Addis Ababa University

Addis Ababa Institute of Technology

School of Chemical & Bio Engineering

Course Title:

ChEg 4190: Introduction to Biochemical Engineering

Credit hour: Lect. 2hr / Tut. 3hr

Instructor: Prof. Eduardo Ojito Cespedes

A/Year: Semester II, 2016

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

Human pancreatic amylase

The active site
One part of an enzyme,
the active site, is
particularly important
The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
more easily
 Cofactors and coenzymes
An additional non-protein
molecule that is needed by
some enzymes to help the
reaction
Tightly bound cofactors are
called prosthetic groups
these includes metal ions,
Mg, Zn, Mn, Fe.

Cofactors that are bound

and released easily are
called coenzymes.
Coenzymes are complex
organic molecules such as
NAD, FAD, CoA or some
vitamins.

Nitrogenase enzyme with Fe, Mo and ADP cofactors

Jmol from a RCSB PDB file 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS

IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)

Components of a holoenzyme
 The substrate
The substrate of an enzyme are the reactants
that are activated by the enzyme
Enzymes are specific to their substrates
The specificity is determined by the active site
 Name of Enzymes
End in ase
Identifies a reacting substance
sucrase reacts sucrose
lipase - reacts lipid
Describes function of enzyme
oxidase catalyzes oxidation
hydrolase catalyzes hydrolysis
Common names of digestion enzymes still use in
pepsin, trypsin

14
 Classification of Enzymes

Class Reactions catalyzed

Oxidoreductoases oxidation-reduction
Transferases transfer group of atoms
Hydrolases hydrolysis
Lyases add/remove atoms
to/from a double bond
Isomerases rearrange atoms
Ligases combine molecules
using ATP
15
 Examples of 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?

Lowering the activation energy

Proximity effect

Orientation effect

Enzyme action models

The mechanism of enzymatic action
 The mechanism of enzymatic action
1. Substrate to active site (Proximity and
orientation effect, collision)
2. Formation of ES complex
3. Substrate transferred by rearrangement of
existing atoms, breakdown or combination with
another substrate (The binding of the substrate
to the enzyme 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.)
4. Product released b/c no longer fit the active site
What bio-molecular magic is happening at the
active site?
What bio-molecular magic is happening here?
"Over the past 50 years or so," says biochemist
David Deerfield, "we've made enormous progress
in filling in the big picture of what enzymes do
and how they do it at an ever increasing level of
detail. But we're a long way from precisely
understanding what happens in the transition
from substrate to product, the atom-by-atom
details of these changes and exactly how
enzymes play their role."
 Chemical reactions
Chemical reactions need an initial input of energy =
THE ACTIVATION ENERGY
During this part of the reaction the molecules are
said to be in a transition state.
 Reaction pathway

2007 Paul Billiet ODWS

 Making reactions go faster
Increasing the temperature make molecules move
faster
Biological systems are very sensitive to temperature
changes.
Enzymes can increase the rate of reactions without
increasing the temperature.
They do this by lowering the activation energy.
They create a new reaction pathway a short cut
 An enzyme controlled pathway

Enzyme controlled reactions proceed 108 to 1011 times faster

than corresponding non-enzymic reactions.
Proximity effect and Orientation effect
In multisubstrate enzyme catalyzed reactions,
enzyme can hold substrates such that reactive
regions of substrates are close to each other and
to the enzymes active site which is known as
proximity effect. How we can achieve this?
Also, enzymes can hold the substrates at certain
positions and angles to improve the reaction rate,
which is known as orientation effect.
 The Lock and Key Hypothesis
Fit between the substrate and the active site of the enzyme is
exact
Like a key fits into a lock very precisely
The key is analogous to the enzyme and the substrate analogous
to the lock.
Temporary structure called the enzyme-substrate complex
formed
usually weak interaction force, van der Waals forces and
hydrogen bonding are responsible for the formation of enzyme-
substrate complex
Products have a different shape from the substrate
Once formed, they are released from the active site
Leaving it free to become attached to another substrate
The Lock and Key Hypothesis

S
E
E
E

Enzyme- Enzyme may

substrate be used again
complex P

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

Hexokinase (a) without (b) with glucose substrate

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

This explains the enzymes that can react with a range

of substrates of similar types
 Modeling of enzymatic reaction
Modeling of simple enzyme kinetics

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?

In chemical reaction engineering (CRE), the

information obtained from kinetics is a means to
determine something about the reactor: size, flow
and thermal configuration, product distribution, etc.
 Enzyme Kinetics
Michaelis-Menten kinetics or saturation kinetics which
was first developed by V.C.R. Henri in 1902 and developed by
L. Michaelis and M.L. Menten in 1913.
This model is based on data from batch reactors with
constant liquid volume.
- Initial substrate, [S0] and enzyme [E0]
concentrations are known.
- An enzyme solution has a fixed number of active
sites to which substrate can bind.
- At high substrate concentrations, all these sites may
be occupied by substrates or the enzyme is
saturated.
Saturation Enzyme Kinetics
 M-M Enzyme Kinetics
Saturation kinetics can be obtained from a simple reaction
scheme that involves a reversible step for enzyme-
substrate complex formation and a dissociation step of
the ES complex.
K1
k2
E+S
K-1
ES P E
where the rate of product formation v (moles/l-s, g/l-min) is
d[ P]
v k 2 [ ES]
dt
Ki is the respective reaction rate constant.
 Enzyme Kinetics
The rate of variation of ES complex is

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

How to use independent variable [S] to represent v?

Enzyme Kinetics
At this point, an assumption is required to
achieve an analytical solution.
- The rapid equilibrium assumption
Michaelis - Menten Approach.

- The quasi-steady-state assumption.

Briggs and Haldane Approach.
 Michaelis - Menten Approach
The rapid equilibrium assumption:
- Assumes a rapid equilibrium between the enzyme
and substrate to form an [ES] complex.

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 '

Substituting [E] in the above equation with enzyme

mass conservation equation
[ E ] [ E0 ] [ ES ]

yields,
([ E0 ] [ ES ])[ S ]
[ ES ]
Km '
 Michaelis - Menten Approach
[ES] can be expressed in terms of [S],
[ E0 ][ S ]
[ ES ]
Km' [S ]

Then the rate of production formation v can be

expressed in terms of [S],
d [ P] k 2 [ E0 ][S ] Vm [S ]
v k 2 [ ES]
dt '
K m [S ] '
K m [S ]
Where Vm k 2 [E0 ]
represents the maximum forward rate of reaction (e.g.moles/L-min).
Michaelis - Menten Approach
K m' is often called the Michaelis-Menten constant, mol/L, mg/L.
- The prime reminds us that it was derived by assuming rapid
equilibrium in the step of enzyme-substrate complex formation.
- Low value indicates high affinity of enzyme to the substrate.

- 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

Solving this algebraic equation yields

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

Using [S] to represent [ES] yields

[ E0 ][ S ]
[ ES ]
k 1 k 2
[S ]
k1
 Briggs-Haldane Approach
Then the product formation rate becomes
d [ P] k 2 [ E0 ][S ]
v k [ ES]
dt 2 k 1 k 2
[S ]
k1
K m K m ' 1
k

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 ]

To determine the rate parameters:

- Predict a specific enzyme catalysis system.
- Design bioreactor
The determination of Vm and Km are typically obtained
from initial-rate experiments.

-A batch reactor is charged with known initial concentration of

substrate [So] and enzyme [Eo] at specific conditions such as
T, pH, and Ionic Strength.
- The product or substrate concentration is plotted against
time.
- The initial slope of this curve is estimated.
v=(d[P]/dt) , or = - (d[S]/dt) .
This value v depends on the values of [E0] and [S0].
- Many such experiments can be used to generate many pairs of
V and [S] data, these data can be plotted as v-[S].
Lineweaver-Burk Plot (Double-Reciprocal Plot)

Vm [S ]
v
K m [S ]

Linearizing it in double-reciprocal form:

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

- the slope is 1/Vm

- y-axis intercept is Km/Vm
- better fit: even weighting of the data
 Vm
Vm k 2 [E0 ]

-The unit of Vm is the same as that of a reaction rate

(moles/l-min, g/l-s)
-The dimension of K2 must reflect the units of [E0]
-if the enzyme is highly purified, it may be possible to
express [E0] in mol/l, g/l, then K2 in 1/time.
- if the enzyme is crude, its concentration is in units.
A unit is the amount of enzyme that gives a predetermined amount of
catalytic activity under specific conditions.
(Textbook, Bioprocessing Engineering, M. Shuler, p.66-67)
If Vm is mmol/ml-min, [E0] is units/ml, then K2 should be in
mmol/unit-min.
 Enzyme Activity
Specific Activity is the number of units of activity per
amount of total protein.

Ex. A crude cell lysate might have a specific activity of 0.2

units/mg or ml protein upon which purification may
increase to 10 units/mg or ml protein.
One unit would be formation of one mol product per
minute at a specific pH and temperature with a substrate
concentration much greater than the value of Km.
Summary of Simple Saturation Kinetics

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

The rate expression in this case is

V=- (d[S]/dt) = Vm[S]n/km+ [S]n
Where n= cooperativity coefficient and n>1
indicates positive cooperativity.
The cooperativity coefficient can be
determined by rearranging as ln (v/vm-v) =
nln[S] lnkm and by plotting ln (v/vm-v) vs
ln[S] yields slope n.
 Inhibited Enzyme Kinetics
Inhibitors may bind to enzyme and reduce
their activity.
Enzyme inhibition may be reversible or
irreversible.
For reversible enzyme inhibition, there are
Competitive Inhibition
Noncompetitive Inhibition
Uncompetitive Inhibition
 Enzyme inhibitors
Enzyme function and inhibition (with audio narration).mp4
Inhibited Enzyme Kinetics
1. Competitive inhibitors (I)
- substrate analogs
- compete with substrate for the active
site of the enzyme
K1
k2
E+S ES P E
K-1
+
I
KI
EI
Inhibited Enzyme Kinetics
Assume rapid equilibrium and with the
definitions of

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

Vm Remains that of Michaelis-Menten equation.

K m,app Is increased.
The net effect of competitive inhibition is an
increased value of Kmapp and therefore reduced
reaction rate.
High substrate concentration could overcome
competitive inhibition.
Inhibited Enzyme Kinetics
2. Noncompetitive inhibitors (I)
- not substrate analogs
- bind on sites other than the active site and
reduce the affinity for the substrate.
Km
k2
E+S ES P E
+ +
I I
KI KI
Km
EI+S ESI
Inhibited Enzyme Kinetics
Assume:
- rapid equilibrium
- same equilibrium constants of inhibitor binding
to E and ES KI
- same equilibrium constants of substrate binding
to E and EI
Km
Inhibited Enzyme Kinetics

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

Km Remains in Michaelis-Menten equation.

Vm ,app is reduced.
Vm, app Vm /(1 [ I ] / K I ) When [I] =0, Vm, app Vm
The net effect of non competitive inhibition is a
reduction in Vm.
High substrate concentration would not
overcome noncompetitive inhibition. Other
reagents need to be added to block the binding if
inhibitor to the enzyme.
In some forms of non competitive inhibition Vm
is reduced and Km is increased. This occurs if the
complex ESI can form product.
Inhibited Enzyme Kinetics
3. Uncompetitive inhibitors (I)
- have no affinity for the enzyme itself
- bind only to the ES complex

Assume rapid equilibrium,

Km
k2
E+S ES P E
+
I
KI
ESI
Inhibited Enzyme Kinetics

d[ P]
v k 2 [ ES]
dt
' k 1 [ E ][ S ]
Km
k1 [ ES ]
[ ES ][ I ]
KI
[ ESI ]

