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Modified and
Modifying Enzymes
(Do not forget to read the handout about
modified enzymes)
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What is an Enzyme?
What is an Modified Enzyme?
What is an Modifying Enzyme?
What is modification of an Enzyme?
How we can use modifying and modified
Enzyme ?
M.H.Modarressi MD.,PhD.
TUMS
Enzymes
What are enzymes?
Types of enzymes:
ALL of the enzymes in a living organism
are made by the cells of that organism.
Specialized proteins
What do they do?
They act as …….. in chemical
reactions in our bodies or…...
Why enzyme?
Enzymes increase the rate of reaction in a
living organism, for example, 1 million
times, without altering the energy
(temperature) of the reaction.
Enzymes increases the rate of reaction by
lowering the activation energy barrier, thus
allowing reactions to proceed without an
input of energy
speed up a chemical reaction by lowering the activation energy
required
MOST of the enzymes made by an
individual cell are used in that cell.
SOME enzymes pass out of the cell to be
used elsewhere (i.e. digestive enzymes)
Enzymes are:
The object enzymes work upon are
called substrates.
Most enzymes are named according to
what they work upon.
The way this is done is by changing the
ending of the substrate to –ase.
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How do enzymes work?
-Enzyme activity relies completely on
the shape of the protein (enzymes are
proteins so they have secondary
structure).
-Enzymes are large proteins with specific 3D
shape
How do enzymes work?
(continued…)
Specificity
• All enzymes operate only on specific
substrate:
• some enzymes will act only on one
particular substrate;
• others act on similar molecules;
• many will break a particular linkage, e.g.
hydrogen bonds
How do enzymes work?
(continued…)
Again, it is the shape of the enzyme that
allows it to work.
More specifically, on the surface of each
enzyme is a region called the active site.
The active site fits the shape of the
substrate the enzyme works on.
How do enzymes work?
(continued…)
Catabolic reaction
Substrate
Activated Complex
Products
Anabolic reaction
Substrates
Activated Complex
Product
Active sites…
When the active site of an enzyme comes
into contact with it’s substrate it may do 2
things:
1) With only 1 substrate, it will break
bonds, to form 2 or more substances.
2) With 2 substrates it will form bonds,
to make 1 compound.
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Enzyme activity
• Enzyme, protein, activity is effected by
environmental conditions
– pH, temperature, salts and presence of
cofactors or inhibitors
– inorganic cofactors, I.e. magnesium
– organic cofactors are called coenzymes
Enzyme Activity
• Temperature
• pH
• Enzyme
Concentration
• Substrate
Concentration
• i.e. vitamins
Enzyme
concentration:
- active sites of an
enzyme can be
used again &
again,
- therefore only a
low
concentration of
the enzyme is
needed
pH
• The precise three-dimensional molecular
shape which is vital to the functioning of
enzymes is partly the result of hydrogen
bonding;
• H+ ions may break these bonding and
change the shape of the molecule.
- At low substrate concentration, the
active sites of the enzyme molecules
are not all used. As the substrate
concentration is increased, more and
more sites come into use and eventually
all sites are fully occupied.
- Increasing the substrate concentration
cannot increase the rate of reaction
because substrate concentration has
now become a limiting factor.
pH and Temperature
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Cofactors and Coenzymes
• Cofactors are necessary for some
enzymes. Most often metal ions
• Coenzymes
– Organic molecules
– Soluble
– Prosthetic groups
• Apoenzyme vs Holoenzyme
Metal Ion Catalysis
• Various metals, all positively charged and
including zinc, iron, magnesium,
manganese and copper, are known to form
complexes with different enzymes or
substrates. This metal-substrate-enzyme
complex can aid in the orientation of the
substrate in the active site, and metals are
known to mediate oxidation-reduction
reactions by reversible changes in their
oxidation states (like Fe3+ to Fe2+).
Cofactors
• Non-protein molecules that help
enzymes function.
• Bind to active site to enhance enzymatic
reactions.
• Cofactors may be inorganic metals such
as zinc, iron, or copper.
