2022 - RS - Lectures03and04 - Enzyme Kinetics - Advanced

2022_RS_Lectures 03 and 04
Enzyme kinetics:
Influences on enzyme activity
(pH, temperature, shear);
Inhibition of enzyme reactions
(competitive/non-competitive/un-competitive);
Industrial applications of enzymes.
Prof. R. Shanthini 1
Simple Enzyme Kinetics (in summary)
[E]
S P
rmaxCS
rP = - r S =
KM + CS
where rmax = k3CE0 = KcatCE0

and KM = f(rate constants)
rmax is proportional to the enzyme concentration
KM is a constant
Kcat is the turnover number (independent of enzyme concentration)

- learn the effects of pH, temperature and substrate
concentration on enzyme activity (or reaction rates)
http://www.youtube.com/watch?v=D2j2KGwJXJc
(accessed on 09 Oct 2022)
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
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm
More energetic collisions:
When molecules collide, the kinetic energy of the molecules can
be converted into chemical potential energy of the molecules.
If the chemical potential energy of the molecules become great
enough, the activation energy of a exergonic reaction can be
achieved and a change in chemical state will result.
Thus the greater the kinetic energy of the molecules in a system,
the greater is the resulting chemical potential energy when two
molecules collide.
As the temperature of a system is increased it is possible that
more molecules per unit time will reach the activation energy.
Thus the rate of the reaction may increase.
Increase in the number of collisions per unit time:
In order to convert substrate into product, enzymes must collide
with and bind to the substrate at the active site.
Increasing the temperature of a system will increase the
number of collisions of enzyme and substrate per unit time.
Thus, within limits, the rate of the reaction will increase.
Denaturation of the enzyme:
Enzymes are very large proteins whose three dimensional shape is
vital for their activity.
When proteins are heated up too much they vibrate.
If the heat gets too intense then the enzymes literally shake
themselves out of shape, and the structure breaks down.
The enzyme is said to be denatured.
A denatured enzyme does not have the correct 'lock' structure.
Therefore it cannot function efficiently by accepting the 'key'
substrate molecule.
http://www.woisd.net/moodle/mod/resource/view.php?id=44
As temperature increases, enzyme activity increases

until its optimum temperature is reached. At higher
temperatures, the enzyme activity rapidly falls to zero.
Denaturation for most human enzymes:
The optimum
Optimal for most
temperature for most
human enzymes
human enzymes to
work at is around
37ºC which is why
this temperature is
body temperature.
Enzymes start to
denature at about
45°C.
http://www.woisd.net/moodle/mod/resource/view.php?id=44
Optimal for most Optimal for some

human enzymes thermophillic
bacterial
enzymes
Reaction rate
Prof. R. Shanthini
Temperature (deg C) 11
https://wikispaces.psu.edu/display/230/Enzyme+Kinetics+and+Catalysis
Effects of pH on enzyme activity:
The structure of the protein enzyme can depends on how

acid or alkaline the reaction medium is, that is, it is pH
dependent.
If it is too acid or too alkaline, the structure of the protein is
changed and it is 'denatured' and becomes less effective.
If the enzyme does not have the correct 'lock' structure, it
cannot function efficiently by accepting the 'key' substrate
molecule.
In the optimum pH range, the enzyme catalysis is at its
most efficient.
Optimal for pepsin Optimal for trypsin

(a stomach (an intestinal
enzyme) enzyme)
Reaction rate
pH
https://wikispaces.psu.edu/display/230/Enzyme+Kinetics+and+Catalysis
Amylase (pancreas) enzyme
Optimum pH: 6.7 - 7.0
Function: A pancreatic enzyme that catalyzes the breakdown

(or hydrolysis) of starch into soluble sugars that can readily be
digested and metabolised for energy generation.
Amylase (malt) enzyme
Function: Catalyzes the breakdown (or hydrolysis) of starch

into soluble sugars in malt carbohydrate extracts.
www.docbrown.info/page01/ExIndChem/ExIndChema.htm
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)
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
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
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.
Maltase enzyme
Function: Breaks down malt sugars.
Pepsin enzyme
Function: Catalyses the breakdown/hydrolysis of proteins

into smaller peptide fragments.
Trypsin enzyme
Function: Catalyses the breakdown/hydrolysis of proteins

into amino acids. Note again, the significantly different
optimum pH to similarly functioning pepsin.
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)
Effects of substrate concentration on enzyme activity:
https://www.youtube.com/watch?v=zN_Jb9kvJ5A
(accessed on 09 Oct 2022)
Complex enzyme kinetics
- learn about inhibited enzyme kinetics

