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 (Figure 2.12).
2. 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
1⁄2Vmax.
3. Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic 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 -1/Km moves closer to zero from a negative value (see Figure 2.12).

2.12

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4.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),1 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 (Figure 2.13).

B. Noncompetitive inhibition
This type of inhibition is recognized by its characteristic effect on Vmax
(Figure 2.14). 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 (Figure 2.15).
1. Effect on Vmax: Noncompetitive inhibition cannot be overcome 2.13
by increasing the concentration of substrate. Thus, noncompetitive
inhibitors decrease the apparent Vmax of the reaction.

2. 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. Km
is unchanged.

3. 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
(see Figure 2.14).

2.14

2.15

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C. IRREVERSIBLE INHIBITION
Irreversible Inhibition An irreversible enzyme inhibitor is a molecule that inactivates enzymes by
forming a strong covalent bond to an amino acid side-chain group at the enzyme’s active site. In
general, such inhibitors do not have structures similar to that of the enzyme’s normal substrate. The
inhibitor–active site bond is sufficiently strong that addition of excess substrate does not reverse the
inhibition process. Thus the enzyme is permanently deactivated. The actions of chemical warfare
agents (nerve gases) and organophosphate insecticides are based on irreversible inhibition.

D. Enzyme inhibitors as drugs

At least half of the ten most commonly dispensed drugs in the United States act as enzyme
inhibitors. For example, the widely prescribed β-lactam antibiotics, such as penicillin and
amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by
inhibiting extracellular reactions. 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. These drugs, which include captopril, enalapril,
and lisinopril, cause vasodilation and a resultant reduction in
blood pressure. Aspirin, a non-prescription drug irreversibly
inhibits prostaglandins and thromboxane synthesis by inhibiting
cyclooxygenase.

VII. ENZYME REGULATION

--------------------------------------------------------------------------------------------
A. Allosteric enzymes
Are regulated by molecules called effectors (also called
modifiers) that bind noncovalently at a site other than the active
site. These enzymes are usually composed of multiple subunits,
and the regulatory (allosteric) site that binds the effector may be
located on a subunit that is not itself catalytic. 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. Positive and negative effectors can affect the
affinity of the enzyme for its substrate (K0.5), modify the maximal
catalytic activity of the enzyme (Vmax), or both (Fig. 2.16).
Allosteric enzymes frequently catalyze the committed step, often
the rate limiting step, early in a pathway.
2.16

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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.

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 (Figure
2.17).

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,
2.17
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. Figure 2.18 summarizes the common ways that enzyme activity is regulated.

2.18

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D. Proteolytic enzyme and zymogen : regulating cellular enzyme activity based on the production of
enzymes in an inactive form. These inactive enzyme precursors are then “turned on” at the
appropriate time. Such a mechanism for control is often encountered in the production of proteolytic
enzymes. Because they would otherwise destroy the tissues that produce them, proteolytic
enzymes are generated in an inactive form and then later, when they are needed, are converted to
their active form. Digestive enzymes needs activator like HCl so that zymogen are converted to
proteolytic enzyme.

• proteolytic enzyme : an enzyme that catalyzes the breaking of peptide bonds that maintain
the primary structure of a protein.
• zymogen or proenzyme: is the inactive precursor of a proteolytic enzyme.

EXAMPLES:
ZYMOGEN PROTEOLYTIC ENZYME
▪ Pepsinogen ------- pepsin
▪ Tyrpsinogen ------- trypsin
▪ Chymotrypsinogen ------ chymotrypsin
▪ Angiotensinogen ------- angiotensin

VIII. ENZYMES IN CLINICAL DIAGNOSIS

---------------------------------------------------------------------------------------------------------------------------------
Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes
are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens
(inactive precursors) of the enzymes involved in blood coagulation. Second, a large number of
enzyme species are released from cells during normal cell turnover. These enzymes almost always
function intracellularly, and have no physiologic use in the plasma. In healthy individuals, the levels
of these enzymes are fairly constant, and represent a steady state in which the rate of release from
damaged cells into the plasma is balanced by an equal rate of removal of the enzyme protein from
the plasma. Increased plasma levels of these enzyme may indicate tissue damage (Figure 5.20).

BIOCHEMICALLY IMPORTANT ENZYMES: (Nemire, 2nd ed)

1.CREATINE KINASE: is an enzyme that is found primarily in skeletal and cardiac muscle and in
smaller fractions in the brain

TYPES OF CK
• muscle (CK-MM), brain (CK-BB),
• cardiac tissue (CK-MB) - important marker in the diagnosis of acute myocardial
infarction (AMI)

