COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.

: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee

MODULE 3: ENZYME AND ENZYME KINETICS

[Ref: Campbell, M.K., & Farrell, S.O. (2009). Biochemistry, Sixth Edition. Belmont: Thomson Higher Education.; Mathews, C.K., Holde, K., & Ahern, K.G. (2000). Biochemistry Third
Edition. San Francisco: Robin Heyden.; Rodwell, V.W., Bender, D.A., Botham, K.M., Kennelly, P.J., & Weil, P.A. (2015). Harper’s Illustrated Biochemistry 30th Edition. Minion: Cenveo
Publisher Services.; Stoker, H.S. (2017). Biochemistry Third Edition. Quezon City: C&E Publishing, Inc.]

INTRODUCTION TO ENZYMES

 An enzyme is a compound, usually a protein, that acts as a catalyst for a biochemical reaction.
 As a catalyst, it speeds up chemical reactions within cellular systems by factors of up to 1020.
 Enzymes are not consumed during the reaction itself.
 Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction.

STRUCTURES OF ENZYMES

 Enzymes can be divided into two general structural classes:

Simple Enzymes Enzymes composed only of protein or amino acid chains.

Enzymes
Conjugated Enzymes that have a nonprotein portion attached to the
Enzyme protein structure.

 In conjugated enzymes, neither the protein nor the nonprotein portion has catalytic properties. Both must come together to
become biochemically active.
o Holoenzyme: It is the biochemically active conjugated enzyme produced from an apoenzyme and a cofactor.
o Apoenzyme: It is the protein part of a conjugated enzyme.
o Cofactor: It is the nonprotein part of a conjugated enzyme.

Note: Cofactors provide additional chemically reactive functional groups besides those present in the amino acid side chains of
apoenzymes.

Classifications of Cofactors:

 Metal Ion Cofactors: This includes Zn2+, Mg2+, Fe2+, Fe+3, Cu+, and Cu2+
which serves as bridging groups that form coordination complexes. Metal
ions are supplied mainly via dietary intake.
 Coenzymes: It is a small organic molecule, such as NAD+ and FAD, that
serves as a cofactor in a conjugated enzyme. Coenzymes are mainly
synthesized within the body.
 Prosthetic Group: An organic substance which is dialyzable and
thermostable firmly attached to the protein or apoenzyme portion.

Binding of Cofactors

 Many cofactors are permanently bonded to the apoenzyme via covalent bonds. Breaking of the covalent bonds deactivates the
enzyme.
 Other times, a coenzyme temporarily binds to the amino acid portion of an enzyme at the time it is needed and subsequently
released after the reaction has occurred.

THE ENZYME’S ACTIVE SITE

 The active site is a small pocket or cleft within the enzyme’s structure that is actually involved in catalysis.
 It is a “crevice-like” three-dimensional entity formed by the folding and bending of amino acid chains.
 Consists of two parts:
o Binding Site: Binds and orients the substrate.
o Catalytic Site: Responsible for reducing the chemical activation energy.
 Electrostatic interactions, hydrogen bonds and hydrophobic interactions all help attract and bind substrate molecules to the
active site.

MODELS OF ENZYME ACTION

 Upon binding with the active site, an enzyme-substrate complex (ES) is formed. This is the intermediate reaction species that is
formed when a substrate binds to the active site of an enzyme.
 Within the enzyme-substrate complex, the substrate encounters more favorable reaction conditions than if it were free. This
results in faster formation of product.
COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee

Models of Action

Lock and Key Model Induced Fit Model

Lock and Key Model

 In the 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.

Induced-Fit Model

 The induced-fit model shows that enzymes are flexible thus allowing for conformational changes when the substrate binds with
the active site.

NOMENCLATURE AND CLASSIFICATION OF ENZYMES

 Recommended Nomenclature: Enzymes are named according to the reaction that they catalyze and/or the substrate upon
which it acts on.
o Substrate: It is the reactant in an enzyme-catalyzed reaction.