- learn about allosteric enzymes and their kinetics
Inhibited enzyme reactions
Inhibitors are substances that slow down the rate of enzyme
catalyzed 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.
Inhibited enzyme reactions - applications
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
Primary constituents of Snake Venom
Enzymes - Spur physiologically disruptive or destructive processes.
Proteolysins - Dissolve cells and tissue at the bite site, causing local
pain and swelling.
Cardiotoxins - Variable effects, some depolarise cardiac muscles and
alter heart contraction, causing heart failure.
Harmorrhagins - Destroy capillary walls, causing haemorrhages near
and distant from the bite.
Coagulation - Retarding compounds prevent blood clotting.
Thromboses - Coagulate blood and foster clot formation throughout
the circulatory system.
Haemolysis - Destroy red blood cells.
Cytolysins - Destroy white blood cells.
Neurotoxins - Block the transmission of nerve impulses to muscles,
especially those associated with the diaphragm and breathing.
http://www.writework.com/essay/biochemistry-snake-venom
Inhibited enzyme reactions
Inhibitors are also classified as competitive and non-competitive
inhibitors.
Competitive inhibition
- The structure of inhibitor molecule closely resembles the

chemical structure and molecular geometry of the substrate.
- The inhibitor competes for the same active site as the
substrate molecule.
- It does not alter the structure of the enzyme.
- The inhibitor may interact with the enzyme at the active site,
but no reaction takes place.
http://www.elmhurst.edu/~chm/vchembook/573inhibit.html
- The inhibitor is "stuck" on the enzyme and prevents any
substrate molecules from reacting with the enzyme.
- However, a competitive inhibition is usually reversible if
sufficient substrate molecules are available to ultimately
displace the inhibitor.
- Therefore, the amount of enzyme inhibition depends upon
the inhibitor concentration, substrate concentration, and the
relative affinities of the inhibitor and substrate for the active
site.
http://www.elmhurst.edu/~chm/vchembook/573inhibit.html
Competitive inhibitors (denoted by I) compete with substrate to
occupy the active site of the enzyme.
k1 k3
E+S ES E+P
k2
k4
E+I EI
k5
where
rP = k3 CES (17)
CE0 = CE + CES + CEI (18)
Assuming rapid equilibrium, we get
k1 CE CS = k2 CES
k2 CE CS
KM = = (19)
k1 CES
k4 CE CI = k5 CEI
k5 CE CI
KI = = (20)
k4 CEI
Combining (17) to (20), we get
k3CE0CS rmaxCS
rP = = (21)
KM (1 + CI / KI) + CS KM,app + CS
where
rmax = k3CE0 (5)
KM,app = KM (1 + CI / KI) (22)
KM = k2 / k1 (6)
Prof. R. Shanthini KM,app > KM 32

The Lineweaver-Burk Plot
1
CI > 0
- rS
1 CI = 0 (no inhibitor)
1 - KM, app
- KM 1
rmax
1
CS
In the presence of a competitive inhibitor,
the maximal rate of the reaction (rmax) is unchanged,
but the Michaelis constant (KM) is increased.
Competitive inhibition – an example
Ethanol is metabolized in the body by oxidation to acetaldehyde,
which is a toxic compound and a known carcinogen.
The enzyme alcohol dehydrogenase (ADH)

converts ethanol into acetaldehyde plus two
hydrogen atoms.
Acetaldehyde is generally short-lived; it is quickly broken down to
a less toxic compound called acetate in a rapid reaction so that
acetaldehyde does not accumulate in the body.
.
The enzyme aldehyde dehydrogenase

