Understanding Enzyme Function
Background Reading: Introduction to Enzyme Function and Applications
Introduction
Enzymes are biological molecules that help accelerate various biochemical reactions in nature;
more specifically, they help catalyze the conversion of substrates to products. Enzymes are
specific to both their substrates and the types of reactions they catalyze; for example, proteases
are enzymes that break down proteins but do not interact with carbohydrates. You can visualize
an enzyme-substrate complex like a lock and key model where the enzyme is the lock that
accepts only a certain shape of substrate key. As the key turns in the lock, it rearranges the lock
so as the door opens, or in our case, the reaction progresses. The interaction between a substrate
and an enzyme occurs in the pocket of an enzyme where the substrate binds, known as the active
site. Binding of the substrate to enzyme can induce a conformational change to accommodate the
substrate and this conformational change is called induced fit, and plays an important role in
catalyzing substrates into products. Most enzyme-catalyzed reactions are pH and temperature
sensitive; an increase or decrease in temperature or pH will affect the rate of enzyme activity.
Enzyme reactions can also be affected by inhibitors, molecules that slow down enzyme activity
by competing with the substrates for binding to the active site.
Applications of enzymes in biotechnology
Enzymes are essential tools within the biotechnology field due to their ability to catalyze
chemical reactions that result in desirable products for human use. Within the pharmaceutical
industry, enzymes can be used to synthesize antibiotics, as well as screen enzyme inhibitors as
potential new drug candidates. The latter can be done by screening for chemicals or natural
products that decrease an enzyme’s activity by competing against the substrate for the enzyme
active site. In the human body, enzymes play a vital role to breaking down chemical compounds
into formats that the body can process; for example, those who are able to digest dairy can only
do so because of the enzyme lactase, which breaks down the milk sugar lactose. Introducing this
enzyme into milk products provides a means for those who do not produce lactase internally to
digest dairy. Similarly, glucose oxidase is an enzyme that is able to detect glucose, and can be
used for diabetes diagnostics to detect the presence of free glucose with in the bloodstream.
Development of testing platforms to assess enzyme function
Developing applications for enzymes within drug discovery, such as using enzymes as models
for screening the effectiveness of various enzyme inhibitors, relies on finding optimal conditions
for enzyme function. A first step often used in screening of optimal conditions is the
development of enzyme arrays to experiment with multiple conditions at the same time.
Encapsulation using hydrogels is becoming an exceedingly popular choice for the development
of such assay or screening platforms. The two main advantages for the use of hydrogels include:
(1) biocompatibility with a wide range of biological entities and (2) relative simplicity of
hydrogel encapsulation.
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Understanding Enzyme Function
Enzymes can be encapsulated in hydrogel systems such as alginate to create enzyme
array platforms. Alginate solutions can be crosslinked using calcium ions to form hydrogels, and
biomolecules such as proteins can be encapsulated in alginate hydrogels by simply adding them
into alginate solutions before the crosslinking step, i.e. the addition of calcium (Figure 1).
Figure 1. Encapsulation of biomolecules in alginate by crosslinking using CaCl2
Colorimetric assays to detect enzyme function
In addition to developing enzyme arrays, it is also important to develop enzymatic assays that are
rapid and efficient to identify optimal conditions for various enzymes. Such assays often utilize
chromogenic substrates, which under appropriate conditions produce colored products (for ease
of detection) in the presence of enzymes and co-substrates. For example, ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) is a commonly known chromogenic substrate that
is oxidized by horseradish peroxidase in the presence of hydrogen peroxide to produce a bluegreen color (Figure 2). Another commonly used chromogenic assay is the ONPG assay, in which
the enzyme β-galactosidase hydrolyzes the synthetic substrate ONPG (o-Nitrophenyl-βgalactoside), in the presence of magnesium to produce a yellow product. The changes in color
intensity in these assays can be quantified by measuring changes in absorbance or using imaging
techniques, which will help determine optimal conditions for the different enzyme applications.
Figure 2. Oxidation of ABTS by HRP in the presence of H2O2 to yield a blue-green product
Glossary of key terms:
• Enzymes – Macromolecules made out of proteins that catalyze (accelerate) biochemical
reactions by lowering the activation energy needed for a reaction to occur.
• Substrate – The chemical species that an enzyme acts upon to catalyze into a product.
• Product – The resulting species of a chemical reaction.
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Understanding Enzyme Function
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Active site – The area in an enzyme where a substrate binds. The active sight consists of a
binding region that holds the substrate, and a catalytic sight, where the activation energy
of the reaction to turn the substrate into a product is reduced.
Induced Fit – A conformational change of an enzyme after the binding of a substrate in
order to produce a snugger fit between the substrate and the active site.
Inhibitors – Chemical species that compete with substrates in binding to the active site of
an enzyme.
Arrays (or microarrays) – A laboratory method wherein the desired biomolecules are
spatially spotted on a solid support (usually glass slides) to allow the high throughput
analysis of biological function and activity.
Hydrogel – A type of water-based, crosslinked polymer biomaterial that is often used as a
supporting matrix for cells or other molecules.
Biocompatibility – A term used to characterize materials that cause no adverse affects to
biological systems and/or can be integrated into a biological system.
Enzymatic assays – Methods for determining enzyme activity, such as the rate of
substrate consumption or product formation.
Chromogenic – Able to produce color or pigments.
Co-substrate – A second substrate that is able to bind to the active site of an enzyme and
is essential to drive the reaction.
Pre-lab questions:
1. What are some commercial applications of different types of enzymes?
2. What is the significance of using bioprinted enzyme arrays?
3. What is the significance of using a chromogenic substrate (e.g. ABTS) in the
development of an enzyme assay?
4. What is the significance of a co-substrate in an enzymatic assay (e.g. H2O2 in the
HRP/ABTS assay)?
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