[E0 ] [E] [ES] [ESI]

 Inhibited Enzyme Kinetics

(Vm /(1 [ I ] / K I ))[ S ]

v
' /(1 [ I ] / K )) [S ]
(K m I

Vm, app[ S ]
v
' , app [ S ]
Km

' ' /(1 [ I ] / K )

Vm, app Vm /(1 [ I ] / K I ) Km , app K m I
' '
K m, app / Vm, app K m / Vm
The net effect of uncompetitive inhibition is a reduction in
both Vm and Km values.
Reduction in Vm has a more pronounced effect than the
reduction in Km, and the net result is a reduction in
reaction rate.
Uncompetitive inhibition occurs when the inhibitor (I)
binds only to the enzyme-substrate complex (ES) and not
free E. One can hypothesize that on binding the substrate
(S), a conformational change in E occurs which presents a
binding site for I.
Inhibition occurs since ESI can not form product. It is a
deadend complex which has only one fate: to return to ES.
This is illustrated in the mechanism at the top of the figure
below and in the molecular cartoon beneath it.
Substrate Inhibition
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors
- can cause inhibition at high substrate
concentration
- bind only to the ES complex
Assume rapid equilibrium,
Km
k2
E+S ES P E
+
S
K SI
ES2
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)
d[ P]
v k 2 [ ES]
dt
' k 1 [ E ][ S ]
Km
k1 [ ES ]
[ ES ][ S ]
K SI
[ ES 2]

[E0 ] [E] [ES] [ES 2]

Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)
we can obtain,
Vm [ S ]
v
' [ S ] [ S ]2 / K
Km SI
At low substrate concentration [S ]2 / K SI <<1
Vm [ S ]
v Michaelis-Menten Equation
Km' [S ]
At high substrate concentration ' /[S ] 1
Km
Vm
v
1 [ S ] / K SI
Inhibited Enzyme Kinetics
Uncompetitive substrate inhibitors (I)

The substrate concentration resulting in the maximum

reaction rate can be determined by setting dv/d[S]=0,
[S]max is given by

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

Determine the type of inhibition.

Determine the parameters for Michaelis-
Menten equation without inhibition.
Determine the parameter of KI for inhibited
kinetics.
 Summary of Inhibited Kinetics
For reversible enzyme inhibition, there are
- competitive
- noncompetitive
- uncompetitive
- substrate inhibition

Determine parameters for all these types of

inhibition kinetics.
 Factors affecting 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

2007 Paul Billiet ODWS

 Substrate concentration: Non-enzymic reactions

Reaction
velocity

Substrate concentration

The increase in velocity is proportional to the

substrate concentration
2007 Paul Billiet ODWS
 Substrate concentration: Enzymic reactions

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

2007 Paul Billiet ODWS

pH
 The effect of pH
Extreme pH levels will produce denaturation
The structure of the enzyme is changed
The active site is distorted and the substrate
molecules will no longer fit in it
At pH values slightly different from the enzymes
optimum value, small changes in the charges of the
enzyme and its substrate molecules will occur
This change in ionisation will affect the binding of the
substrate with the active site.

2007 Paul Billiet ODWS

 The effect of temperature
Q10 (the temperature coefficient) = the increase in
reaction rate with a 10C rise in temperature.
For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with every
10C rise in temperature)
Enzyme-controlled reactions follow this rule as they
are chemical reactions
BUT at high temperatures proteins denature
The optimum temperature for an enzyme controlled
reaction will be a balance between the Q10 and
denaturation.

2007 Paul Billiet ODWS

 The effect of temperature

Q10 Denaturation
Enzyme
activity

0 10 20 30 40 50
Temperature / C

2007 Paul Billiet ODWS

 The effect of temperature
For most enzymes the optimum temperature is
about 30C
Many are a lot lower,
cold water fish will die at 30C because their
enzymes denature
A few bacteria have enzymes that can withstand very
high temperatures up to 100C
Most enzymes however are fully denatured at 70C

2007 Paul Billiet ODWS