Enzymology
Dr. Samra Khalid
ASAB,
National University of Sciences and
Technology
Course content
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Introduction and history of enzymes •
Historical aspects
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Discovery of enzymes
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Chemistry of enzymes
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Function and importance
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Enzymes in biotechnology
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Characteristics and properties
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Catalytic power and specificity
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Enzyme substrate complex
Catayltic cycle of enzyme
Nomenclature / Classification and
Activity Measurements
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Oxidoreductase-dehydrogenase
Transferase
Hydrolase
Lyase
Isomerase
Ligase
Activity measurements
Enzyme Purification and Assay
Initial velocity measurements
Assay types
Enzyme units of activity
Turnover number and properties
Purification and assessment
Methods for measurement
Enzyme kinetics
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Michaelis-Menten Kinetics
Introduction
Assumptions
Derivation
Description of vo versus [S]
Michaelis constant (KM)
Course content
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Enzyme inhibition and kinetics
Classification of inhibitors
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ATP Synthase
ATP Synthase with Tethered Actin
Myosin-V
Kinesin motor attached to a fluorescent bead
Single Molecule Studies of Cholesterol Oxidase
β-galactosidase: a model Michaelis-Menten
enzyme?
Reversible, Irreversible, Iodoacetamide, DIFP
Classification of Reversible Inhibitors
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Competitive, Uncompetitive, Noncompetitive,
Substrate
Multi-substrate Reactions and Substrate Binding
Analysis
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Single Molecule Enzymology
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Specificity/Substrate constant (SpC)
Graphical Analysis of Kinetic Data, pH and Temp •
Dependence
Graphical Analysis
Lineweaver-Burk Analysis
Hanes-Woolf Analysis
Eadie-Hofstee Analysis
Direct Linear Plot (Eisenthal/Cornish-Bowden Plot)
Nonlinear Curve Fitting
pH-dependence of Michaelis-Menten Enzymes
Temperature-Dependence of Enzyme Reactions
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Substrate Binding Analysis
Single Binding Site Model
Binding Data Plots
Direct Plot
Reciprocal Plot
Scatchard Plot
Determination of Enzyme-Substrate Dissociation
Constants
Kinetics
Equilibrium Dialysis
Equilibrium Gel Filtration
Ultracentrifugation
Spectroscopic Methods
Mechanism of enzyme catalysis
Engineering More Stable Enzymes
Incorporation of Non-natural Amino Acids into
Enzymes
Protein Engineering by Combinatorial Methods
DNA Shuffling
Enzymes
 Biological catalyst…
 Biomolecules catalyze, increase the rates of chemical reactions
 Almost all enzymes are proteins.
 act only upon a specific substrate (or substrate group)
 do not change the energetics of the reaction
 Living systems use enzymes to accelerate and control the rates
of vitally important biochemical reactions.
Historical Background
2100 BC
700 BC
1700s
Late 1800s
1903
1913
1950s-1960s
1965
Codex of Hammurabi-description of wine making
Homer’s Iliad: “As the juice of fig tree curdles milk, and
thickens it in a moment though it be liquid, even so instantly
did Paeeon cure fierce Mars”
Réaumur - studies on the digestion of buzzardsdigestion is a chemical rather than a physical process
Kühne - term 'enzyme': Greek "in yeast"
Hans & Eduard Buchner – filtrates of yeast extracts
could catalyse fermentation! No need to living cells
E. Fischer – “lock and key” hypothesis
Henri – first successful mathematical model
Michaelis and Menten – NZ rate equation....
Koshland – “Induced fit” model
Monod, Wyman and Changeux – allosteric regulation
History of Enzymes
 As early as the late 1700s and early 1800s, the
digestion of meat by stomach secretions and the
conversion of starch to sugars by plant extracts and
saliva were known. However, the mechanism by
which this occurred had not been identified.
History of Enzymes
In the 19th century, when studying the fermentation
of sugar to alcohol by yeast, Louis Pasteur came to
the conclusion that this fermentation was catalyzed
by a vital force contained within the yeast cells called
"ferments", which were thought to function only
within living organisms. He wrote that "alcoholic
fermentation is an act correlated with the life and
organization of the yeast cells, not with the death or
putrefaction of the cells.
 In 1878 German physiologist Wilhelm Kühne (1837–1900) first
used the term enzyme, which comes from Greek ενζυμον "in
leaven", to describe this process. The word enzyme was used
later to refer to nonliving substances such as pepsin, and the
word ferment used to refer to chemical activity produced by
living organisms.
 In 1897 Eduard Buchner began to study the ability of yeast
extracts that lacked any living yeast cells to ferment sugar. In a
series of experiments at the University of Berlin, he found that
the sugar was fermented even when there were no living yeast
cells in the mixture.
 He named the enzyme that brought about the fermentation of
sucrose "zymase". In 1907 he received the Nobel Prize in
Chemistry“ for his biochemical research and his discovery of cellfree fermentation".
