Transcript Document

Enzymes I
Reactions, Kinetics, Inhibition, Applications
Enzymes as Biological Catalysts
The Kinetic Properties of Enzymes
Substrate Binding and Enzyme Action
Enzyme Inhibition
Applications of Enzyme Action
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Enzymes as biological catalysts
Biological catalysts
• Typically are very large proteins.
• Permit reactions to ‘go’ at conditions
that the body can tolerate.
• Can process millions of molecules
every second.
• Are very specific - react with one or
only a few types of molecules
(substrates).
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Introduction to enzymes
Consider the following reaction:
2 H2O 2
2 H2 O + O 2
• The reaction is thermodynamically favored but
occurs very slowly.
• Slow reaction rate is due to the high activation
energy for the reaction.
• Only a small portion of the molecules have
sufficient energy to overcome this energy.
• We could increase the energy of the system but
this is not an option for biological systems.
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Energy diagram
H O
O H
H O
Energy
O H
transition
state
activation
energy
reactants
2 H2O2
products
H
2 H2O + O2
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Enzymatic reactions
• Enzymes act by providing an alternate,
easier pathway for a reaction.
• Same reactants, products and equilibrium.
• Increase reaction rates by having a lower
activation energy barrier.
Some enzymes require an additional
component to function properly - cofactor.
This can be an organic or organometallic
molecule or metal ion like Cu2+, Zn2+ or Mg2+
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Enzymatic reactions
Energy
enzymatic
activation
energy
2 H2O2
H
2 H2O + O2
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Naming of enzymes
Name is based on:
- what it reacts with
- how it reacts
- add -ase ending
Examples
lactase - enzyme that reacts with lactose.
pyruvate decarboxylase - removes carboxyl from
pyruvate.
Each enzyme has an official name ending in
ase and a four digit classification number
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Classification of enzymes
Based on type of reaction
Oxidoreductase
catalyze a redox reaction
Transferase
transfer a functional group
Hydrolase
cause hydrolysis reactions
Lyase
break C-O, C-C or C-N bonds
Isomerases
rearrange functional groups
Ligase
join two molecules
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Enzyme classes
Absolutely specific
Only reacts with a single substrate.
Group specific
Works with similar molecules with the same
functional group.
Linkage specific
Catalyzes a specific combination of bonds.
Stereochemically specific
Only will work with the proper D- or L- form.
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The kinetic properties of enzymes
For non-catalyzed reactions
Reaction rate increase with concentration.
Enzyme catalyzed reactions
Also increase but only to a certain point.
Vmax
maximum velocity
This catalytic behavior is observed for most
enzymes.
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Rate of reaction
(velocity)
Effect of substrate concentration
A plot of initial reaction
rates at various
concentrations shows
that a maximum
reaction rate is observed
if all other conditions
are held constant.
Substrate concentration
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Michaelis-Menten equation
The mechanism for this type of reaction
was originally formulated by Michaelis
and Menten.
In the simplest case, it involves the reaction
of a substrate (S) with an enzyme (E) to
initially form an activated complex (ES).
The complex can then decompose to a
product (P) and the enzyme or back to the
substrate.
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Michaelis-Menten equation
E+S
ES
energy
E+S
ES
P+E
Although the presence of
an enzyme will reduce the
activation energy for a
reaction, it does not
eliminate it.
E+P
As a result,
product tends
to accumulate.
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Michaelis-Menten equation
E+S
k1
k2
ES
k3
k4
P+E
Three rate expressions are used to describe
the enzymatic reaction:
ratef = k1[Eo-ES][S]
rated = k2[ES]
ratep = k3[ES]
formation of ES
decomposition of ES
formation of product
Eo = initial enzyme concentration
k4 is neglected because its effect is very small
during the initial stages of the reaction.
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Michaelis-Menten equation
Typically, as this type of reaction proceeds,
it reaches an equilibrium like condition
where [ES] remains constant.
ratef = rated + ratep
If we substitute in our rate expressions and
rearrange, we end up with:
[ES] =
k1 [Eo] [S]
(k2 + k3)/k1 + k1[S]
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Michaelis-Menten equation
Michaelis constant - Km
We can simplify our equation by including
all of the rate constants in a single term.
k2 + k 3
Km =
k1
The rate of product formation is then:
[S]
ratep = k3 [Eo]
KM + [S]
This is the step of greatest analytical interest.
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Michaelis-Menten equation
Typically, it is the substrate that is to be
measured so k3[Eo] will control the rate.
Maximum velocity - Vmax = k3[Eo]
This represents the maximum attainable
reaction rate based on the initial enzyme
concentration.
Our rate of product formation is then:
[S]
ratep = Vmax
KM
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Michaelis-Menten equation
Rate of reaction
(velocity)
Vmax
KM = [S] where v = 1/2 Vmax
1/2 Vmax
KM
Substrate concentration
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Michaelis-Menten equation
If k2 is much greater than k3 then:
KM =
k2
k1
In this form, KM is the dissociation constant
for the ES complex.
A large KM indicates that ES complex is held
together rather weakly
A small KM indicates that the forces holding
the ES complex together are strong.
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Turnover number
This is a measure of how rapidly an enzyme
can process a substrate.
Vmax
turnover number = k3 =
[ET]
Example. A 10-9 M solution of catalase causes the
breakdown of 0.4 M H2O2 per second.
k3 =
0.4 moles/liter H2O2 per second
10-9 moles/liter catalase
k3 = 40,000,000 H2O2 per mole of catalase per second
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Lineweaver-Burk equation
Using the Michaelis-Menten equation can be
difficult to determine Vmax from
experimental data.
An alternate approach was proposed by
Lineweaver and Burk that results in a
linear plot of data.
1
= KM
vo
Vmax
.
1
[S]
+
1
Vmax
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1/vo
Lineweaver-Burk equation
slope of line
KM / Vmax
y intercept
1 / Vmax
-1 / KM
1 / [S]
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Factors that influence
enzyme activity
Other conditions and species can alter the
performance of an enzyme.
Environmental factors
Temperature, pH
Cofactors
Metal ions, organic and organometallic
species
Effectors
Species that alter enzyme activity
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Factors that influence
enzyme activity
Effect of pH on enzyme activity
vo
pepsin
trypsin
2
4
6
pH
8
10
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Examples of optimum pH
Enzyme
Source
pepsin
sucrase
catalase
arginase
alkaline
phosphatase
gastric mucosa
intestine
liver
beef liver
bone
Optimum
pH
1.5
6.2
7.3
9.0
9.5
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Effect of temperature
on enzymatic reactions
Exceeding normal temperature ranges
always reduces enzyme reaction rates.
temperature
Optimum temperature is usually 25 - 40oC
but not always.
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Substrate binding
and enzyme action
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Steps in an
enzymatic reaction
1.
Enzyme and substrate combine to form a
complex.
2.
Complex goes through a transition state
- not quite substrate or product
3.
A complex of the enzyme and the
product is produced
4.
Finally the enzyme and product separate
All of these steps are equilibria.
Lets review each step
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The players
Binding
site
Catalytic
site
Enzyme
Substrate
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Formation of the
enzyme-substrate complex
First step in an enzyme catalyzed reaction
E
Enzyme
+
S
Substrate
ES
Complex
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Formation of the
transition state
An intermediate species is then formed.
ES
ES*
scissile
bond
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Formation of the
enzyme-product complex
The enzyme-product complex is then formed.
ES*
EP
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Formation of the product
The product is finally made and the
enzyme is ready for another substrate.
EP
E + P
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The active site
Enzymes are typically HUGE proteins, yet only
a small part is actually involved in reaction.
The active site has two
basic components.
catalytic site
binding site
Model of
triose-p-isomerase
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Characteristics of
enzyme active sites
Catalytic site
Where the reaction actually occurs.
Binding site
Area that holds substrate in proper place.
Enzymes use weak, non-covalent interactions to
hold the substrate in place based on R groups of
amino acids.
Shape is complementary to the substrate and
determines the specificity of the enzyme.
Sites are pockets or clefts on the enzyme surface.
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Characteristics of
enzyme active sites
Lock and key model
1890 picture by Emil Fisher. This model
assumed that only a substrate of the
proper shape could fit with the enzyme.
Induced-fit model
Proposed by Daniel Koshland in 1958.
This model assumes continuous changes
in active site structure as a substrate
binds.
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Lock and key model
This model assumes that an enzyme active
site will only accept a specific substrate.
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Induced fit model
This new model recognizes that there is
much flexibility in an enzyme’s structure.
According to the model, an enzyme is able
to conform to a substrate.
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General acid-base catalysis
Enzyme functional groups in the active site region can
serve as acids (-COOH) or bases (-COO-, -NH2).
O
H
+ H
O
+
+H
R C N
R C N
R'
+ H2NR'
R C
OH
H
2
O
R'
H+
O
H
O
H
+ H
R C N H
R'
O
H
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Metal-ion catalysts
Metal ions associated with an enzyme or
substrate often participate in catalysis.
Common metal ions:
Na+, K+, Mg2+, Mn2+, Cu2+, Zn2+, Fe2+, Fe3+, Ni2+
They assist by one of the following actions.
• Properly holding substrate in place using
coordinate covalent bonds
• Enhance a reaction by polarizing the scissile bond
or stabilizing a negatively charged intermediate.
• Participate in an oxidation-reduction reaction.
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Covalent catalysis
This occurs when a nucleophilic functional
group on an enzyme reacts to form a
covalent bond with the substrate.
This leads to an intermediate form that is
highly reactive.
Serine proteases are a group of enzymes
that rely on this approach.
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Covalent catalysis
Step 1
E
O
+ RN
Ser OH
..
C
O
R'
slow
E
O
E
C
Ser O
C
R' + RNH2
covalent
intermediate
H
Step 2
Ser O
O
R' + H2 O
fast
E
Ser OH +
R
C
OH
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Enzyme inhibition
Many substances can inhibit enzyme activity.
substrate analogs, toxins,
drugs, metal complexes
Inhibition studies can provide:
• Information on metabolic pathways.
• Insight on how drugs and toxins exert their
effects.
• Better understanding of enzyme reaction
mechanisms.
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Reversible and
irreversible inhibitors
Two broad classes of inhibitors have been
identified based on the extent of interaction.
Irreversible
Forms covalent or very strong noncovalent bonds.
The site of attack is an amino acid group that
participates in the normal enzymatic reaction.
Reversible
Forms weak, noncovalent bonds that readily
dissociate from an enzyme. The enzyme is only
inactive when the inhibitor is present.
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Inhibitors
Competitive inhibitor.
Resembles the normal substrate and
competes with it for the same site.
competitive
inhibitor
normal
substrate
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Inhibitors
Noncompetitive inhibitors.
Materials that bind at a location other than
the normal site. This results in a change
in how the enzyme performs.
inhibitor
noncompetitive
site
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Inhibitors
Uncompetitive inhibitor.
Similar to a noncompetitive inhibitor but
only binds to the ES complex.
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Acetylcholinesterase and
nerve transmission
This enzyme is needed to transmit a nerve signal at
a neuromuscular junction.
Arrival of a nerve signal causes Ca2+ levels to
increase.
This causes acetylcholine containing vesicles to
move to end of the nerve cell and is released.
Acetylcholine then diffuses across synapse to
pass the signal to the muscle.
Acetylcholinesterase then destroys the
acetylcholine to stop the signal.
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Acetylcholinesterase and
nerve transmission
synaptic
cleft
Presence of
acetylcholine at receptor
causes a flow of sodium
and potassium ions.
This causes a muscle
contraction.
acetylcholine
receptor protein
acetylcholinesterase
- destroys excess
acetylcholine
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Acetylcholinesterase
Stick model of
acetylcholinesterase.
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Acetylcholinesterase and
nerve transmission
Without the enzyme, muscles would continue
to contract causing spasms.
Acetylcholinesterase inhibitors are used as
drugs and poisons.
Organo fluorophosphates
Bind to the enzyme. Death can occur.
Succinylcholine
Acts like acetylcholine and binds to sites
on the muscle. Used as a muscle relaxant.
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Analytical methods for
determination of substrates
Two approaches can be used.
• Add a large amount of enzyme and
measure the product after complete
reaction of the substrate.
• Not a good choice because enzymes are
relatively expensive.
• Add a small amount of enzyme and
determine the initial rate of reaction.
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Analytical methods for
determination of substrates
Other factors to consider.
• Temperature, pH and other conditions
must be held constant.
• Other materials may compete for either
your enzyme or substrate. These should
be masked, removed or at least held
constant.
• Any loss of products or interactions of
the products with other materials must be
addressed.
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Examples
Determination of urea
Based on the catalyzed hydrolysis of urea
by urease.
urease
NH2CONH2 + 2H2O + H+
2NH4+ + HCO3-
Potential species to measure
H+
All are pH dependent
NH4+
HCO3-
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Examples
Determination of urea
An easy approach would appear to be to
measure the pH - using a pH electrode.
Unfortunately, as H+ is consumed, the
reaction rate changes.
pH stat - a device that monitors a solution
and adds acid or base to keep pH
constant.
A plot of acid added at a fixed time verses
[S] produces a linear relationship.
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Examples
Determination of glucose.
This is a common material to assay for in
clinical laboratories.
Enzymatic reaction used:
peroxidase
glucose + H2O + O2
gluconic acid + H2O2
Two approaches are used for measure the
rate based on measurement of O2 or
H2 O 2 .
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Examples
Determination of glucose - H2O2
Peroxide can’t be measured directly by
any rapid, convenient method.
A coupled reaction is used to produce a
detectable species.
H2O2 + reduced dye
colorless
peroxidase
H2O + oxidized dye
colored
The increase in absorbance can be
measured which is proportional to the
concentration of glucose.
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Examples
Determination of glucose - O2
Polarographic method
With this approach, [O2] is directly
measured at an electrode using the
following reaction:
O2 + 4H+ + 4e- = H2O
A commercial glucose analyzer has been
developed using this approach.
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Examples
Determination of glucose - O2
Polarographic method
Only a VERY small amount of O2 is
actually used and it is VERY rapid
(~10sec) so conditions don’t change very
much.
The sample must NOT be in rapid
equilibrium with the atmosphere in order
to get reliable results.
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