CP504 PPT Set 03 DeterminationOfKineticParameters EnzymeReactions OK

CP504 ppt_Set 03
Enzyme kinetics and associated reactor design:
Determination of the kinetic parameters of enzyme-induced reactions

- learn about the meaning of kinetic parameters - learn to determine the kinetic parameters - learn the effects of pH, temperature and substrate concentration on enzyme activity (or reaction rates) - learn about inhibited enzyme kinetics - learn about allosteric enzymes and their kinetics
Prof. R. Shanthini Updated: 23 Nov 2012
Simple Enzyme Kinetics (in summary)

E+S
k1 k2 which is equivalent to
ES
k3
E+P
[E]
S
S E ES
for substrate (reactant) for enzyme for enzyme-substrate complex
P
for product

[E]
P
rmaxCS
rP = - rS =
KM + CS
where rmax = k3CE0 = kcatCE0
and KM = f(rate constants)

rmax is proportional to the initial concentration of the enzyme
KM is a constant
Simple Enzyme Kinetics (in summary) -rs

rmax rmaxCS KM + CS
uncatalyzed reaction Catalyzed reaction
rmax 2
- rS =
KM
Cs
An exercise
Consider an industrially important enzyme, which catalyzes the conversion of a protein substrate to form a much more valuable product. The enzyme follows the Briggs-Haldane mechanism:
An initial rate analysis for the reaction in solution, with E0 = 0.10 M and various substrate concentrations S0, yields the following Michaelis-Menten parameters: Vmax = 0.60 M/s; KM = 80 M. A different type of experiment indicates that the association rate constant, k1, is k1 = 2.0 x 106 M-1s-1 (2.0 M-1s-1). a. Estimate the values of k2 and k-1.
b. On average, what fraction of enzyme-substrate binding events result in product formation?

Source: Jason Haugh, Department of Chemical & Biomolecular Engineering, North Carolina State University
Catalytic step
E+S
k1
ES
k3
E+P
k2
Substrate binding step
k3 = kcat
How to determine the kinetic parameters rmax and KM ?

Carry out an enzyme catalysed experiment, and measure the substrate concentration (CS) with time.
t 0 10
15
Cs given given
given
- rs given given
given
- rS =
rmaxCS KM + CS
How to determine the M-M kinetics rmax and KM ?

Carry out an enzyme catalysed experiment, and measure the substrate concentration (CS) with time.
t 0 10
15
Cs given given
given
- rs given given
given
- rS =
rmaxCS KM + CS
We could rearrange
- rS =
rmaxCS KM + CS
to get the following 3 linear forms:
CS - rS
1 - rS - rS
KM rmax
1
1 rmax
KM rmax KM
CS
1 CS - rS CS
(14)
rmax
(15)
rmax -
(16)
The Langmuir Plot CS

- rS CS - rS rmax - KM
KM
rmax
1
rmax
CS
(14)
CS
The Langmuir Plot CS

- rS CS - rS rmax - KM
KM
rmax
1
rmax
CS
(14)
Determine rmax more accurately than the other plots. CS
The Lineweaver-Burk Plot

1 - rS 1 - rS rmax KM 1
rmax
KM rmax
1 CS
(15)
1 - KM
1 CS

1 - rS 1 - rS 1
rmax
KM rmax
1 CS
(15)
1 - KM
- Gives good estimates of rmax, but not necessarily KM KM - Data points at low substrate rmax concentrations influence the slope and intercept more than data points at high Cs 1 CS
The Eadie-Hofstee Plot - rS

= rmax -
KM
- rS
CS
(16)
- rS
KM
rmax
KM
-rS CS
The Eadie-Hofstee Plot - rS

= rmax -
KM
- rS
CS
(16)
- rS - Can be subjected to large errors since both coordinates contain (-rS) KM - Less bias on point at low Cs than rmax with Lineweaver-Burk plot K
M
-rS CS
Data:
CS
(mmol/l)
- rS
-(mmol/l.min)
1 2 3
0.20 0.22 0.30
5
7 10
0.45
0.41 0.50
Determine the M-M kinetic parameters for all the three methods discussed in the previous slides.
25 20
The Langmuir Plot
CS/(-rS) min
15 10 5 0 0 2 4 6 CS (mmol/l) 8 10 y = 1.5866x + 4.6417 R2 = 0.9497
rmax = 1 / slope = 1 / 1.5866 = 0.63 mmol/l.min

Prof. R. Shanthini M Updated: 23 Nov 2012
K = rmax x intercept = 0.63 x 4.6417 = 2.93 mmol/l
1/(-rS) l.min/mmol
5 4 3 2 1 0 0 0.2 0.4 0.6 1/CS l/mmol 0.8 1 y = 3.4575x + 1.945 R2 = 0.8463
rmax = 1 / intercept = 1 / 1.945 = 0.51 mmol/l.min