(ALDH) converts acetaldehyde to acetyl
Prof. R. Shanthini (acetate) radical and a hydrogen atom. 36
A drug, disulfiram (Antabuse), inhibits the aldehyde
dehydrogenase.
Such inhibition results in the accumulation of acetaldehyde in
the body.
High levels of acetaldehyde act directly on the heart and blood
vessels, causing flushing, a racing heartbeat and a drop in
blood pressure that causes dizziness. Other unpleasant
symptoms include headache, shortness of breath, palpitations,
nausea and vomiting.
This drug is sometimes used to help people overcome the
drinking habit.
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 complete 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.
https://ibhumanbiochemistry.wikispaces.com/C.7.5
- Non competitive inhibitors are usually reversible,

- but are not influenced by concentrations of the substrate as
is the case for a reversible competitive inhibitor.
https://ibhumanbiochemistry.wikispaces.com/C.7.5
k1 k3
E+S ES E+P
k2
k4
E+I EI
k5
k6
EI + S ESI
k7
k8
ES + I ESI
k9
We could drive the rate equation (given on the next page)
assuming the following:
k2 k7
= KM = = KIM
k1 k6
k5 k9
= KI = = KMI
k4 k8
Exercise: Drive the rate equation (given on the next page)
rmax,appCS
rP = (23)
KM + CS
where
rmax
rmax,app = (24)
(1 + CI / KI)
rmax = k3CE0 (5)
KM = k2 / k1 (6)
Prof. R. Shanthini rmax,app < rmax 42

1
CI > 0
- rS
1
rmax,app
CI = 0 (no inhibitor)
1
- KM 1
rmax
1
CS
In the presence of a non-competitive inhibitor,
the maximal rate of the reaction (rmax) is lower
but the Michaelis constant (KM) is unchanged.
Uncompetitive inhibition
k1 k3
E+S ES E+P
k2
k4
ES + I ESI
k5
Inhibitor can only bind to the

enzyme-substrate complex,
reversibly forming a
nonproductive complex.
An uncompetitive inhibitor binds only to the enzyme-substrate
complex preventing the formation or release of the enzymatic
products.
Unlike with competitive inhibition an uncompetitive inhibitor
need not resemble the structure of the enzymes natural
substrate.
An uncompetitive inhibitor is most effective at high substrate
concentration as there will be more enzyme-substrate
complex for it to bind.
Unlike with competitive inhibitors the effects of an
uncompetitive inhibitor cannot be overcome by increasing the
concentration of substrate.
rmax,appCS
rP = (25)
KM,app + CS
where
rmax
rmax,app = (24) rmax,app < rmax
(1 + CI / KI)
KM,app = KM / (1 + CI / KI) (26) KM,app < KM
rmax = k3CE0 (5)
KM = k2 / k1 (6)
KM is reduced
rmax is also reduced
This is because the total ‘pool’ of enzymes available to react

has been reduced, effectively our enzyme concentration has
reduced.
Can be explained by rmax = k3CE0 = kcatCE0
1
- rS CI > 0
1
rmax,app
CI = 0 (no inhibitor)
1
- KM, app
1 1
1 rmax CS
Prof. R. Shanthini - KM 49
Competitive versus Uncompetitive inhibition
Exercise:
The kinetic properties of the ATPase enzyme, isolated from yeast,
which catalyzes the hydrolysis of ATP to form ADP and Pi, are
assessed by measuring initial rates in solution, with various ATP
concentrations S0 and a total ATPase concentration E0 = 0.60 μM.
From these experiments, it is determined that
Vmax = 1.20 μM/s; KM = 40 μM.
a. Calculate the values of kcat and the catalytic efficiency for ATPase
under these conditions.
b. An inhibitor molecule is added at a concentration of 0.1 mM, and
the experiments are repeated. The apparent Vmax and KM are now
found to be 0.6 μM/s, and 20 μM, respectively. Speculate on how
this inhibitor works (i.e., specify which species are engaged by the
inhibitor).
Prof. R. Shanthini Source: Jason Haugh, Department of Chemical & 51

Biomolecular Engineering, North Carolina State University
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.
Substrate / Product inhibition
Exercise:
Get the rate equations for substrate and product inhibition
“Food for Thought”
Problem 3.13 from Shuler & Kargi: CS -rS
The following substrate reaction rate (-rS) (mg/l) (mg/l.h)
data were obtained from enzymatic 10 5
oxidation of phenol by phenol oxidase at 20 7.5
different phenol concentrations (CS). 30 10
By plotting (-rS) versus (CS) curve, or
otherwise, determine the type of inhibition 50 12.5
described by the data provided? 60 13.7
80 15
90 15
110 21.5
130 9.5
140 7.5
Prof. R. Shanthini 150 5.755
Sigmoid/Hill kinetics
A particular class of enzymes exhibit kinetic properties that
cannot be studied using the Michaelis-Menten equation.
The rate equation of these unique enzymes is characterized

by Sigmoid/Hill kinetics as follows:
rmaxCSn
rP = (27) The Hill
K + C Sn equation
Hill constant Hill coefficient
n = 1 gives Michaelis-Menten kinetics

n > 1 gives positive cooperativity
n < 1 gives negative cooperativity
http://chemwiki.ucdavis.edu/Biological_Chemistry/Catalysts/Enzymatic_Kinetics/Sigmoid_Kinetics
Sigmoid/Hill kinetics
Examples of the “S-shaped” sigmoidal/Hill curve, which is
different from the hyberbolic curve of M-M kinetics.
n=6
n=4
n=2
Sigmoid kinetics
For an alternative formulation of Hill equation, we could
rewrite (27) in a linear form as follows:
rP CSn
θ = =
rmax K + CSn
θ
ln = n ln(CS) – ln (K) (28)
1-θ
Allosteric enzyme
https://www.youtube.com/watch?v=UWrJssS76XE
https://www.youtube.com/watch?v=ttiduKb_DQ4
https://www.youtube.com/watch?v=6qvIPMzS_iY

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2022_RS_Lectures 03 and 04

where rmax = k3CE0 = KcatCE0

rmax is proportional to the enzyme concentration

Kcat is the turnover number (independent of enzyme concentration)

Increases in the temperature of a system results from

Kinetic energy increase has the following effects on the

As temperature increases, enzyme activity increases

Optimal for most Optimal for some

The structure of the protein enzyme can depends on how

Optimal for pepsin Optimal for trypsin

Amylase (pancreas) enzyme

Optimum pH: 6.7 - 7.0

Function: A pancreatic enzyme that catalyzes the breakdown

Amylase (malt) enzyme

Optimum pH: 4.6 - 5.2

Function: Catalyzes the breakdown (or hydrolysis) of starch

Optimum pH: ~7.0

Function: Catalyses the breakdown of potentially harmful

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

Optimum pH: 4.5

Function: Catalyses the breakdown/hydrolysis of sucrose

C12H22O11 + H2O ==> C6H12O6 + C6H12O6

Lipase (pancreas) enzyme

Optimum pH: ~8.0

Function: Lipases catalyse the breakdown dietary fats, oils,

Lipase (stomach) enzyme

Optimum pH: 4.0 - 5.0

Function: As above, but note the significantly different

Optimum pH: 6.1 - 6.8

Function: Breaks down malt sugars.

Optimum pH: 1.5 - 2.0

Function: Catalyses the breakdown/hydrolysis of proteins

Optimum pH: 7.8 - 8.7

Function: Catalyses the breakdown/hydrolysis of proteins

Optimum pH: ~7.0

Function: Catalyzes the breakdown of urea into ammonia

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

- learn about inhibited enzyme kinetics

- The structure of inhibitor molecule closely resembles the

CE0 = CE + CES + CEI (18)

Assuming rapid equilibrium, we get

Combining (17) to (20), we get

KM,app = KM (1 + CI / KI) (22)

Prof. R. Shanthini KM,app > KM 32

The enzyme alcohol dehydrogenase (ADH)

The enzyme aldehyde dehydrogenase

- Non competitive inhibitors are usually reversible,

Exercise: Drive the rate equation (given on the next page)

rmax = k3CE0 (5)

Prof. R. Shanthini rmax,app < rmax 42

Inhibitor can only bind to the

KM,app = KM / (1 + CI / KI) (26) KM,app < KM

rmax = k3CE0 (5)

rmax is also reduced

This is because the total ‘pool’ of enzymes available to react

Can be explained by rmax = k3CE0 = kcatCE0

Prof. R. Shanthini Source: Jason Haugh, Department of Chemical & 51

Get the rate equations for substrate and product inhibition

The rate equation of these unique enzymes is characterized

n = 1 gives Michaelis-Menten kinetics

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