• Coenzymes are organic cofactors (e.g.
vitamins)
Inhibition of Enzyme Activity
Block enzyme action
• competitive inhibition - competes with
substrate for binding to active site
– increasing substrate concentration can dilute
or prevent inhibition
• noncompetitive inhibitor - binding to
sites such as the allosteric site
Enzyme Nomenclature (cont)
Some pesticides and antibiotics inhibit
enzymes
• pesticide malathion
• antibiotic penicillin interferes with an
enzyme that aids in bacteria cell wall
preparation
• prosthetic group - a metal or other co-enzyme
covalently bound to an enzyme
• holoenzyme - a complete, catalytically active
enzyme including all co-factors
• apoenzyme - the protein portion of a
holoenzyme minus the co-factors
• isozyme - (or iso-enzyme) an enzyme that
performs the same or similar function of another
enzyme. This generally arises due to similar
but different genes encoding these enzymes
and frequently is tissue-type specific or
dependent on the growth or developmental
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Enzyme Nomenclature
• active site - a region of an enzyme comprised
of different amino acids where catalysis occurs
(determined by the tertiary and quaternary
structure of each enzyme)
• substrate - the molecule being utilized and/or
modified by a particular enzyme at its active site
• co-factor - organic or inorganic molecules that
are required by some enzymes for activity.
These include Mg2+, Fe2+, Zn2+ and larger
molecules termed co-enzymes like nicotinamide
adenine dinucleotide (NAD+), coenzyme A, and
many vitamins.
•Allosteric site - binding site which modulates the activity of the enzyme,
can alter an enzyme’s 3D shape
Cooperativity
Feedback
Inhibition
Enzymes:
Modifying enzyme used in Genetic
Engineering (read Handout)
Restriction Enzymes:
Biology and Activity of Restriction Endonucleases
Cutting DNA with Restriction Endonucleases
-Modified and Modifying enzymes
-Catalytic enzymes (such as digestive
enzymes)
Restriction Mapping
Factors that Influence Restriction Enzyme Activity
DNA Ligation
Polymerases (DNA modifying enzymes)
oE. coli DNA Polymerase I
oKlenow Fragment of E. coli DNA Polymerase I
oT4 DNA Polymerase
-Diagnostic enzymes
-Industrial enzymes
oT7 DNA Polymerase
oThermostable DNA Polymerases (Taq, Pfu, Vent, etc.)
oTerminal Transferase
oReverse Transcriptases
oBacteriophage RNA Polymerases
-……..
Other DNA Modifying Enzymes
oNucleases: DNase and RNase
oDNA Ligase
oAlkaline Phosphatase
oPolynucleotide Kinase
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Nucleases
• Enzymes degrade DNA by
breaking the phosphodiester
bonds.
• Endonucleases, Exonucleases.
• Restriction enzymes (type II
endonucleases) cut DNA at
defined sites.
DNA Modifying Enzymes
• AKA: Restriction Enzymes
• The restriction/modification system functions
as a type of immune system for individual
bacterial strains, protecting them from
infection by foreign DNA (e.g. viruses).
• Methylation occurs at very specific sites in
the DNA.
Nomenclature
EcoRI
coli
strain
• Another example:
BamHI from Bacillus amyloliquefaciens.
A characteristic feature of the sites of
methylation, was that they involved
palindromic DNA sequences.
(EcoR1 methylase specificity. Rubin and Modrich, 1977)
In addition to possessing a particular methylase,
individual bacterial strains also contained accompanying
specific endonuclease activities.
• The endonucleases cleaved at or near the methylation
recognition site.
• These specific nucleases, however, would not cleave at
these specific palindromic sequences if the DNA was
methylated.
Restriction Enzymes
• In the bacterial strain EcoR1, the sequence
GAATTC will be methylated at the internal adenine
base (by the EcoR1 methylase).
• The EcoR1 endonuclease within the same
bacteria will not cleave the methylated DNA.
• Foreign viral DNA, which is not methylated at the
sequence "GAATTC" will therefore be recognized
as "foreign" DNA and will be cleaved by the
EcoR1 endonuclease.
• Cleavage of the viral DNA renders it nonfunctional.