Functions of Enzymes
 Break down nutrients into useable molecules. (Lehninger et al.,
1993, p. 198)
 Store and release energy (ATP). (Lehninger et al., 1993, p. 198;
Campbell & Reece, 2002, pp. 162-163)
 Create larger molecules from smaller ones. (Lehninger et al.,
1993, p. 198; Campbell & Reece, 2002, pp. 295, 316-317)
 Coordinate biological reactions between different systems in an
organism. (Lehninger et al., 1993, p. 198; Campbell & Reece,
2002, pp. 101-102)
Importance of Enzymes
They are catalysts so they make reactions easier
to increases productivity and yield
As catalysts they are not consumed by the reaction
may be used over and over again
Most enzyme reaction rates are millions of times
faster than those of un-catalyzed reactions.
Enzymes show specificity to the reaction they control
Enzymes are sensitive to their environment so they
can be controlled by adjusting the temperature, the
pH or the substrate concentration
However, enzymes do differ from most other
catalysts by being much more specific.
Properties of enzymes as
catalysts
Catalytic Power of Enzyme
Most enzyme reaction rates are millions of times faster
than those of comparable uncatalyzed reactions. As with all
catalysts, enzymes are not consumed by the reactions they
catalyze, nor do they alter the equilibrium of these
reactions. However, enzymes do differ from most other
catalysts by being much more specific.
The ratio of uncatalyzed to catalyzed reaction rate is
called the catalytic power. For uncatalyzed hydrolysis of
urea the reaction rate is 3x104 and for catalyzed reaction it
is 3X10-10
The Catalytic power is therefore 3x1014 .
Specificity
Enzymes are highly specific to their substrate and
reaction catalysed
Complementary shape, charge and
hydrophilic/hydrophobic characteristics of enzymes
and substrates are responsible for this specificity.
Most enzymes can be denatured that is, unfolded
and inactivated by heating, which destroys the threedimensional structure of the protein.
 Depending on the enzyme, denaturation may be
reversible or irreversible.
Enzymes and biotechnology
 Almost all processes in a biological cell need
enzymes in order to occur at significant rates.
Since enzymes are extremely selective for their
substrates and speed up only a few reactions
from among many possibilities, the set of
enzymes made in a cell determines which
metabolic pathways occur in that cell.
 Every cell in every plant and animal contains
many different types of enzyme. Each enzyme
catalyzes a different reaction.
Biotechnology – an old art
Can you think of some products that have been made using biotechnology for thousands
of years?
bread
beer and wine
cheese and yoghurt
What is Fermentation?
Yeast cells contain enzymes that converts sugars (such as glucose
and sucrose) into alcohol (ethanol) and carbon dioxide. This
reaction is called fermentation.
glucose
C6H12O6 (aq)
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
ethanol
C2H5OH (l)
+
+
carbon
dioxide
CO2 (g)
Fermentation usually takes place at 20-30°C. It must take place in anaerobic conditions
(without oxygen) otherwise the ethanol would react with oxygen and turn into vinegar.
Fermentation and beer-making
Fermentation and yoghurt
Biotechnology – a new science
Can you think of more recent uses of biotechnology?
using enzymes to improve
detergents
manufacturing
medicines such as
penicillin
transferring disease-resistance genes into
plants
Biological washing powder
Biological washing powders contain enzymes to help remove stains.
 Proteases break down proteins in
stains such as grass, blood and
sweat.
 Lipases break down stains
containing fat and oil.
 Carbohydrases break down stains
containing carbohydrates, such as
starch.
wax coat
The enzymes are coated with a special wax. This melts in the wash,
releasing the enzymes. Once the stains have been broken down, they are
easier to remove by the detergent.
Modern Biotechnology
Enzymes in DNA-technology
DNA is basically a long chain of deoxyribose sugars
linked together by phosphodiester bonds. Organic
bases, adenine, thymine, guanine and cytosine are
linked to the sugars and form the alphabet of genes.
The specific order of the organic bases in the chain
constitutes the genetic language.
Genetic engineering means reading and modifying
this language. Enzymes are crucial tools in this
process.
Modern Biotechnology
Enzymes in DNA-technology
DNA-technology has revolutionized both traditional
biotechnology and opened totally new fields for
scientific study.
Recombinant DNA-technology allows one to produce
new enzymes in traditional overproducing and safe
organisms
Protein engineering is used to modify and improve
existing enzymes.
Enzymes and Industry
 Enzymes are becoming more common as catalysts for industrial
processes. Why is this the case?
Enzymes work at fairly low temperatures – this saves energy and
money, and reduces pollution.
Enzymes work in fairly mild conditions (normal pressure, in water
and pH close to 7) – this reduces the need for potentially dangerous
chemicals.
Enzyme-reactions can be easily controlled – by slightly changing the
temperature or pH.
Enzymes are biodegradable – they reduce pollution and
environmental problems.
There are thousands of different enzymes in your body.
Enzyme action
 Enzymes are large molecules that have a small section dedicated to a
specific reaction. This section is called the active site.
 The active site reacts with the desired substance, called the substrate.
