Enzymology
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.