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2.CARDIAC TROPONIN

Description
• Troponin I and T are sensitive markers of cardiac injury.
• Troponin I is found solely in the cardiac muscle, and
• Troponin T is found in both cardiac and skeletal muscle.
Clinical Significance
• Troponin levels begin to rise within 4 hours of onset of chest pain. Levels should be
drawn on admission and within 8 to 12 hours thereafter. Patients with elevated troponin
levels are considered at high risk for a significant cardiac event.
3. GASTROINTESTINAL TESTS
A.Alanine Aminotransferase/ serum glutamic pyruvic transaminase (SGPT).
i. - liver tissue. It is also located in myocardial, muscle, and renal tissue
ii. - considered a specific marker for liver disease
B.Aspartate Aminotransferase/ serum glutamic oxaloacetic transaminase (SGOT
i. - found in the liver. It is also present in the heart, kidney, pancreas, lungs,
and skeletal muscle
ii. For diagnosis of liver disease
C.g-Glutamyl Transpeptidase
o an enzyme found in the liver, kidney, and pancreas. GGT levels are useful in
the diagnosis and monitoring of alcoholic liver disease
o Increased GGT may be seen in alcoholic liver disease, metastatic liver disease,
obstructive jaundice, cholelithiasis, and pancreatitis
D.Lactate Dehydrogenase
o enzyme involved in the interconversion of lactate and pyruvate.
o is found in many tissues, including heart, brain, liver, skeletal muscle, kidneys,
lungs, and RBCs.
o LDH4 and LDH5 are present in liver tissue, and elevations may be seen in liver
disease such as hepatitis and cirrhosis.
o LDH1 and LDH2 may be useful in the diagnosis of myocardial infarction
E. Lipase
• enzyme that aids in the digestion of fat. It is primarily secreted by the pancreas.
• useful in the diagnosis of pancreatitis and is considered a more specific marker
for acute pancreatitis than amylase
F.Amylase
• enzyme that aids in digestion by breaking down complex carbohydrates into
simple sugars.
• The majority of amylase is produced in the pancreas and salivary glands, and
lesser amounts are secreted by the fallopian tubes, lungs, thyroid, and tonsils
• Serum amylase levels are most often used in the diagnosis of acute pancreatitis

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Condition Isoenzyme pattern Serum enzyme Major diagnostic use

Muscular dystrophy Elevation of LDH1-3 Acid phosphate Prostate cancer

Megaloblastic Large elevation of LDH1 Alkaline phosphatase Liver and bone disease
anemia
Sickle cell anemia Moderate elevation of LDH1 and Creatine phosphokinase Myocardial infarction
LDH2 and muscle disorders
Arthritis with joint Elevation of LDH5 Lactate dehydrogenase Myocardial infarction,
infections leukemia, anemia
Acute hepatitis Moderate elevation of LDH1, slight Renin Hypertension
elevation of LDH2

Activity 3: Skill-building Activities (with answer key) (25 mins + 5 mins checking)

A. Identification. Write the correct answer to the following before the number

Yeast
____________1. The meaning of “zyme” in enzyme.

Ribozyme 2. RNAs with catalytic activity are called _____

____________

Active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction.
____________3.
Enzyme-Substrate
Complex
____________ 4. is the intermediate reaction species that is formed when a substrate binds to the active site
of an enzyme.
Substrate 5. is the reactant in an enzyme-catalysed reaction
____________

B. Matching Type. Write the correct answer to the following before the number

Column A1 Column B1

A. Absolute
A
________1. enzyme will catalyze only one reaction
B. Group
C. Linkage B
________2. the enzyme will act only on molecules that
have a specific functional group, such as hydroxyl, amino,
D. Stereochemical
or phosphate groups

D
________3. the enzyme will act on a particular
stereoisomer
C
________4. the enzyme will act on a particular type of
chemical bond, irrespective of the rest of the molecular
structure

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Column A2 Column B2

A. Oxidoreductases
A
________1. Oxidases
B. Transferases E
C. Hydrolases ________2. Mutases
D. Lyases D
E. Isomerases ________3. Decarboxylase
F. Ligases B
________4. Kinases
C
________5. Phosphatases
F
________6. Carboxylases

Column A3

A. Substrate
Concentration
B. Temperature
C. pH
D. Enzyme concentration

Column B3
B
________1. A 3.
_______

C
______2. D
________ 4.

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Column A4

A. Competitive inhibition
B. Non-competitive
inhibition
C. Irreversible inhibition

Column B4
A
________1.
\ B 3.

C
________ 2.

Column A5 Column B5
A. CK – MM A
________1. Creatinine kinase found in muscle
B. CK – BB
C. Troponin I E
________2. Also known as alanine aminotransferase
D. Troponin T
E. SGPT D
________3. found in both cardiac and skeletal muscle.
F. SGOT
F
________4. Also known as aspartate aminotransferase

B
________5. Creatinine kinase found in brain

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 Course Code: BIO 024 (Biochemistry/Biomolecules)
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Section: ____________ Schedule: ____________________________________ Date: _______________

Activity 4: What I Know Chart, part 2 (2 mins)

Instruction: To review what was learned from this session, please go back to Activity 1 and
answer the “What I Learned” column. Notice and reflect on any changes in your answer

Activity 5: Check for Understanding (20 mins)

Instruction: Now it’s time for you to figure this one out on your own! Take time to read, analyze, and
understand the following questions. For this instance, you will not have the chance to check if you have the
correct answers since there are no more keys to correction. Write the letter of your choice. Good luck!