Extra-chromosomal element
Eschreichia
Frequency of Digestion
Nucleotide Specificity
Example
Frequency of
Occurrence
Four
Alu I
256 (0.25 Kb)
Five
Nci I
1024 (1.0 Kb)
Six
EcoR I
4096 (4.1 Kb)
Seven
EcoO109I
16384 (16.4 Kb)
Eight
Not I
65536 (65.5 Kb)
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Types of Ends Generated by Different
Restriction Enzymes
Overhanging and Sticky ends used for cloning
HindIII
Haemophilus influenzae
Cos
5’ overhanging
Sticky ends
Pst1
Providencia stuartii
3’overhanging
Generation of Recombinant DNA Using
Restriction Enzymes
Uses of Restriction Enzymes
• RFLP
– Restriction Fragment Length Polymorphism
• DNA finger printing
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DNA Polymerase I
Molecular diagnostics
Cloning
cDNA library construction
Population studies
………….
The Klenow Fragment
• 5’-3’ Exonuclease Activity
• 3’-5’ Exonuclease Activity
• DNA Polymerase Activity
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Uses of DNA Polymerase Enzymes
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Types of endonuclease
PCR (Molecular diagnostics, ….)
Labeling
Cloning
………….
Catalytic enzymes (such as
digestive enzymes)
Diagnostic enzymes
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Introduction
Enzymes are
used in industry
Non-recombinant Sources
• Industrial vs Medicinal/Therapeutic
• Types
• $1.5 billion industry
Recombinant Sources
Most industrial enzymes are produced recombinantly
GRAS
Bacillus
protein is secreted into fermentation medium
easier purification
Aspergillus
Yeast
Why?
A. Higher expression
B. Higher purity (%protein:other junk)
C. cheap
D. can engineer protein
E. can express enzymes which are found
in pathogenic organisms
Heterologous Protein Expression
Homologous Protein Expression
Classes
• Lipases
• Proteases
Proteolytic Enzymes
Largest group of Industrial Enzymes (50%)
Function - degrade proteins
• Carbohydrases
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They can be used in Detergents
Removes biological and non-biological stains
Safety allergic reactions from microbial enzymes?
Protease for Cheese
manufacture
Rennin (Chymosin) is a aspartic protease
coagulation of milk
cleave casein to form curds (micelles)
Preprorennin
Prorennin
Rennin (active)
First food product produced by rDNA technology approved
for human use (1990)
Meat Tenderizers
Collagen makes meat tough
Younger animal less cross linking of collagen
Papain -cysteine protease
degrades muscle fiber and connective proteins
active at high temperature (cooking)
Enzymes use in Molecular
biology
1. Restriction endonucleases
defense
specific sequence
methylation
blunt vs staggered
4, 6, 8 base cutters
2. Ligase catalyze formation of bonds of nucleic acids (DNA)
3. DNA polymerase
taq
Deep Vent DNA polymerase
Lipase
• Lipase enzyme preparation derived from Penicillium
camembertii
• Intended Use: Production of fatty acids from fats and
oils
• Similar to other lipases used in food processing
• Source Organism (published information)
– Nontoxigenic
– Nonpathogenic
– Long used in the production of Camembert
cheese
Lipase
• Lipase enzyme preparation from
Penicillium camembertii
• Chymosin enzyme preparation from
bioengineered Escherichia coli K-12
• amylase enzyme preparation from
bioengineered Bacillus licheniformis
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Bioengineered -Amylase
• Amylase enzyme preparation derived
from a bioengineered strain of Bacillus
licheniformis
• Bioengineered for enhanced stability at
low pH, low calcium concentration and
high temperature
• Intended use: starch hydrolysis in the
production of syrups (e.g., high-fructose
corn syrup) and alcohol
Bioengineered -Amylase: The Enzyme
• Gene encoding -amylase derives from B.
licheniformis.
– Modifications for enhanced stability and a lower
calcium requirement (as compared with other amylases):
• 35 amino acids at amino-terminal region from
B. amyloliquefaciens - amylase
• Five additional altered amino acids
– DNA sequence: highly homologous to those
encoding other -amylases
– Same enzymatic function as other -amylases that
have a history of safe use in food
Thanks
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