Suffix Prefix Additional

The suffix -ase identifies a substance as The prefix often contains the type of The identity of the substate is sometimes
an enzyme. Earlier discoveries have been reaction catalyzed by the enzyme such noted along with the reaction such as glucose
designated with the suffix -in such as as hydrolase for hydrolysis reaction or oxidase. The substrate may also take the
trypsin and pepsin. oxidase for oxidation reactions. place of the prefix such as in urease.

The International Union of Biochemistry and Molecular Biology (IUBMB) devised a system of classification and identification of
enzymes in terms of the reactions they catalyze. This relies on a numerical system (the EC number) to classify enzymes in groups
according to the types of reaction catalyzed and systematic naming that describes the chemical reaction involved.

The EC number of Enzyme Commission number is a four-component identifier which classifies an enzyme according to class, subclass,
sub-subclass, and the final component being a serial number within that sub-subclass.

 Systematic Nomenclature: Enzymes are grouped into six major classes on the basis of the types of reactions they catalyze.

Class Definition Subclasses Type of Reactions Catalyzed

Oxidases • Oxidation of a substrate
Reductases • Reduction of a substrate
An enzyme that catalyzes an oxidation- • Introduction of a double bond by
1. Oxidoreductase
reduction reaction. formal removal of two H atoms
Dehydrogenases
from a substrate, with one H being
accepted by a coenzyme.
• Transfer of an amino group
Transaminases
An enzyme that catalyzes the transfer of a between substrates
4. Transferase
functional group from one molecule to another. • Transfer of a phosphate group
Kinases
between substrates
Lipases • Hydrolysis of ester linkages in lipid
• Hydrolysis of amide linkages in
Proteases
proteins
An enzyme that catalyzes a hydrolysis reaction • Hydrolysis of sugar-phosphate
Nucleases
6. Hydrolase in which the addition of a water molecule to a ester bonds in nucleic acids.
bond causes the bond to break. • Hydrolysis of glycosidic in
Carbohydrases
carbohydrates
• Hydrolysis of phosphate-ester
Phosphatases
bonds
An enzyme that catalyzes the addition of a Dehydratases • Removal of H2O from a substrate
group to a double bond or the removal of a Decarboxylases • Removal of CO2 from a substrate
11. Lyase
group to form a double bond without hydrolysis Deaminases • Removal of NH3 from a substrate
or oxidation. Hydratases • Addition of H2O to a substrate
• Conversion of a D isomer to L
An enzyme that catalyzes the isomerization Racemases
isomer or vice versa
(rearrangement of atoms) of a substrate in a
15. Isomerase • Transfer of a functional group from
reaction, converting it into a molecule isomeric
Mutases one position to another in the
with itself.
same molecule
17. Ligase An enzyme that catalyzes the bonding together • Formation of a new bond between
of two molecules into one with the participation Synthetases two substrates with participation of
of ATP. ATP
Carboxylases • Formation of a new bond between
COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee

a substrate and CO2 with

participation of ATP

PROPERTIES OF ENZYMES

 Catalytic Efficiency
o Enzyme-catalyzed reactions are highly efficient proceeding from 103-108 times faster than uncatalyzed reactions. The
exact efficiency may vary depending upon the reaction itself but will always proceed faster than reactions done
without the aid of enzymes.
 Specificity
o Enzyme specificity is the extent to which an enzyme’s activity is restricted to a specific substrate, a specific group of
substrates, a specific type of chemical bond, or a specific type of chemical reaction.
o The degree of enzyme specificity is determined by the active site.

Types of Enzyme Specificity

TYPE DESCRIPTION EXAMPLE

Absolute Specificity The enzyme will catalyze only one reaction. Catalase
Group Specificity The enzyme will act only on a specific functional group. Carboxypeptidase
The enzyme will act on a particular type of chemical bond irrespective of
Linkage Specificity Phosphatases
the rest of the molecular structure.
Stereochemical
The enzyme will act only on a particular stereoisomer. L-amino acid oxidase
Specificity

 Regulation
o Enzyme activity can be increased or decreases so that the rate of product formation responds to cellular need.
o Continuous production of either product or enzyme is a waste of energy.

FACTORS THAT AFFECT ENZYME ACTIVITY

 Enzyme activity is a measure of the rate at which an enzyme converts substrate to products in a biochemical reaction.