5. Inactive precursor of a proteolytic enzyme:

For number 1 a. Holoenzyme c. Apoenzyme
b. Zymogen d. Coenzyme

6. Enzymes, which are produced in inactive form in the

living cells, are called
a. Papain c. Lysozymes
b. Apoenzymes d. Proenzymes
1. The enzyme-substrate complex and enzyme is
represented by diagram
a. A;C b.B;D c.C:B d.D:B e. C;A 7. Enzyme that is very useful in the diagnosis of infectious
For numbers 2-4 hepatitis
a. SGOT c. TPP
b. SGPT d. NAD

8. The following are examples of a proteolytic enzyme,

except?
a. Angiotensin c. Pepsinogen
b. Chymotrepsin d. Trypsin

9. Major diagnostic use of Creatine phosphokinase:

2. In the area marked A on the graph, many of the a. Hypertension D. Wilson’s disease
enzyme molecules have active sites that are b. Prostate cancer
a. Available for substrate binding c. Myocardial infarction and muscle disorders
b. Occupied by the inhibitors
c. Damaged by denaturants 10. This type of enzyme specificity states that some
d. Saturated by the substrate enzymes are specific to only one isomer even if the
e. Increasingly interacting with the substrate compound is one type of molecule
a. Absolute specificity
3. What phenomenon explains the flat area between b. Group specificity
points B and C? Still referring to the illustration. c. Stereochemical specificity
a. The reaction has come to stop. d. Linkage specificity
b. All of the enzyme’s active site are occupied
c. The reaction has run out of substrate
d. The enzyme has stopped working. 11. competitive inhibitor can
e. the enzymes saturation curve is achieved. a. be nullified by increasing product concentration
f. the reaction has dropped. b. be reversed by increasing substrate concentration
c. speed up reaction decreasing the temperature
4. Which of these changes might increase the rate of d. be overcome by maintaining pH level
the reaction beyond point C? Increase…
a. Substrate concentration
b. Enzyme concentration
c. temperature
d. water concentration
e. nonspecific inhibitors

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 Course Code: BIO 024 (Biochemistry/Biomolecules)
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Name: ____________________________________________________________ Class number: _______

Section: ____________ Schedule: ____________________________________ Date: _______________

12. It increases enzyme activity; the shape of the active site is changed such that it can more readily accept
substrate.
A. Competitive enzyme inhibitor
B. Noncompetitive enzyme inhibitor
C. Irreversible enzyme inhibitor
D. Positive regulator

13. Fischer’s ‘lock and key’ model of the enzyme action implies that:
a. The active site is complementary in shape to that of the substance only after interaction.
b. The active site is complementary in shape to that of substance.
c. Substrates change shape prior to active site interaction
d. The active site is flexible and adjusts to substrate.

14. The following are examples of enzymes that exhibit group specificity, EXCEPT for
a. Phosphatases c. Esterases
b. Carboxypeptidase d. Glycosidases

1. Found solely on the cardiac muscles

a. Troponin C
b. Troponin T
c. Troponin I
d. Troponin I and T

A. LESSON WRAP-UP

1) Activity 6: Thinking about Learning (5 mins)

A. Work Tracker: You are done with this session! Let’s track your progress. Shade the session
number you just completed.
P1 P2
1 2 3 4 5 6 7 8 9 10

B. Think about your Learning:

Tell me about your thoughts! Today’s topic is all about the enzymes. What interests you about the
lesson today? What are the things that confuses you from the topic?

What interests me about today’s topic is that each cell

___________________________________________________________
in the human body contains thousands of enzymes. In
___________________________________________________________
addition to that, it also helps speed up chemical
___________________________________________________________
________________________________________________________
reactions in the human body.

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 Course Code: BIO 024 (Biochemistry/Biomolecules)
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Section: ____________ Schedule: ____________________________________ Date: _______________

KEY TO CORRECTIONS FOR ACTIVITY 3

A. Identification

1. Yeast 5. Substrate
2. Ribozyme
3. Active site
4. ESC or Enzyme - Substrate Complex

B. Matching Type

A1. A2. A3. A4. A5.

1. A 1. A 1. B 1. A 1. A
2. B 2. E 2. C 2. C 2. E
3. D 3. D 3. A 3. B 3. D
4. C 4. B 4. D 4. F
5. C 5. B
6. F

SUGGESTED VIDEOS:

Enzymes overview
https://www.youtube.com/watch?v=qgVFkRn8f10

How enzymes work (energy of activation)?

https://www.youtube.com/watch?v=wiIUS2LDCl8
https://www.youtube.com/watch?v=j00Ep0Byu0Y

Factors affecting enzymatic activity:

https://www.youtube.com/watch?v=dhAiTiqUv3w

Michaelis-Menten Kinetics: Part 1(These are quite long or about 20min videos)
https://www.youtube.com/watch?v=4eLjRcHnMCk
Michaelis-Menten and Lineweaver-Burk Plot: Part 2
https://www.youtube.com/watch?v=y43pIHUtjeM

Enzyme inhibition: (This is also quite long or about 20min videos)

https://www.youtube.com/watch?v=jJUoQMLMV2E

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