- learn about inhibited enzyme kinetics

- learn about allosteric enzymes and their kinetics

S
 COMPLEX ENZYME KINETICS
Inhibition

o The prevention of an enzyme process as a result of

interaction of inhibitors with the enzyme.
INHIBITORS:
Any substance that can diminish the
velocity of an enzyme catalyzed reaction is called an
inhibitor.

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 Types of inhibition

Inhibition

Reversible Irreversible

Competitive Uncompetitive Mixed Non-competitive

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 Inhibited enzyme reactions
There are two distinct types of inhibitors:
- Irreversible inhibitors form a stable complex with enzymes
and reduce enzyme activity (e.g. lead, cadmium,
organophosphorous pesticide)
- Reversible inhibitors interact more loosely with enzymes
and can be displaced.

Many drugs and poisons are inhibitors of enzymes in the

nervous system.
Poisons: snake bite, plant alkaloids and nerve gases
Medicines: antibiotics, sulphonamides, sedatives and
stimulants
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 Competitive inhibition

In this type of inhibition, the inhibitors compete with the

substrate for the active site. Formation of E.S complex is
reduced while a new E.I complex is formed.

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 competes with the
substrate for the active
site of an enzyme.
inhibitor (I) occupies
the active site and it
prevents binding of the
substrate to the enzyme.
compounds that
resemble the substrate
and combine with the
enzyme to form an EI
complex.

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Assuming rapid equilibrium and with the definition of

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Km,app > Km
Maximum rate of reaction is unchanged
Increases Km by a factor (1+[I]/KI) i. e. the affinity of the
enzyme to the substrate decreases and also reduces reaction
rate.
The net effect of competitive inhibition is an increased value of
Km app and, therefore it can be overcome by high
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concentration of substrate.
 Non-competitive inhibition

- The structure of inhibitor molecule is entirely different from

that of the substrate molecule.
- The inhibitor forms complex at a point other than the active
site (remote from or very close to the active site).
- It does not compete with the substrate.
- It alters the structure of the enzyme in such a way that the
substrate can no longer interact with the enzyme to give a
reaction.

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Non-competitive inhibition

We could drive the rate

equation (given on the
K3 next page) assuming the
K-3 following:

K-1 K-3
= KM =
k1 k3

KI Same for both cases

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With the definition of

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Non-competitive inhibition
In the presence of a non-competitive inhibitor,
the maximal rate of the reaction (Vmax) is lower
but the Michaelis constant (KM) is unchanged.

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The net effect of noncompetitive inhibition is in
reduction in Vm, therefore, high concentration of
substrate would not overcome noncompetetitive
inhibition.
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Uncompetitive inhibition

In this type of inhibition, inhibitor does not compete

with the substrate for the active site of enzyme instead
it binds to another site known as allosteric site.
Binds to the ES complex only and have no affinity for the
enzyme

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k1
k-1 k2

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The net effect of uncompetitive inhibition is in reduction
of both Vm and Km, therefore, net result in reduction of
reaction rate.

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Substrate / Product inhibition

Either the substrate or product of an enzyme reaction

inhibit the enzyme's activity.
This inhibition may follow the competitive, uncompetitive or
mixed patterns.
In substrate inhibition there is a progressive decrease in
activity at high substrate concentrations.
Product inhibition is often a regulatory feature in
metabolism and can be a form of negative feedback.

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Substrate / Product inhibition

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UNCOMPETITIVE SUBSTRATES
Uncompetitive Substrates Inhibition
Inhibition

At low substrate concentrations, [S]2/Ks1 1, and

inhibition effect is not observed. The rate is

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At high substrate concentrations, Km[S] 1, and inhibition is
dominant. The rate in this case is

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21
 Optimum Substrate Concentration

The substrate concentration resulting in the maximum

reaction rate can be determined by setting dv/d[S] = 0.
The [S]max or [S]opt is given by

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Competitive versus Uncompetitive inhibition

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 Allosteric Enzymes

Some enzymes have more than one substrate binding

site.

The binding of one substrate to the enzyme facilitates

binding of other substrate molecules.

This behavior is known as allostery or cooperative

binding, and regulatory enzymes show this behavior.

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 Allosteric Enzymes Kinetics
Kinetic properties cannot be studied by using the Michaelis-
Menten equation.

The rate equation of these unique enzymes is

characterized by Sigmoid/Hill kinetics as follows:

The Hill
equation
Hill constant Hill or cooperative
coefficient
n = 1 gives Michaelis-Menten kinetics
n > 1 gives positive cooperativity
n < 1 gives negative cooperativity
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http://chemwiki.ucdavis.edu/Biological_Chemistry/Catalysts/Enzymatic_Kinetics/Sigmoid_Kinetics
Allosteric Enzymes Kinetics
Examples of the S-shaped sigmoidal/Hill curve, which is
different from the hyberbolic curve of M-M kinetics.

[S]=Km [S]n=Km
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 Allosteric Enzymes Kinetics
For an alternative formulation of Hill equation, we could
rewrite it in a linear form as follows:

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Example 1
An inhibitor (I) is added to the enzymatic reaction at a level of 1.0
g/l. The following data were obtained for Km=9.2 g S/l.
a. Is the inhibitor competitive or noncompetitive?
b. Find KI

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29
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 Effects of pH on enzyme activity:
Three dimensional structure of proteins is based on the interaction
between R groups

R groups can carry a charge: Asp (-), Glu (-), Cys (-), Tyr (-),
His (-), Arg (+), Lys (+)

Terminal groups can carry a charge: (-COOH-, -NH3+)

It makes sense that pH can cause a change in enzyme activity

pH of medium may affect vmax, Km, and enzyme stability.

