Enzymology (Lecture 1)
Rumeza Hanif
Course Content
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Introduction and history of enzymes
Historical aspects
Discovery of enzymes
Chemistry of enzymes
Function and importance
Enzymes in biotechnology
Characteristics and properties
Catalytic power and specificity
Enzymes as catalysts
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Enzyme - substrate interactions
Lock & key model
Induced fit model
Transition state model
Quantum tunnelling model
Enzymes as proteins
Non-protein cofactors
Metal ions
Organic cofactors
Nomenclature / Classification and Activity
Measurements
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Oxidoreductase-dehydrogenase
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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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Specificity/Substrate constant (SpC)
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Graphical Analysis of Kinetic Data, pH and Temp
Dependence
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Graphical Analysis
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Substrate Binding Analysis
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Lineweaver-Burk Analysis
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Single Binding Site Model
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Hanes-Woolf Analysis
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Binding Data Plots
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Eadie-Hofstee Analysis
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Direct Plot
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Direct Linear Plot (Eisenthal/Cornish-Bowden Plot)
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Reciprocal Plot
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Nonlinear Curve Fitting
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Scatchard Plot
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pH-dependence of Michaelis-Menten Enzymes
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Temperature-Dependence of Enzyme Reactions
Determination of Enzyme-Substrate Dissociation
Constants
Single Molecule Enzymology
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Kinetics
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ATP Synthase
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Equilibrium Dialysis
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ATP Synthase with Tethered Actin
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Equilibrium Gel Filtration
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Myosin-V
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Ultracentrifugation
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Kinesin motor attached to a fluorescent bead
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Spectroscopic Methods
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Single Molecule Studies of Cholesterol Oxidase
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β-galactosidase: a model Michaelis-Menten enzyme? 
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Enzyme inhibition and kinetics
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Classification of inhibitors
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Reversible, Irreversible, Iodoacetamide, DIFP
Classification of Reversible Inhibitors
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Competitive, Uncompetitive, Noncompetitive,
Substrate
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Multi-substrate Reactions and Substrate Binding
Analysis
Mechanism of enzyme catalysis
Engineering More Stable Enzymes
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Incorporation of Non-natural Amino Acids into
Enzymes
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Protein Engineering by Combinatorial Methods
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DNA Shuffling
Marks distribution
Total marks 100
 Final exam 40-50
 1st OHT 15
 2nd OHT 15
 Presentation/Assignments/Project/Practical/
Class evaluation10-20
 Quiz 10 (Minimum no of quiz 3)
 75% attendance is needed to sit in the final
exam
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Terminologies
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Enzyme: Greek enzumouz or en-zume (leaven)
any of a group of complex proteins or conjugated prot
eins that areproduced by living cells and act as catalyst
s in specific biochemicalreactions
Catalyst: A substance that increases the rate of a
chemical reaction without itself undergoing any
permanent chemical change.
Substrate: A substance or material on which an
enzyme act.
Active site: A part of an enzyme on which catalysis
of a substrate occur.
Product: A chemical substance formed as a result of
a chemical reaction.
Mechanism of enzyme action
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Substrate (or substrates) binds to the active site
on the enzyme.
This binding causes changes in the distribution of
electrons in the chemical bonds of the substrate
and ultimately causes the reactions that lead to the
formation of products.
The products are released from the enzyme surface
to regenerate the enzyme for another reaction
cycle.
The active site has a unique geometric shape that
is complementary to the geometric shape of a
substrate molecule, similar to the fit of puzzle
pieces.
This means that enzymes specifically react with only
one or a very few similar compounds.
Lock and Key Theory
First postulated in 1894 by Emil Fischer
Induced fit theory
•Substrate plays a role in determining the final shape of the
enzyme and that the enzyme is partially flexible.
•This explains why certain compounds can bind to the
enzyme but do not react because the enzyme has been
distorted too much.
•Other molecules may be too small to induce the proper
alignment and therefore cannot react.
•Only the proper substrate is capable of inducing the proper
alignment of the active site.
Enzyme catalysis
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An active site directly binds to a substrate and carries a reaction.
It contains catalytic groups which are amino acids that promote
formation and degradation of bonds.
By forming and breaking these bonds, enzyme and substrate interaction
promotes the formation of the transition state structure.
Enzymes help a reaction by stabilizing the transition state intermediate.
This is accomplished by lowering the energy barrier or activation
energy- the energy that is required to promote the formation of
transition state intermediate.
The active site is only a small part of the total enzyme volume.
It enhances the enzyme to bind to substrate and catalysis by many
different weak interactions because of its nonpolar microenvironment.
The weak interactions includes the Van der Waals, hydrogen bonding, and
electrostatic interactions.
The overall result is the acceleration of the reaction process and
increasing the rate of reaction. Furthermore, not only do enzymes contain
catalytic abilities, but the active site also carries the recognition of
substrate.
Transition state
Enzymatic activity
Enzymes are evaluated according to their
activity.
 Example
Group A: 10 workers saw 10 cubic meter of
wood in one hour
Group B: 20 workers saw 10 cubic meter of
wood in one hour
 An enzyme can be more active than another.
 ‘’A measure of conversion per unit time is
the amount of product formed per minute
under well-defined, standardised conditions.’’
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Optimal conditions for enzymatic activity
An optimal supply of substrate is needed by
enzyme.
 Substrate saturate the enzyme.
Example: The workers can reach their maximum
performance only when there is sufficient wood
available to be sawed.
 Enzyme and substrate must have a constant and
unimpaired contact for maximum enzymatic
activity.
 This occurs when the enzyme and substrate are
present in dilute aqueous solutions.
 Insoluble substrate and dry solids are enzymatically
inert.
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Relative amount
Enzymes work at a constant rate
•As long as the reaction conditions do not change, twice
the yield of the product will be generated in twice the
time.
•The conversion rate is reduced when there is
insuffcicient substrate available to saturate the enzyme.
pH dependence of enzymatic activity
Temperature dependence of enzyme
activity