Temperature pH

Substrate Concentration Enzyme Concentration

COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee

ENERGY LEVELS AND THE BASIC ENZYME REACTION

 In order for a chemical reaction to proceed, some form of energy is needed. This quantity of energy is termed as “activation
energy.”
 It is the magnitude of the activation energy which determines how fast the reaction will proceed.
 Enzymes lower the activation energy for the reaction they are catalyzing. It reduces the “path” of the reaction thus requiring less
energy for each molecule of substrate converted to product.
 Given a total amount of available energy, more molecules of substrate would be converted when the enzyme is present than
when it is absent. Hence, the reaction is said to go faster in a given period of time.

 The basic enzymatic reaction can be represented as follows:

S + E - P + E

 E, represents the enzyme catalyzing the reaction; S, the substrate, the substance being modified; and P, the product of the
reaction.
 Savante Arrhenius proposed that the substrate and enzyme formed some intermediate substance which is known as the
enzyme/substrate complex (ES). The reaction can be represented as:

S + E  ES  P + E

 However, most chemical reactions do not go to true completion. This is due to the reversibility of most reactions.
COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee

ENZYME KINETICS

 The equation above can be plotted into a graph known as the Michaelis-Menten Plot.

Michaelis-Menten Plot
2.5

Reaction Rate (μM/min.)

2
f(x) = 0.24 ln(x) + 0.94
1.5
R² = 0.93
1
0.5
0
0 10 20 30 40 50 60
[S] (μM)

 The Michaelis-Menten plot is an effective way to show the effect of substrate concentration on reaction velocity. It is the based
on the steady-state assumption which states that the concentration of enzyme-substrate complex remains nearly constant
through much of the reaction.
 Re-arranging the basic enzyme reaction, we can derive the Michaelis-Menten Equation:

 Km, the Michaelis-Menten constant, measures the substrate concentration at which the reaction rate is Vmax/2.
o It is the concentration at which half of the active sites are occupied.
o It is associated with the affinity of enzyme for the substrate.
o ↑ Km = ↓ Affinity
o ↓ Km = ↑ Affinity
 Vmax is the reaction rate when the enzyme is saturated with substrate.
o This will be affected by different factors and even the presence of inhibitors.
 [S] is the concentration of the substrate.
 Kcat, the turnover number, measures the rate of the catalytic process.

 Despite the usefulness of the Michaelis-Menten plot, determining the actual values of the kinetic parameters becomes a difficult
task.
 Moreover, estimating Vmax is quite difficult because it is an asymptote, and the value cannot be reached unless substrate
concentrations are infinite.
 The solution is to rearrange the equation which will give the Lineweaver-Burk double-reciprocal plot. Plotting 1/V against 1/[S]
will provide a straight line. More importantly, Km and Vmax can be derived.

Lineweaver-Burke Plot
1.6
1.4 f(x) = x + 0.5
1/Vmax (min./μM)

1.2 R² = 0.95
1
0.8
0.6
0.4
0.2
0
0 0.2 0.4 0.6 0.8 1 1.2
1/[S] (μM)
COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee

 The Lineweaver-Burke Equation is:

 Using this slope equation, 1/Vmax corresponds to the intercept of the line with the 1/V axis.

ENZYME INHIBITION

 An enzyme inhibitor is a substance that slows or stops the normal catalytic function of an enzyme by binding to it.

Enzyme Inhibition

Noncompetitive
Competitive Inhibitor Uncompetitive Inhibitor Irreversible Inhibitor
Inhibitor

COMPETITIVE INHIBITOR NONCOMPETITIVE INHIBITOR

• A molecule closely resembling the substrate.
• Binds to the active site and temporarily prevents the
substrates from occupying it, thus blocking the reaction.
• A molecule that binds to a site on an enzyme that is not the
active site.
• The normal substrate still occupies the active site but the
enzyme cannot catalyze the reaction due to the presence of
the inhibitor.

UNCOMPETITIVE INHIBITOR IRREVERSIBLE INHIBITOR

• An inhibitor that binds to the enzyme-substrate complex • A molecule that forms a covalent bond to a part of the active
itself, but not to a free enzyme. site, permanently preventing substrates from occupying it.

No reaction occurs.
COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee