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1
Chapter 3: Enzyme Kinetics
Outline
1. Introduction to enzyme
Definition
How enzymes work?
2. Modeling of enzymatic reaction
Modeling of simple enzyme kinetics
Modeling of complex enzyme kinetics
Allosteric enzymes
Inhibited enzyme kinetics
3. Factors affecting enzyme kinetics (pH and
Temperature)
ENZYMES
Enzymes are usually proteins of high molecular weight (15,000
several millions of Daltons) with catalytic properties due to its
power of specific activation
Recently it has been shown that some RNA molecules are also
catalytic- Ribozymes
Enzymes are specific, versatile, very effective biological catalysts,
resulting in much higher reaction rates as compared to chemically
catalyzed reactions under ambient condition.
More than 2000 enzymes are known
Enzyme reactions are different from chemical reactions, as
follows:
1. An enzyme catalyst is highly specific, and catalyzes
only one or a small number of chemical reactions. A
great variety of enzymes exist, which can catalyze a
very wide range of reactions.
2. The rate of an enzyme-catalyzed reaction is usually
much faster than that of the same reaction when
directed by non-biological catalysts. Only a small
amount of enzyme is required to produce a desired
effect.
3. The reaction conditions (temperature, pressure, pH,
and so on) for the enzyme reactions are very mild.
4. Enzymes are comparatively sensitive or unstable
molecules and require care in their use.
Nomenclature of Enzymes
Originally enzymes were given non-descriptive
names such as:
Rennin -curding of milk to start cheese-making
process
Pepsin hydrolyzes proteins at acidic pH
Trypsin - hydrolyzes proteins at mild alkaline pH
The nomenclature was later improved by adding the suffix -
ase to the name of the substrate with which the enzyme
functions, or to the reaction that is catalyzed
For example:
Name of substrate + ase
-amylase starch glucose + maltose +oligosaccharides
lactase lactose glucose + galactose
Lipase fat fatty acids + glycerol
Maltase maltose glucose
Urease urea + H2O 2NH3 + CO2
Cellobiase cellobiose glucose
Reaction which is catalyzed + ase
alcohol dehydrogenase ethanol+NAD+ acetaldehyde + NADH2
glucose isomerase glucose fructose
glucose oxidase D-glucose + O2 + H2O gluconic acid
lactic acid dehydrogenase lactic acid pyruvic acid
As more enzymes were discovered, this system
generated confusion and resulted in the formation
of a new systematic scheme by the International
Enzyme Commission in 1964.
The new system categorizes all enzymes into six
major classes depending on the general type of
chemical reaction which they catalyze.
Each main class contains subclasses,
Subsubclasses, and subsubsubclasses.
Therefore, each enzyme can be designated by a
numericalcode system.
As an example, alcohol dehydrogenase is assigned
as 1.1.1.1.
Enzyme structure
Enzymes are
proteins
They have a globular
shape
A complex 3-D
structure
14
Classification of Enzymes
Oxidoreductoases
oxidases - oxidize ,reductases reduce
Transferases
transaminases transfer amino groups
kinases transfer phosphate groups
Hydrolases
proteases - hydrolyze peptide bonds
lipases hydrolyze lipid ester bonds
Lyases
carboxylases add CO2
hydrolases add H2O
16
How enzymes work?
Proximity effect
Orientation effect
S
E
E
E
Reaction coordinate
The Lock and Key Hypothesis
This explains enzyme specificity
This explains the loss of activity when enzymes
denature
The Induced Fit Hypothesis
Some proteins can change their shape (conformation)
When a substrate combines with an enzyme, it induces
a change in the enzymes conformation
The active site is then moulded into a precise
conformation
Making the chemical environment suitable for the
reaction
The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)
Induced Fit Model
Enzyme structure flexible, not rigid
Enzyme and active site adjust shape to bind
substrate
Increases range of substrate specificity
Shape changes also improve catalysis during
reaction
32
The induced-fit theory assumes that the
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.
The Induced Fit Hypothesis
What is kinetics?
is the study of the rate and mechanism in the
reactor
gives us a quantitative description of how fast
chemical reactions occur, and the factors affecting
these rate
Why Kinetics?
d[ES]
k1[E][S ] k1[ES] k2[ES]
dt
Since the enzyme is not consumed, the
conservation equation on the enzyme yields
[ E] [ E0 ] [ES]
Enzyme Kinetics
d[ P]
v k 2 [ ES]
dt
d[ES]
k1[ E][S ] k1[ ES] k2[ES]
[ E] [ E0 ] [ES]
dt
K1
k2
E+S
K-1
ES P E
k1[ E ][ S ] k1[ ES ]
Michaelis - Menten Approach
The equilibrium constant Kcan ' be expressed by the
m
following equation in a dilute system.
K1
k2
E+S ES P E
K-1
' k 1 [ E ][ S ]
Km
k1 [ ES ]
Michaelis - Menten Approach
Then rearrange the above equation,
[ E ][ S ]
[ ES ]
Km '
yields,
([ E0 ] [ ES ])[ S ]
[ ES ]
Km '
Michaelis - Menten Approach
[ES] can be expressed in terms of [S],
[ E0 ][ S ]
[ ES ]
Km' [S ]
- It corresponds
to the substrate concentration, giving the
Half maximum reaction velocity.
1 Vm [ S ]
k 1 v Vm
K m' 2 Km' [S ]
k1
Re-arrange the above equation, 1
' [S ]
Km When v Vm
2
Michaelis - Menten Approach
Vm is maximum forward velocity (e.g.mol/L-s)
Vm k 2 [E0 ]
It increases with initial enzyme concentration.
It is determined by the rate constant k2 of the product
formation and the initial enzyme concentration.
Briggs-Haldane Approach
The quasi-steady-state assumption:
- A system (batch reactor) is used in which the initial
substrate concentration [S0] greatly exceeds the
initial enzyme concentration [E0].
since [E0] was small,
d[ES]/dt 0
- It is shown that in a closed system the quasi-steady-
state hypothesis is valid after a brief transient if
[S0]>> [E0].
The quasi-steady-state hypothesis is valid after a
brief transient in a batch system if [S0]>> [E0].
Briggs-Haldane Approach
With such assumption, the equation representing the
accumulation of [ES] becomes
d[ES]
k1[ E][S ] k1[ES] k2[ES] 0
dt
k1[ E ][ S ]
[ ES ]
k1 k2
Briggs-Haldane Approach
Substituting the enzyme mass conservation equation
[ E ] [ E0 ] [ ES ]
in the above equation yields
k1([ E0 ] [ ES ])[S ]
[ ES ]
k 1 k 2
Then, Vm [S ] k1
v
K m [S ]
Where Vm k [E0 ] same as that for rapid equilibrium assumption.
2
k 1 k 2
Km when K2 << k-1,
k1
Comparison of the Two Approaches
Michaelis-Menten Briggs-Haldane
Assumption: k1[ E ][ S ] k1[ ES ] d[ES]/dt 0
Vm [ S ] Vm [S ]
Equation: v v
' k 1
Km Km' [S ] K m [S ]
k1
Maximum forward
reaction rate: Vm k 2 [E0 ] Vm k 2 [E0 ]
k 1 k 2
Constant: Km
k1
k 1 k 2
when k2 << k-1, Km Km '
k1
Experimentally Determining Rate Parameters for
Michaelis-Menten Type Kinetics.
Vm [S ]
v
K m [S ]
Vm [S ]
v
K m [S ]
1 1 Km 1
v Vm Vm S
a slope equals to Km/Vm
y-intercept is 1/Vm.
More often used as it shows the independent variable [S] and
dependent variable v.
1/v approaches infinity as [S]
decreases
Eadie-Hofstee Plot
v
v Vm K m
[S ]
- the slope is Km
- y-axis intercept is Vm.
Hanes-Woolf (Langmuir) Plot
[S ] K m 1
[S ]
v Vm Vm
Michaelis-Menten Approach
Briggs-Haldane Approach
Use experimental data to obtain parameters
of Michaelis-Menten kinetics.