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 Effects of pH on enzyme activity:

Optimal for pepsin Optimal for trypsin

(a stomach (an intestinal
enzyme) enzyme)
Reaction rate

pH
Prof. R. Shanthini 32
Updated: 23 Nov 2012 https://wikispaces.psu.edu/display/230/Enzyme+Kinetics+and+Catalysis
Effects of pH on enzyme activity:

Amylase (pancreas) enzyme

Optimum pH: 6.7 - 7.0

Function: A pancreatic enzyme that catalyzes the

breakdown/hydrolysis of starch into soluble sugars that can
readily be digested and metabolised for energy generation.

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www.docbrown.info/page01/ExIndChem/ExIndChema.htm
Effects of pH on enzyme activity:

Catalase enzyme

Optimum pH: ~7.0

Function: Catalyses the breakdown of potentially harmful

hydrogen peroxide to water and oxygen. Important in
respiration/metabolism chemistry.

2H2O2(aq) ==> 2H2O(l) + O2(g)

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www.docbrown.info/page01/ExIndChem/ExIndChema.htm
 Effects of pH on enzyme activity:

Invertase enzyme

Optimum pH: 4.5

Function: Catalyses the breakdown/hydrolysis of sucrose

into fructose + glucose, the resulting mixture is 'inverted
sugar syrup'.

C12H22O11 + H2O ==> C6H12O6 + C6H12O6

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www.docbrown.info/page01/ExIndChem/ExIndChema.htm
 EFFECT of pH on ENZYME KINETICS
Cause:
Some of the Enzyme has ionic groups on their active sites and this
ionic group must be in suitable form (acid/base) to function.
Variation of the pH indicates the results in changes in the ionic
forms.
Changes of pH also alter the shape of 3-D structure of enzyme.

Results:
Enzymes are only actives over certain pH ranges and medium may
affect the maximum reaction rate, Km and the stability of the
enzyme.

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Effect of pH on Enzyme Kinetics
(Ionizing Enzyme)

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We can derive the following rate expression:

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For the case of ionizing substrate, the following scheme and rate
expression can be developed:

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Effects of temperature on enzyme activity:

Increases in the temperature of a system results from

increases in the kinetic energy of the system.

Kinetic energy increase has the following effects on the

rates of reactions:
1) More energetic collisions
2) Increase in the number of collisions per unit time
3) Denaturation of the enzyme or substrate

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http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm
Effects of temperature on enzyme activity:
Denaturation of the enzyme:

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Effects of temperature on enzyme activity:
Denaturation for most human enzymes:
The optimum
Optimal for most
temperature for most
human enzymes
human enzymes to
work at is around
37C which is why
this temperature is
body temperature.

Enzymes start to
denature at about
40C.

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http://www.woisd.net/moodle/mod/resource/view.php?id=44
 Effects of temperature on enzyme activity:

Optimal for most Optimal for some

human enzymes thermophillic
bacterial
Reaction rate

enzymes

Temperature (deg C) 43
Prof. R. Shanthini
Updated: 23 Nov 2012 https://wikispaces.psu.edu/display/230/Enzyme+Kinetics+and+Catalysis
Effects of temperature on enzyme activity:
The rate of enzyme-catalyzed reactions increases with
temperature to a certain limit.
Above a certain temperature, enzyme activity decreases
with temperature because of enzyme denaturation.

The ascending part of Fig. 3.15 is

known as temperature activation.
The rate varies according to the
Arrhenius equation in this region.

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temperature inactivation or thermal
denaturation. The kinetics of thermal
denaturation can be expressed as

where [Eo] is the initial enzyme

concentration and kd is the
denaturation constant. kd also
varies with temperature according
to the Arrhenius equation

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 The activation energies of enzyme-catalyzed
reactions are within the 4 to 20 kcal/g mol range
(mostly about 11 kcal/g mol).

Deactivation energies Ed vary between 40 and

130 kcal/g mol (mostly about 70 kcal/g mol).

Affect both Vm and Km values of enzymes

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Notes

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48
Effects of pH on enzyme activity:

Lipase (pancreas) enzyme

Optimum pH: ~8.0

Function: Lipases catalyse the breakdown dietary fats, oils,

triglycerides etc. into digestible molecules in the human
digestion system.

Lipase (stomach) enzyme

Optimum pH: 4.0 - 5.0

Function: As above, but note the significantly different

optimum pH in the acid stomach juices, to optimum pH in
the alkaline fluids of the pancreas.
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www.docbrown.info/page01/ExIndChem/ExIndChema.htm
Effects of pH on enzyme activity:

Maltase enzyme

Optimum pH: 6.1 - 6.8

Function: Breaks down malt sugars.

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www.docbrown.info/page01/ExIndChem/ExIndChema.htm
Effects of pH on enzyme activity:

Pepsin enzyme

Optimum pH: 1.5 - 2.0

Function: Catalyses the breakdown/hydrolysis of proteins

into smaller peptide fragments.

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www.docbrown.info/page01/ExIndChem/ExIndChema.htm
Effects of pH on enzyme activity:

Trypsin enzyme

Optimum pH: 7.8 - 8.7

Function: Catalyses the breakdown/hydrolysis of proteins

into amino acids. Note again, the significantly different
optimum pH to similarly functioning pepsin.

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www.docbrown.info/page01/ExIndChem/ExIndChema.htm
Effects of pH on enzyme activity:

Urease enzyme

Optimum pH: ~7.0

Function: Catalyzes the breakdown of urea into ammonia

and carbon dioxide.

(NH2)2(aq) + H2O(l) ==> 2NH3(aq) + CO2(aq)

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