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Enzyme Inhibition
Enzyme inhibition means decreasing or cessation in the enzyme activity.
The inhibitor is the substance that decreases or abolishes the rate of enzyme action.
According to the similarity between the inhibitor and the substrate, enzyme inhibition is
classified into:
1. Competitive inhibition
2. Noncompetitive inhibition
I. Competitive Inhibition
In this type of inhibition, there is structural similarity between the inhibitor and
substrate.
The inhibitor and the substrate compete with each other to bind to the same catalytic site
of the enzyme.
The inhibition is reversible.
It can be relieved by increasing substrate concentration.
It does not affect Vmax.
It increases Km.

Competitive inhibition increases Km but does not affect Vmax

Some examples of competitive inhibition are illustrated in the following table

Enzymatic process Substrate Inhibitor

Succinate Succinic acid Malonic acid
dehydrogenase
Folic acid synthesis in Para aminobenzoic acid Sulfanilamide
bacteria (PABA)
Prothrombin synthesis Vitamin K Dicumarol

Carbonic anhydrase Carbonic acid Acetazolamide (diamox)

Xanthine oxidase Xanthine Allopurinol (zyloric)

Choline esterase Acetyl choline Physostigmine

The formulae of malonic and succinic acids show the structural similarity between them.
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II- Noncompetitive inhibition

Non-competitive inhibition may be specific or non-specific.
A- Specific noncompetitive inhibition
In this type of enzyme inhibition:
There is no structural similarity between the inhibitor and the substrate.
The inhibitor does not bind to the catalytic site as the substrate but it binds to another
site.
It can bind to enzyme or to enzyme substrate complex
The inhibition is irreversible.
It cannot be relieved by increasing substrate concentration.
It decreases Vmax.
It does not affect Km.

Noncompetitive inhibition decreases Vmax but does not affect Km

Noncompetitive inhibition may be caused by:

1. Inhibition of sulphahydryl group.
2. Inhibition of cofactors.
3. Inhibition of specific ion activator.

1)- Inhibition of Sulphahydryl (-SH) group

Many enzymes depend on free sulphahydryl group for its activity.
Inhibition of this group leads to loss of the enzyme activity.
Sulphahydryl group can be inhibited by:
a- Oxidizing agents as potassium ferricyanide.
2 E-SH E-S-S-E

Potassium ferricyanide Potassium Ferrocyanide

Where E-SH is an enzyme containing free sulphahydryl group.

b- Alkylating agents as iodoacetic acid (I-CH2-COOH) and iodoacetamide

E-SH E-S-CH2-COOH

I-CH2-COOH HI (iodic acid)

Enzyme inhibition by iodoacetic acid

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c- Effect of heavy metals: Heavy metal ions as mercury (Hg++) and Lead
(Pb++) block sulphahydryl group of enzymes forming mercaptides.
2 E-SH + Pb++ E-S-Pb-E-S

2)- Inhibition of cofactors

The inhibitors block an active group in coenzymes or the prosthetic group:
a. Coenzyme inhibition e.g. hydrazine and hydroxylamine
block the aldehyde group in the pyridoxal phosphate, which
is a coenzyme needed for transamination, decarboxylation
and desulfhydration of amino acids.
b. Inhibitors of prosthetic group e.g. carbon monoxide
(CO), cyanide and bisulphate block the iron in the haeme
which is the prosthetic group of cytochrome oxidase
enzyme
3)- Inhibition of metal ion activator
Removal of calcium ions from blood prevents its coagulation as Ca++ is needed to
activate thrombokinase enzyme which converts the inactivate prothrombin to
active thrombin that causes blood clotting.
B- Non-specific non-competitive inhibition
As enzymes are protein in nature, any factor that causes protein denaturation will inhibit
enzyme activity e.g. strong acids, strong alkalis severe agitation and repeated freezing
and thawing.

