Enzymes

AYESHA SHAFI
Factors affecting rate of enzyme
catalyzed reactions
Activity of enzymes is markedly affected by several factors such as
Temperature,
pH
conc. of other substances
presence of activators or inhibitors, etc.
Effect of temperature
Raising the temperature increases the rate of enzyme catalyzed reaction by increasing kinetic
energy of reacting molecules.
Enzymes work maximum over a particular temperature known as optimum temperature. Enzymes
for humans generally exhibit stability temperature up to 35-45 ᵒC.
The temperature coefficient is a factor Q₁₀ by which the rate of biological processes increases for a
10 ᵒC increase in temperature.
For most biological processes Q₁₀ = 2.
Activity of enzyme progressively decreases when the temperature of reaction is below or above
the optimum temperature. However, increase in temperature also causes denaturation of enzyme.
However some times heat energy can also increase kinetic energy to a point that exceed the
energy barrier which results in denaturing of enzymes.
 5- 40oC Temperature
Increase in Activity

40oC - denatures

Rate of Reaction

0 10 20 30 40 50 60

<5oC - inactive
 Effect of pH
Rate of almost all enzymes catalyzed reactions depends on pH. The enzymatic activity is
maximum at a particular pH which is called its optimum pH. Most enzymes exhibit optimal
activity at pH value between 4 and 9.High or low pH value than optimum value will cause
ionization of enzyme which result in denaturation of enzyme. At a very low or high pH the H-
bonds may be inactivated in the protein structure, destroying its 3D structure.
Effect of enzyme concentration
In the beginning velocity of the enzymatic reaction is directly proportional to the enzyme
concentration. When the substrate conc. is in large excess exceeding that of Vmax, because
enzyme is the limiting factor in the enzyme-substrate reaction and providing more enzyme
molecules enables the conversion of progressively larger numbers of substrate molecules into
the product.
Effect of product formation
Products formed as a result of enzymatic reaction may accumulate and this excess of product
may lower the enzymatic reaction by occupying the active site of the enzyme. It is also possible
that under certain conditions of high concentration of products a reverse reaction may be
favored forming back the substrate.
Effect of substrate concentration
As already described a known quantity of enzyme, the reaction is directly proportional to the
substrate concentration. However, this is true only up to a certain concentration after which the
increasing concentration of substrate does not further increase the velocity of reaction .
Effect of Activators and inhibitors
The activity of certain enzymes is greatly dependent of metal ion activators and coenzymes.
Whenever the active site is not available for the binding of the substrate the enzyme activity
may be reduced. The substances which stop or modify the enzymatic reaction are called
inhibitors or modulators. Presence of these substances in reaction medium can adversely affect
the rate of enzymatic reaction.
INHIBITION
 Inhibitions
The chemical substances which inactivate the enzymes and also reduce the rate of enzymatic
reactions are called as inhibitors and the process is called as enzyme inhibition. Enzymes are
protein and they can be inactivated by the agents that denature them. Inhibitors are sometimes
referred to as negative modifier. They may be small inorganic ions, or organic substances.
Enzyme inhibition is of two types
Reversible inhibition (substance binds to the enzyme through non-covalent bond).
Irreversible inhibition (substance binds to the enzyme through covalent bond).
Enzyme inhibition is classified into three major types.
 Inhibition (continue)
Competitive inhibition (Reversible).
Non-competitive inhibition (Irreversible or reversible).
Allosteric inhibition.
1. Competitive Inhibition :
When the active site or catalytic site of an enzyme is occupied by a substance other than the
substrate of that enzyme, its activity is inhibited. The type of inhibition of this kind is known
as competitive inhibition. This is a type of reversible inhibition.
 Competitive Inhibition
 A competitive I combines with the free enzyme to form an EI complex
in a manner that prevents S binding
 Binding of S & I is mutually exclusive
 Inhibition can be reversed by increasing the concentration of S at a
constant [I]
 Degree of inhibition will depend on the concentrations of S & I and on
the relative affinities of the enzyme for S & I

E+I EI
Reversible Enzyme inhibitor
reaction complex
 Competitive inhibition
So the affinity of the substrate for the enzyme is progressively decreased with the increase in
conc. of inhibitor lowering the rate of enzymatic reaction. Thus, the Km is high, but Vmax is the
same in competitive inhibition. However, when the concentrated substrate is increased, the
effect of inhibitor can be reversed forcing it out from EI complex.

Example of competitive inhibition:

1. Statin drugs such as Lipitor (atorvastatin) compete with HMG-CoA(substrate) and inhibit the
active site of HMG CoA-REDUCTASE (that bring about the catalysis of cholesterol synthesis).
Competitive inhibition (example continue)
2. Sulphonamide: A very commonly used antibacterial agent.
Para-amino benzoic acid (PABA) is essential for synthesis of folic acid by the enzyme action. Folic
acid is needed for bacterial growth and survival. Bacterial wall is impermeable to folic acid.
Sulphonamide drugs are structurally similar to PABA and competitively inhibit enzyme action.
Thus, folic acid is not synthesized and growth of bacteria suffers and they die.
 Non competitive inhibition
` This is of two different types namely (i) reversible and (ii) irreversible. This occurs when the
substances not resembling the geometry of the substrate do not exhibit mutual competition. Most
probably the sites of attachment of the substrate and inhibitor are different. The inhibitor binds reversibly
with a site on enzyme other than the active site. Since I doesn't bear structural resemblance to the S, it
must bind to the enzyme at a site distinct from the S binding site

The presence of I does not affect S bonding but does interfere with the catalytic functioning of the
enzyme .The binding of I often deforms the E so that it doesn’t form ES complex at a normal rate and once
formed, ES complex doesn’t decompose at normal rate to yield products. A Non Competitive I doesn’t
affect the Km because the binding of I does not block S binding or vice-versa

 I effectively lowers the concentration of active enzyme and hence decreases the apparent V max. .since
there is no competition between S & I, the inhibition is not reversed by increasing the [S]
Binding of I to a distinct inhibitor site causes a conformational change in the enzyme that distorts or masks the
S binding site or vice versa. This probably brings about the changes in 3D structure of the enzyme
inactivating it catalytically. In noncompetitive inhibition Vmax is lowered, but Km is kept constant. If the
inhibitor can be removed from its site of binding without affecting the activity of the enzyme, it is called as
Reversible-Non-competitive Inhibition. However, if the inhibitor can be removed only at the loss of
enzymatic activity, it is known as Irreversible Non-competitive Inhibition.
I S

Enzyme

S I
I S

Enzyme Enzyme
 Examples for Non- Competitive Inhibition

1.Enzymes requiring divalent metal ions (e.g. Mg 2+ & Ca2+ etc.) for their activity are inhibited non-competitively
by chelating agents like EDTA(ethylene di amine tetra acetic acid) which removes metal ions from the enzyme

2.Enzymes with -SH groups that participate in the maintenance of the three dimensional conformation of the
molecule are non-competitively inhibited by heavy metal ions.

E SH + Hg2+ E S Hg+ + H+
 Suicide Inhibition
It is a special type of irreversible noncompetitive inhibition. In this type of inhibition, substrate
analogue is converted to a more effective inhibitor with the help of the enzyme to be inhibited.
The so formed new inhibitor binds irreversibly with the enzyme.
Examples :
Allopurinol The best example of suicide inhibition. The drug is used in treatment of gout, as it
inhibits the enzyme xanthine oxidase thus decreasing the uric acid formation. But allopurinol
gets oxidized by the enzyme xanthine oxidase itself to form “alloxanthine” a more potent
effective and stronger inhibitor of xanthine oxidase thus potentiating the action of allopurinol.
 Allosteric inhibition
There is a mixed kind of inhibition when the inhibitor binds to the enzyme at a site other than the active
site but on a different region in the enzyme molecule called allosteric site. Allosteric inhibition does not
follow the Michaelis-Menten hyperbolic kinetics. Instead it gives a sigmoid kinetics.
When the inhibitor is present it fits into its site and there is a conformational change in the enzyme
molecule
The enzyme’s molecular shape changes
The active site of the substrate changes
The substrate cannot bind with the substrate.
The reaction slows down
This is not competitive inhibition but it is reversible
When the inhibitor concentration diminishes the enzyme’s conformation changes back to its active form
Switching off
These enzymes have two receptor
sites
One site fits the substrate like other
enzymes
The other site fits an inhibitor Inhibitor
molecule
Substrate molecule
cannot fit into
the active
site Inhibitor fits into
allosteric site

© 2008 Paul Billiet ODWS

The allosteric site the enzyme “on-off” switch
Active
site

E Allosteric
Substrate site Conformational E
fits into empty change Inhibitor
the active molecule
Substrate
site is present
cannot fit
The inhibitor into the
molecule is active site Inhibitor fits
absent into allosteric
site

© 2008 Paul Billiet ODWS

Phosphofructokinase
This enzyme an active site for fructose-6-phosphate molecules to bind with another phosphate
group
It has an allosteric site for ATP molecules, the inhibitor
When the cell consumes a lot of ATP the level of ATP in the cell falls
No ATP binds to the allosteric site of phosphofructokinase
The enzyme’s conformation (shape) changes and the active site accepts substrate moleculesThe
respiration pathway accelerates and ATP (the final product) builds up in the cell
As the ATP increases, more and more ATP fits into the allosteric site of the phosphofructokinase
molecules
The enzyme’s conformation changes again and stops accepting substrate molecules in the active site
Respiration slows down.

© 2008 Paul Billiet ODWS