Transcript History of enzymology

Enzymology
Dr. Nasir Jalal
ASAB,
National University of Sciences and Technology
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
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Induced fit model
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Transition state model
Quantum tunnelling model
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Hydrolase
Lyase
Isomerase
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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
Enzymes as proteins
Non-protein cofactors
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Introduction
Assumptions
Metal ions
Organic cofactors
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Derivation
Description of vo versus [S]
Michaelis constant (KM)
Nomenclature / Classification and Activity
Measurements
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Oxidoreductase-dehydrogenase
Transferase
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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Mechanism of enzyme catalysis
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β-galactosidase: a model Michaelis-Menten enzyme?
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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
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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
Substrate
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Multi-substrate Reactions and Substrate Binding
Analysis
My office hours
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Thursdays
Fridays
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[email protected]
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9:00-11:00
2:00-4:00
Introduction to enzymology
 Enzymes are Biomolecules that catalyze, increase the rates of chemical
reactions by 1015 to 1017 fold.
 Almost all enzymes are proteins.
 In enzymic reactions, the molecules at the beginning of the process are
called substrates, and the enzyme converts them into different molecules, the
products.
 Living systems use enzymes to accelerate and control the rates of vitally
important biochemical reactions.
Brief history
Earliest known use of enzymes comes from the Egyptian civilization
which used yeast for fermentation and called the product Boza.
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.
Fermentation
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.
Meaning of “enzyme” (Greek)
In 1878 German physiologist Wilhelm Kühne (1837–
1900) first used the term enzyme , which comes from
Greek ενζυμον (enzymon)"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 cell-free
fermentation".
History of
Biotechnology
Stages of Biotech
evolution
Ancient
Classical
Modern
Ancient Biotechnology
Ancient Biotech
Begins
with early civilization
Developments in
agriculture and food
production
Few records exist
Ancient Biotech
Archeologists
research
Ancient carvings and
sketches sources of
information
Ancient Biotech
Not
known when biotech
began exactly
Focused on having food and
other human needs
Ancient Biotech
 Useful
plants brought from
the wild, planted near caves
where people lived
 As food was available, ability
to store and preserve
emerged
Ancient
Food
preservation most
likely came from unplanned
events such as a fire or
freeze.
Domestication
 15,000
years ago, large animals
were hard to capture
 People only had meat when
they found a dead animal
 Came up with ways of
capturing fish and small
animals
Domestication
 Food
supplies often seasonal
 Winter food supplies may get
quite low
 Domestication is seen by
scientists as the beginning of
biotech
Domestication
Adaptation
of organisms so
they can be cultured
Most likely began 11,000 –
12,000 years ago in the
middle east
Domestication
 Involved
the collecting of seed
from useful plants and growing
crude crops from that seed
 Involved the knowledge that the
seed had to properly mature.
 A most recent find in Peru,
documents the first civilization
at 15000 years.
Domestication
 Proper
planting
 Need for water, light and
other conditions for plant
growth
 Earliest plants likely grains and
other seeds used for food
Domestication
 Raising
animals in captivity
began about the same time in
history.
 Easier to have an animal close
by than to hunt and capture a
wild one.
Domestication
 Learned
that animals need
food and water.
 Learned about simple
breeding.
 How to raise young.
 Cattle, goats and sheep were
the first domesticated food
animals.
Domestication
 About
10,000 years ago,
people had learned enough
about plants and animals to
grow their own food
 The beginning of farming.
Food
 Domestication
resulted in
food supplies being greater in
certain times of the year.
 Products were gathered and
stored.
Food
 Some
foods rotted
 Others changed form and
continued to be good to eat
 Foods stored in a cool cave
did not spoil as quickly
Food
Foods
heated by fire also
did not spoil as quickly
Immersing in sour liquids
prevented food decay
Food preservation
Using
processes that
prevent or slow spoilage
Heating, cooling, keeps
microorganisms (mo’s)
from growing
Food preservation
 Stored
in bags of leather or jars
of clay
 Fermentation occurs if certain
mo’s are present
 Creates an acid condition that
slows or prevents spoilage
Classical Biotechnology
Classical Biotech
 Follows ancient practices.
 Makes wide spread use of
methods from ancient
practices, especially
fermentation.
 Methods adapted to industrial
production e.g., salting,
canning.
Classical Biotech
 Produce
large quantities of food
products and other materials in
short amount of time.
 Meet demands of increasing
population.
 Many methods developed
through classical biotech are
widely used today.
Cheese
 One
of the first food products
made through biotechnology
 Began some 4,000 years ago
 Nomadic tribes in Asia
Cheese
 Strains
of bacteria were added
to milk
 Caused acid to form
 Resulting in sour milk
Cheese
 Enzyme
called “rennet” was
added
 Rennet comes from the lining
of the stomachs of calves
Cheese
 Rennet
is genetically engineered
today.
 Not all cheese is made from
produced rennet.
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Rennet is a complex of enzymes produced in mammalian
stomach, and is used in the production of cheese. Rennet
contains several enzymes, including the proteolytic enzyme
protease that coagulates the milk, causing it to separate into
solids (curds) and liquid (whey). They are also very important
in the stomach of young mammals as they digest their
mothers' milk. The active enzyme in rennet is called chymosin
or rennin.
Yeast
 Long
used in food preparation
and preservation
 Bread baking
 Yeast produces a gas in the
dough causing the dough to
rise
Yeast
 Fermented products
 Vinegar
 Require the use of yeast
stage of production.
in at least one
 Sugar
(glucose or fructose) → alcohol
(ethanol) + carbon dioxide
C6H12O6 → 2 CH3CH2OH + 2 CO2
Yeast
 Species
of fungi
 Some are useful
 Some may cause diseases
Vinegar
 Ancient
product used to
preserve food
 Juices and extracts from fruits
and grains can be fermented
Fermentation
 Process
in which yeast
enzymes chemically change
compounds into alcohol
 In making vinegar the first
product of fermentation is
alcohol
Fermentation
 Alcohol
is converted to acetic
acid by additional microbe
activity
 Acid gives vinegar a sour taste
 Vinegar prevents growth of some
bacteria
Vinegar
 Keeps
foods from spoiling
 Used in pickling
 Biblical references to wine
indicate the use of
fermentation some 3,000
years ago
Fermentation control
 In
ancient times, likely
happened by accident
 Advancements occurred in
the 1800’s and early 1900’s
Fermenters
 Used
to advance fermentation
process
 Specially designed chamber
that promotes fermentation
Fermenters
 Allowed
better control,
especially with vinegar
 New products such as
glycerol, acetone, and citric
acid resulted
Development
 Of
yeasts that were
predictable and readily
available led to modern baking
industry
Modern Biotechnology
Modern Biotech
 Manipulation
of genetic
material within organisms e.g.,
pruteen.
 Based on genetics and the use
of microscopy, biochemical
methods, related sciences and
technologies.
Modern Biotech
Often
known as genetic
engineering
Roots involved the
investigation of genes
Antibiotics
 Use
of fermentation hastened
the development of antibiotics
 A drug used to combat
bacterial infections
Antibiotics
 Penicillin
 Developed
in the late1920’s
 Introduced in the 1940’s
 First drug produced by
by microbes
Antibiotics
 Many
kinds available today
 Limitations in their use keep
disease producing organisms
from developing immunity to
antibiotics
Antibiotics
 Use
antibiotics only when
needed.
 Overuse may make the
antibiotic ineffective when
really needed later
Antibiotics
 Some
disease organisms are
now resistant to certain
antibiotics e.g., MDG
Tuberculosis.
 Used in both human and
veterinary medicine.
Modern Biotech
 Deals
with manipulating genetic
info
 Microscopy and advanced
computer technology are used
 In-depth knowledge of science
 Based on genetics research from
the mid 1800’s
Genetics
 Study
of heredity
 Most work has focused on
animal and plant genetics
 Genes – determiners of
heredity
Genes
 Carry
the genetic code
 Understanding genetic
structure essential for genetic
engineering
Heredity
 How
traits are passed from
parents to offspring
 Members of the same species
pass the characteristics of that
species
Heredity
 Differences
exist within each
species.
 Differences are known as
variability
Heredity &variability
 Are
used in modern
biotechnology
 Sources of Variation include:
1. Independent assortment
2. Crossing over
3. Random fertilization
Modern Biotech
 Use
of biotech to produce
new life forms
 Emerged in mid 1900’s
 Made possible by rDNA
technology
Research
 Use
of systematic methods to
answer questions.
 Problems may be basic or
applied
Basic
 Basic--
requires generating
new info to gain
understanding
 Applied – involve use of
knowledge already acquired.
Research
 Supplies
facts that can be used
to improve a process or
product
 Settings range from elaborate
labs to field plots
Field Plot
 Small
area of land that is used
to test questions or
hypothesis
 Belief is that same result
would be obtained if carried
out on larger scale.
 Often tested several times
 Known as replication
Research
 Done
by agencies, universities,
private companies, individuals
 Biotech research in agriculture is
carried out by experiment
stations and large corporations.
Development
 Creation
of new products or
methods based on findings of
research
 Carefully studied before being
put into full scale use
Development
 New
products tested before
approval
 Government agencies such as the
FDA are involved
 Prototype is developed –
research model that is carefully
tested
Prototype
 Becomes
a pattern for the
production of similar products
 After being fully tested, full
scale production begins.
Specificity
Almost all processes in a biological cell need enzymes in order to occur
at significant rates. Enzymes are extremely specific for their substrates
and as such speed up only specific reactions. There are no side or byproducts, which means that the products are also very specific.
False discovery rate FDR
Josh Elias from Stanford agrees. He says, “Partial tryptic search, or semi-tryptic searching, is ideal. And the
reason for this is that it helps to distract away all the incorrect identifications”. Josh Elias from Stanford
agrees. He says, “Partial tryptic search, or semi-tryptic searching, is ideal. And the reason for this is that it
helps to distract away all the incorrect identifications”. this slide, which shows numbers of hits for a
proteomics experiment against a target database and against a decoy database of assumed noise, using the
target-decoy strategy presented in his talk and published in Elias & Gygi, “Target-decoy search strategy for
increased confidence in large-scale protein identifications by mass spectrometry”, Nature Methods – 4, 207 –
214 (2007).
Catalytic power
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 .
E-S Complex
the concentration of substrate. Some enzymes are used commercially, for
example, in the synthesis of antibiotics. In addition, some household products use
enzymes to speed up biochemical reactions (e.g. , enzymes in biological washing
powders break down protein or fat stains on clothes)
Catalytic cycle of an enzyme