Enzyme Kinetics
Kinetics are concerned with the rates of reactions. This is a very important matter for the
living organism which maintains its steady state by adjusting reaction rates in response
to the environment and to hormonal controls.
The study of the rate at which an enzyme acts is called enzyme kinetics.
Michaelis-Menten hyperbolic plot

Enzyme kinetics are studied by

plotting the initial velocity (Vi) on the
Y axis and the substrate concentration
[S] on the X axis, we find that:
• At low values of [S], the initial
velocity (Vi) rises almost linearly
with increasing [S].
• A further increase in [S] produces
a less than proportional increase
in reaction rate.
• Eventually, the enzyme becomes saturated Michaelis Menten hyperbolic plot
with the substrate and the reaction attains
its maximal velocity (Vmax)
• Any further increase in [S] does not affect the velocity of the reaction.
• The substrate concentration that produces 1/2 Vmax is the Michaelis-Menten
constant, Km
• The produced curve attains the shape of a rectangular hyperbola. So the curve is
called Michaelis-Menten hyperbolic curve
• It is hard to draw accurately and hard to determine Vmax and Km precisely.
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Lineweaver-Burk plot

Plotting the reciprocals of Vi and [S] (plotting 1/Vi on the Y axis and 1/[S] on the X
axis) yields a "double-reciprocal" or Lineweaver-Burk plot.
• Vmax is determined by the point where the line crosses the Y axis as the [S] is infinite
at that point.
• Km is easily determined from the intercept on the X axis.
• The curve is linear, so it is widely used as it is easy to draw and provides a more
precise way to determine Vmax and Km.

Lineweaver-Burk plot
(Double reciprocal plot)

The Effects of Inhibitors on enzyme kinetics

The distinction between competitive and noncompetitive enzyme inhibition can be
determined by plotting enzyme activity with and without the inhibitor present.

Enzyme behavior in presence of competitive inhibitor:

• Vmax is unchanged
• Km is increased, more substrate is needed to keep the reaction going at 1/2 Vmax
• Michaelis Menten hyperbolic plot shows that the initial velocity (vi) of the
enzyme reaction rises more slowly when competitive inhibitor is present, but
eventually reaches normal Vmax when [S] is very high.
• Lineweaver-Burk plot shows a steeper slope to the line when a competitive
inhibitor is present. The series of lines pivot on the y intercept, since Vmax is not
changed for competitive inhibition. The X-intercept becomes smaller as Km
increases in competitive inhibition.

Enzyme behavior in presence of non-competitive inhibitor:

• Vmax is decreased
• Km remains unchanged
• Michaelis Menten hyperbolic plot shows the initial velocity (vi) of the enzyme
reaction rises more slowly when noncompetitive inhibitor is present and levels
off at reduced Vmax
• Lineweaver-Burk plot shows that the series of lines pivot on the negative X
intercept, since Km is unchanged for non-competitive inhibition. Y-intercept and
slope increase due to the reciprocal dependence on Vmax, which decreases.
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Michaelis Menten plot in presence of Lineweaver-Burk plot in presence of

competitive and noncompetitive inhibitors competitive and noncompetitive inhibitors

Enzyme units
The amount of enzyme present or used in a process is difficult to determine in absolute
terms (e.g. grams), as its purity is often low and a portion of the enzyme may be in an
inactive, or partially active, state.
For these reasons, enzymes are usually measured in terms of activity rather than weight.
The term activity means the number of micromoles of substrate converted to product per
unit of time.
Many ways are used to measure enzyme activity:
1- The activity unit (U), which also is called the international unit (IU), is defined as the
amount of the enzyme that catalyses the conversion of one micromole of substrate to
product in one minute under standard conditions. Standard conditions refer to optimal
conditions, especially with regard to pH, ionic strength, temperature, substrate
concentration and the presence and concentration of cofactors.
2- The katal Unit (kat) is defined as the amount which will catalyse the transformation
of one mole of substrate per second (kat = 60,000,000 IU). It is an impracticable unit and
has not yet received widespread acceptance.
3- Volume activity is the enzyme Activity in international units per ml of extract (the
solution in which the enzyme is present). Its unit is IU/ml.
4- The specific activity is used to monitor the purity of an enzyme during a purification
procedure.
Specific activity of an enzyme in extract equals volume activity (measured in IU/ml)
divided by the protein concentration in the same extract (measured in mg/ml). The unit
of specific activity is IU/mg.
5- Catalytic Efficiency (Kcat) is a direct measure of the catalytic activity under
optimum conditions (i.e. fully saturated enzyme).
It is also called the turnover number as it equals the number of substrate molecules
that are turned over (converted to product) in one second when the enzyme acts at
optimum conditions (i.e. at its maximum velocity, Vmax). The unit of kcat is the number
of substrate molecules turned over per enzyme molecule per second
6- Specificity constant equals the catalytic efficiency of the enzyme (kcat) divided by its
Michaelis constant (Km) It provides a direct measures of enzyme efficiency and
specificity of different enzymes.
7- Sometimes arbitrary (non-standard) units are used to measure enzyme activity.