 The substrate may need an environment different from the mostly
neutral environment of the cell in order to react. Thus, the active site
can be more acidic or basic, or provide opportunities for different types
of bonding to occur, depending on what type of side chains are present
on the amino acids of the active site.
Enzyme action
Why are there so many different enzymes?
Each enzyme has its own unique protein structure and
shape, which is designed to match or COMPLEMENT
on its one type of SPECIFIC substrate. Products have a
different shape from the substrate.
The shape of the active site (binding site) of the enzyme,
matches the shape of the substrate. Allowing the two
molecules to bind during the chemical reaction.
Enzyme Action Theories
Lock & Key Hypothesis
Fit between the substrate and the active site of the enzyme is
exact
The key is analogous to the enzyme and the substrate analogous
to the lock.
Temporary structure called the enzyme-substrate complex formed
Once formed, they are released from the active site
Leaving it free to become attached to another substrate
This theory of enzyme action is called the ‘lock-and-key’
hypothesis
Lock and Key Hypothesis
S
E
E
E
Enzymesubstrate
complex
Enzyme may
be used again
P
P
Reaction coordinate
The Lock and Key Hypothesis
• This explains enzyme specificity
• This explains the loss of activity when
enzymes denature
Enzyme Action
E + S <---> [ES] <---> E + P
enzymes catalyze reactions by lowering the energy of activation (Ea)
Induced Fit Hypothesis
Some proteins can change their shape
(conformation)
When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
The active site is then moulded into a precise
conformation
Making the chemical environment suitable for the
reaction
The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)
© 2007 Paul Billiet ODWS
Induced Fit Hypothesis
Enzymes can form to the shape of its substrate
Enzyme activity and inhibition
 The “normal” way an enzyme functions is when the specific
substrate binds to the active site and creates the products.
 A similar substrate can also bond to the active site covalently
and irreversibly. This prevents the enzyme from functioning.
 A similar substrate can bind to the active site, not
permanently, and prevents the desired substrate from
entering the active site. This changes the products and
functioning of the enzyme. This is called competitive inhibition.
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 A molecule can bond to another part of the enzyme and cause
a change in conformation. This change causes the active site to
change shape as well. This change in shape prevents the
desired substrate from entering the active site. This is called
non-competitive inhibition.
Catalytic cycle of an enzyme
Enzyme cofactors
 A cofactor is a substance that is not a protein that must bind to
the enzyme in order for the enzyme to work.
 A cofactor can be of organic origin. An organic cofactor is called
a coenzyme.
 Cofactors are not permanently bonded. Permanently bonded
cofactors are called prosthetic groups.
 An enzyme that is bonded to its cofactor is called a holoenzyme.
 An enzyme that requires a cofactor, but is not bonded to the
cofactor is called an apoenzyme. Apoenzymes are not active
until they are complexed with the appropriate cofactor.
Factors That Affect Enzyme Activity
Enzyme activity can be affected by other
molecules.
Inhibitors are molecules that decrease
enzyme activity. Many drugs and poisons are
enzyme inhibitors.
 activators are molecules that increase
activity.
 Activity is also affected by temperature,
chemical environment (e.g. pH)
Enzymes and temperature
Enzymes are mostly affected by changes in temperature and pH.
(Campbell & Reece, 2002, pp. 99-102)
Enzyme-catalyzed reactions, usually 20-45°C.
All enzymes work best at only one particular optimum
temperature. Different enzymes have different optimum
temperatures.
As the temperature decreases below the optimum the enzyme
will eventually become inactive. The reaction will stop.
Too high of a temperature will denature the protein
components, rendering the enzyme useless.
Enzymes and pH
Enzyme reactions occur across a range of pH values.
Like for temperature, each enzyme will work best at only one
particular pH.
Some enzymes, for example, those in the stomach, work best in
acidic conditions.
Other enzymes work best in alkaline conditions.
pH ranges outside of the optimal range will protonate or
deprotonate the side chains of the amino acids involved in the
enzyme’s function which may make them incapable of catalyzing a
reaction.
Summary of Enzymes
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Enzymes are mostly proteins
They are highly specific to a reaction
They catalyze many reactions including breaking down nutrients, storing and
releasing energy, creating new molecules, and coordinating biological
reactions.
Enzymes use an active site, but can be affected by bonding at other areas of
the enzyme.
Some enzymes need special molecules called cofactors to carry out their
function.
Cofactors that are organic in nature are called coenzymes.
Coenzymes are usually derived from vitamins.
Coenzymes transfer functional groups for the enzyme they work with.
Enzymes are affected by changes in pH, temperature, the amount of
substrate, cofactors and inhibitors, as well as the amount of allosteric
inhibitors and activators and concentration of products that control feedback
inhibition.
Glossary
 active site – The part of the enzyme into which the reactant
molecule fits.
 catalyst – A substance that changes the rate of a reaction without
being used up.
 denatured – The state of an enzyme when it has been irreversibly
damaged and has changed shape.
 enzyme – A biological catalyst.
 fermentation – The conversion of sugar to ethanol and carbon
dioxide by enzymes in yeast.
 lock and key – A model of how enzymes work and the importance
of their shape.