Enzyme reactor with simple kinetics
Batch Reactor
CSTR
Next week
Modeling of complex enzyme
kinetics
Allosteric enzymes
Inhibited enzyme kinetics
Factors affecting enzyme kinetics
(pH and Temperature)
Allosteric enzymes
Allosteric regulators bind to a specific site called the
allosteric site on the enzyme (not the active site)
Enzymes having allosteric regulation are usually
multimeric (more than one polypeptide subunit)
They oscillate between active and inactive
conformations
Binding of an activator stabilizes the active form
Binding of an inhibitor stabilizes the inactive form
Allosteric enzymes
d[ P]
v k 2 [ ES]
dt
' k 1 [ E ][ S ]
Km
k1 [ ES ]
[ E ][ I ] [E0 ] [E] [ES] [EI ]
KI
[ EI ]
Inhibited Enzyme Kinetics
we can obtain,
Vm [S ]
v
' (1 [ I ] / K ) [S ]
Km I
Vm [ S ]
v
'
Km , app [S ]
' ' (1 [ I ] / K ) When [I] =0, ' '
Km , app K m I K m,app K m
d[ P]
v k 2 [ ES]
dt
' k 1 [ E ][ S ] [ EI ][ S ]
Km
k1 [ ES ] [ ESI ]
[ E ][ I ] [ ES ][ I ]
KI
[ EI ] [ ESI ]
[ E0 ] [ E] [ ES] [ EI ] [ ESI ]
Inhibited Enzyme Kinetics
Vm [S ]
v
' [ S ])
(1 [ I ] / K I )( K m
Vm, app[S ]
v
' [S ]
Km
d[ P]
v k 2 [ ES]
dt
' k 1 [ E ][ S ]
Km
k1 [ ES ]
[ ES ][ I ]
KI
[ ESI ]
Vm, app[ S ]
v
' , app [ S ]
Km
Vm [ S ]
dv / d [ S ] d ( ) / d[S ] 0
' [ S ] [ S ]2 / K
Km SI
' K )1/ 2
[S ]max (Km SI
Uncompetitive substrate inhibitors (I)
Determine [S]max:
Vm [ S ]
dv / d [ S ] d ( ) / d[S ] 0
' [ S ] [ S ]2 / K
Km SI
Vm [ S ](1 2[ S ] / K SI )
Vm
( )0
' [ S ] [ S ]2 / K
Km ( K ' [ S ] [ S ]2 / K ) 2
SI m SI
' [ S ] [ S ]2 / K ) V [ S ](1 2[ S ] / K )
Vm ( K m SI m SI
( )0
(K m' [ S ] [ S ]2 / K ) 2
SI
' V [ S ]2 / K
Vm K m m SI
)0
' [ S ] [ S ]2 / K ) 2
(K m SI
' V [ S ]2 / K 0
Vm K m m SI
' K )1 / 2
[ S ]max ( K m SI
Inhibition Estimation
Product formation rate v ~ [S]: v has a peak?
If yes, then its substrate inhibition.
- get [S]max from the plot of v~[s].
- at low substrate concentration, obtain Vm and Km
graphically or through direct calculation.
- calculate KI through
' K )1/ 2
[S ]max (Km SI
If no, then examine the data with and without inhibitors in 1/v
~ 1/[S] plot (Lineweaver-Burk Plot).
Estimation of Inhibited Enzyme Kinetics
Estimation of Inhibited Enzyme Kinetics
Enzyme concentration
Substrate concentration
Temprature
pH
Enzyme concentration
Substrate concentration
Vmax and Km
Temperature
pH
Graphs for inhibition
Factors affecting Enzymes
substrate concentration
pH
temperature
inhibitors
Reaction
velocity
Substrate concentration
Vmax
Reaction
velocity
Substrate concentration
Faster reaction but it reaches a saturation point when all the
enzyme molecules are occupied.
If you alter the concentration of the enzyme then Vmax will
change too.
2007 Paul Billiet ODWS
The effect of pH
Optimum pH values
Enzyme
activity Trypsin
Pepsin
1 3 5 7 9 11
Q10 Denaturation
Enzyme
activity
0 10 20 30 40 50
Temperature / C