Professional Documents
Culture Documents
Module 3 - Enzyme and Enzyme Kinetics
Module 3 - Enzyme and Enzyme Kinetics
: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee
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
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 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.
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
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.
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.
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.
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.
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.
Enzyme activity is a measure of the rate at which an enzyme converts substrate to products in a biochemical reaction.
Temperature pH
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.
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
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
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
No reaction occurs.
COURSE: Biochemistry for Medical Laboratory Science DOCUMENT NO.: MLS 221_M01
TOPIC: Enzyme and Enzyme Kinetics PREPARED BY: Academic Committee