Prof. R. Shanthini M Updated: 23 Nov 2012
K = rmax x slope = 0.51 x 3.4575 = 1.78 mmol/l
0.6
The Eadie-Hofstee Plot

y = -1.8923x + 0.5386 2 R = 0.6618
(-rS) mmol/l.min
0.5 0.4 0.3 0.2 0.1 0 0 0.05
0.1 0.15 (-rS)/CS per min
0.2
0.25
rmax = intercept = 0.54 mmol/l.min

KM = - slope = 1.89 mmol/l
Comparison of the results
The Langmuir Plot rmax

KM R2
The LineweaverBurk Plot
The EadieHofstee Plot
rmax
KM R2
The Langmuir Plot 0.63

2.93 94.9%
The LineweaverBurk Plot 0.51

1.78 84.6%
The EadieHofstee Plot 0.54

1.89 66.2%
rmax
KM R2
The Langmuir Plot 0.63

2.93 94.9%
Determine rmax more accurately than the other plots
The LineweaverBurk Plot 0.51

1.78 84.6%
Gives good estimates of rmax, but not necessarily KM
The EadieHofstee Plot 0.54

1.89 66.2%
Can be subjected to large errors
http://www.youtube.com/watch?v=D2j2KGwJXJc
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 Prof. R. Shanthini temperatures, the enzyme activity rapidly falls to zero. Updated: 23 Nov 2012

Denaturation for most human enzymes:
The optimum temperature for most human enzymes to work at is around 37C which is why this temperature is body temperature. Enzymes start to denature at about 45C. Optimal for most human enzymes
http://www.woisd.net/moodle/mod/resource/view.php?id=44

Optimal for most human enzymes Reaction rate Optimal for some thermophillic bacterial enzymes
Temperature (deg C)
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 (a stomach enzyme) Reaction rate Optimal for trypsin (an intestinal enzyme)
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/hydrolysis of starch into soluble sugars that can readily be digested and metabolised for energy generation.
Amylase (malt) enzyme

Optimum pH: 4.6 - 5.2 Function: Catalyzes the breakdown/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 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.

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:
Effect of shear
Complex enzyme 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.
E+S E+I
where
k1 k2 k4 k5
ES EI
k3
E+P
rP = k3 CES
CE0 = CE + CES + CEI
(17) (18)
Assuming rapid equilibrium, we get
k1 CE CS = k2 CES
KM = k2 k1
CE CS
CES
(19)
k4 CE CI = k5 CEI
KI =
k5
k4
CE CI CEI
(20)
Combining (17) to (20), we get
rP =
where
k3CE0CS KM (1 + CI / KI) + CS rmax = k3CE0
rmaxCS KM,app + CS (5) (22) (6)
(21)
KM,app = KM (1 + CI / KI) KM = k2 / k1
KM,app > KM
The Lineweaver-Burk Plot 1 - rS CI > 0
1 1 - KM
CI = 0 (no inhibitor) 1 rmax 1 CS
- KM, app
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 (acetate) radical and a hydrogen atom.

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
E+S E+I
k1 k2 k4 k5
ES EI
k3
E+P
EI + S
k6
k7
ESI
ES + I
k8 k9
ESI
We could drive the rate equation (given on the next page) assuming the following:
k2
k1 k5
k4
= KM =
k7
k6 k9
k8
= KIM
= KI =
= KMI
rmax,appCS
rP =
where
KM + CS
rmax (1 + CI / KI)
(23)
rmax,app =
(24) (5) (6)
rmax = k3CE0 KM = k2 / k1
rmax,app < rmax
Non-competitive inhibition The Lineweaver-Burk Plot

1 - rS 1 CI > 0
rmax,app
1 - KM
CI = 0 (no inhibitor) 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
E+S ES + I
k1 k2
k4
ES ESI
k3
E+P
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 =
where
KM + CS
rmax (1 + CI / KI)
(23)
rmax,app =
(24) (5) (6)
rmax = k3CE0 KM = k2 / k1
rmax,app < rmax
rP =
where
rmax,appCS KM,app + CS rmax (1 + CI / KI)
(25)
rmax,app =
(24) rmax,app < rmax (26) KM,app < KM (5) (6)
KM,app = KM / (1 + CI / KI) rmax = k3CE0 KM = k2 / k1

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
The Lineweaver-Burk Plot 1 - rS 1 rmax,app 1 - KM, app 1 1 Prof. R. Shanthini - K Updated: 23 Nov 2012 M 1 CS CI > 0
CI = 0 (no inhibitor)
rmax
Competitive versus Uncompetitive inhibition
Mixed inhibition
An 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 M. From these experiments, it is determined that
Vmax = 1.20 M/s; KM = 40 M. 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 M/s, and 20 M, respectively. Speculate on how this inhibitor works (i.e., specify which species are engaged by the inhibitor).
Source: Jason Haugh, Department of Chemical & 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
Assignment
Get the rate equations for substrate and product inhibition
Food for Thought

Problem 3.13 from Shuler & Kargi: The following substrate reaction rate (-rS) data were obtained from enzymatic oxidation of phenol by phenol oxidase at different phenol concentrations (CS). By plotting (-rS) versus (CS) curve, or otherwise, determine the type of inhibition described by the data provided? CS (mg/l) 10 20 30 50 60 -rS (mg/l.h) 5 7.5 10 12.5 13.7
80 90 110 130 140 150
15 15 21.5 9.5 7.5 5.7
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:
rP =
Hill constant
rmaxCSn K + CS n
(27) The Hill

Hill coefficient
equation
n = 1 gives Michaelis-Menten kinetics
n > 1 gives positive cooperativity

n < 1 gives negative cooperativity
Prof. R. Shanthini Updated: 23 Nov 2012 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 (25) in a linear form as follows:
rP rmax
CSn
K + CSn (28)
ln
1-
= n ln(CS) ln (K)
Allosteric enzyme
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CP504 ppt_Set 03

Enzyme kinetics and associated reactor design:

Determination of the kinetic parameters of enzyme-induced reactions

Simple Enzyme Kinetics (in summary)

for substrate (reactant) for enzyme for enzyme-substrate complex

Simple Enzyme Kinetics (in summary)

where rmax = k3CE0 = kcatCE0

and KM = f(rate constants)

Simple Enzyme Kinetics (in summary) -rs

b. On average, what fraction of enzyme-substrate binding events result in product formation?

Simple Enzyme Kinetics (in summary)

Prof. R. Shanthini Updated: 23 Nov 2012

Prof. R. Shanthini Updated: 23 Nov 2012

How to determine the kinetic parameters rmax and KM ?

Prof. R. Shanthini Updated: 23 Nov 2012

How to determine the M-M kinetics rmax and KM ?

Prof. R. Shanthini Updated: 23 Nov 2012

to get the following 3 linear forms:

The Langmuir Plot CS

The Langmuir Plot CS

Determine rmax more accurately than the other plots. CS

The Lineweaver-Burk Plot

The Lineweaver-Burk Plot

The Eadie-Hofstee Plot - rS

Prof. R. Shanthini Updated: 23 Nov 2012

The Eadie-Hofstee Plot - rS

Prof. R. Shanthini Updated: 23 Nov 2012

0.20 0.22 0.30

Prof. R. Shanthini Updated: 23 Nov 2012

The Langmuir Plot

15 10 5 0 0 2 4 6 CS (mmol/l) 8 10 y = 1.5866x + 4.6417 R2 = 0.9497

rmax = 1 / slope = 1 / 1.5866 = 0.63 mmol/l.min

K = rmax x intercept = 0.63 x 4.6417 = 2.93 mmol/l

The Lineweaver-Burk Plot

5 4 3 2 1 0 0 0.2 0.4 0.6 1/CS l/mmol 0.8 1 y = 3.4575x + 1.945 R2 = 0.8463

rmax = 1 / intercept = 1 / 1.945 = 0.51 mmol/l.min

K = rmax x slope = 0.51 x 3.4575 = 1.78 mmol/l

The Eadie-Hofstee Plot

0.5 0.4 0.3 0.2 0.1 0 0 0.05

0.1 0.15 (-rS)/CS per min

rmax = intercept = 0.54 mmol/l.min

KM = - slope = 1.89 mmol/l

Comparison of the results

The Langmuir Plot rmax

The LineweaverBurk Plot

The EadieHofstee Plot

Prof. R. Shanthini Updated: 23 Nov 2012

Comparison of the results

The Langmuir Plot 0.63

The LineweaverBurk Plot 0.51

The EadieHofstee Plot 0.54

Prof. R. Shanthini Updated: 23 Nov 2012

Comparison of the results

The Langmuir Plot 0.63

The LineweaverBurk Plot 0.51

The EadieHofstee Plot 0.54

Prof. R. Shanthini Updated: 23 Nov 2012

Prof. R. Shanthini Updated: 23 Nov 2012

Effects of temperature on enzyme activity:

Prof. R. Shanthini Updated: 23 Nov 2012

Effects of temperature on enzyme activity:

Prof. R. Shanthini Updated: 23 Nov 2012

Effects of temperature on enzyme activity: