You are on page 1of 83

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
Prof. R. Shanthini Updated: 23 Nov 2012

for product

Simple Enzyme Kinetics (in summary)


[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
Prof. R. Shanthini Updated: 23 Nov 2012

Simple Enzyme Kinetics (in summary) -rs


rmax rmaxCS KM + CS
uncatalyzed reaction Catalyzed reaction

rmax 2

- rS =

KM
Prof. R. Shanthini Updated: 23 Nov 2012

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?


Prof. R. Shanthini Updated: 23 Nov 2012

Source: Jason Haugh, Department of Chemical & Biomolecular Engineering, North Carolina State University

Simple Enzyme Kinetics (in summary)

Catalytic step

E+S

k1

ES

k3

E+P

k2
Substrate binding step

k3 = kcat

Prof. R. Shanthini Updated: 23 Nov 2012

- 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

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

Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

We could rearrange

- rS =

rmaxCS KM + CS

to get the following 3 linear forms:

CS - rS
1 - rS - rS
Prof. R. Shanthini Updated: 23 Nov 2012

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
Prof. R. Shanthini Updated: 23 Nov 2012

KM
rmax

1
rmax

CS

(14)

CS

The Langmuir Plot CS


- rS CS - rS rmax - KM
Prof. R. Shanthini Updated: 23 Nov 2012

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
Prof. R. Shanthini Updated: 23 Nov 2012

1 CS

The Lineweaver-Burk Plot


1 - rS 1 - rS 1

rmax

KM rmax

1 CS

(15)

1 - KM
Prof. R. Shanthini Updated: 23 Nov 2012

- 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

Prof. R. Shanthini Updated: 23 Nov 2012

-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

Prof. R. Shanthini Updated: 23 Nov 2012

-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.

Prof. R. Shanthini Updated: 23 Nov 2012

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

The Lineweaver-Burk Plot

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


Prof. R. Shanthini Updated: 23 Nov 2012

KM = - slope = 1.89 mmol/l

Comparison of the results

The Langmuir Plot rmax


KM R2

The LineweaverBurk Plot

The EadieHofstee Plot

Prof. R. Shanthini Updated: 23 Nov 2012

Comparison of the results

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%

Prof. R. Shanthini Updated: 23 Nov 2012

Comparison of the results

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

Prof. R. Shanthini Updated: 23 Nov 2012

- 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

http://www.youtube.com/watch?v=D2j2KGwJXJc

Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

Effects of temperature on enzyme activity:


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.

Prof. R. Shanthini Updated: 23 Nov 2012

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

Effects of temperature on enzyme activity:


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.

Prof. R. Shanthini Updated: 23 Nov 2012

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm

Effects of temperature on enzyme activity:


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.

Prof. R. Shanthini Updated: 23 Nov 2012

http://www.woisd.net/moodle/mod/resource/view.php?id=44

Effects of temperature on enzyme activity:


Denaturation of the enzyme:

Prof. R. Shanthini Updated: 23 Nov 2012

Effects of temperature on enzyme activity:


Denaturation of the enzyme:

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

Effects of temperature on enzyme activity:


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

Prof. R. Shanthini Updated: 23 Nov 2012

http://www.woisd.net/moodle/mod/resource/view.php?id=44

Effects of temperature on enzyme activity:


Optimal for most human enzymes Reaction rate Optimal for some thermophillic bacterial enzymes

Prof. R. Shanthini Updated: 23 Nov 2012

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.

Prof. R. Shanthini Updated: 23 Nov 2012

Effects of pH on enzyme activity:


Optimal for pepsin (a stomach enzyme) Reaction rate Optimal for trypsin (an intestinal enzyme)

Prof. R. Shanthini Updated: 23 Nov 2012

pH
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.

Amylase (malt) enzyme


Optimum pH: 4.6 - 5.2 Function: Catalyzes the breakdown/hydrolysis of starch into soluble sugars in malt carbohydrate extracts.
Prof. R. Shanthini Updated: 23 Nov 2012

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)

Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

www.docbrown.info/page01/ExIndChem/ExIndChema.htm

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.
Prof. R. Shanthini Updated: 23 Nov 2012

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.

Prof. R. Shanthini Updated: 23 Nov 2012

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.

Prof. R. Shanthini Updated: 23 Nov 2012

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.

Prof. R. Shanthini Updated: 23 Nov 2012

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)

Prof. R. Shanthini Updated: 23 Nov 2012

www.docbrown.info/page01/ExIndChem/ExIndChema.htm

Effects of substrate concentration on enzyme activity:

Prof. R. Shanthini Updated: 23 Nov 2012

www.docbrown.info/page01/ExIndChem/ExIndChema.htm

Effect of shear

Prof. R. Shanthini Updated: 23 Nov 2012

Complex enzyme kinetics


- 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

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.

Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

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.
Prof. R. Shanthini Updated: 23 Nov 2012

http://www.writework.com/essay/biochemistry-snake-venom

Inhibited enzyme reactions


Inhibitors are also classified as competitive and non-competitive inhibitors.

Prof. R. Shanthini Updated: 23 Nov 2012

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.

Prof. R. Shanthini Updated: 23 Nov 2012

http://www.elmhurst.edu/~chm/vchembook/573inhibit.html

Competitive inhibition
- 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.

Prof. R. Shanthini Updated: 23 Nov 2012

http://www.elmhurst.edu/~chm/vchembook/573inhibit.html

Competitive inhibition
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)

Prof. R. Shanthini Updated: 23 Nov 2012

Competitive inhibition
Assuming rapid equilibrium, we get

k1 CE CS = k2 CES
KM = k2 k1

CE CS

CES

(19)

k4 CE CI = k5 CEI

KI =
Prof. R. Shanthini Updated: 23 Nov 2012

k5
k4

CE CI CEI

(20)

Competitive inhibition
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
Prof. R. Shanthini Updated: 23 Nov 2012

KM,app > KM

Competitive inhibition
The Lineweaver-Burk Plot 1 - rS CI > 0

1 1 - KM

CI = 0 (no inhibitor) 1 rmax 1 CS

- KM, app

Prof. R. Shanthini Updated: 23 Nov 2012

Competitive inhibition
In the presence of a competitive inhibitor,
the maximal rate of the reaction (rmax) is unchanged, but the Michaelis constant (KM) is increased.

Prof. R. Shanthini Updated: 23 Nov 2012

Competitive inhibition an example


Ethanol is metabolized in the body by oxidation to acetaldehyde, which is a toxic compound and a known carcinogen.

Prof. R. Shanthini Updated: 23 Nov 2012

The enzyme alcohol dehydrogenase (ADH) converts ethanol into acetaldehyde plus two hydrogen atoms.

Competitive inhibition an example


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. .

Prof. R. Shanthini Updated: 23 Nov 2012

The enzyme aldehyde dehydrogenase (ALDH) converts acetaldehyde to acetyl (acetate) radical and a hydrogen atom.

Competitive inhibition an example


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.

Prof. R. Shanthini Updated: 23 Nov 2012

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.

Prof. R. Shanthini Updated: 23 Nov 2012

https://ibhumanbiochemistry.wikispaces.com/C.7.5

Non-competitive inhibition
- Non competitive inhibitors are usually reversible, - but are not influenced by concentrations of the substrate as is the case for a reversible competitive inhibitor.

Prof. R. Shanthini Updated: 23 Nov 2012

https://ibhumanbiochemistry.wikispaces.com/C.7.5

Non-competitive inhibition
E+S E+I
k1 k2 k4 k5

ES EI

k3

E+P

EI + S

k6
k7

ESI

ES + I

k8 k9

ESI

Prof. R. Shanthini Updated: 23 Nov 2012

Non-competitive inhibition
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

Prof. R. Shanthini Updated: 23 Nov 2012

Non-competitive inhibition
rmax,appCS

rP =
where

KM + CS
rmax (1 + CI / KI)

(23)

rmax,app =

(24) (5) (6)

rmax = k3CE0 KM = k2 / k1
Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

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.
Prof. R. Shanthini Updated: 23 Nov 2012

Uncompetitive inhibition
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.
Prof. R. Shanthini Updated: 23 Nov 2012

Non-competitive inhibition
rmax,appCS

rP =
where

KM + CS
rmax (1 + CI / KI)

(23)

rmax,app =

(24) (5) (6)

rmax = k3CE0 KM = k2 / k1
Prof. R. Shanthini Updated: 23 Nov 2012

rmax,app < rmax

Uncompetitive inhibition
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


Prof. R. Shanthini Updated: 23 Nov 2012

Uncompetitive inhibition

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

Prof. R. Shanthini Updated: 23 Nov 2012

Uncompetitive inhibition
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

Prof. R. Shanthini Updated: 23 Nov 2012

Mixed inhibition

Prof. R. Shanthini Updated: 23 Nov 2012

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).

Prof. R. Shanthini Updated: 23 Nov 2012

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.

Prof. R. Shanthini Updated: 23 Nov 2012

Substrate / Product inhibition

Prof. R. Shanthini Updated: 23 Nov 2012

Assignment
Get the rate equations for substrate and product inhibition

Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

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

Prof. R. Shanthini Updated: 23 Nov 2012

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)

Prof. R. Shanthini Updated: 23 Nov 2012 http://chemwiki.ucdavis.edu/Biological_Chemistry/Catalysts/Enzymatic_Kinetics/Sigmoid_Kinetics

Allosteric enzyme

Find out what it is on your own

Prof. R. Shanthini Updated: 23 Nov 2012 http://chemwiki.ucdavis.edu/Biological_Chemistry/Catalysts/Enzymatic_Kinetics/Sigmoid_Kinetics