1. Enzyme Inhibition
By
Prof. V.K. Gupta
Department of Biochemistry
Kurukshetra University, Kurukshetra
email: vkgupta59@rediffmail.com

2. Enzyme
Inhibitor
 An Enzyme inhibitor is a compound that decreases or
tends to decrease the rate of an enzyme catalyzed
reaction by influencing the binding of S and /or its
turnover number.

3. Type of
Inhibitors
Type of Enzyme Inhibitors
Reversible
Irreversible
Competitive
Active Site Uncompetitive
Directed
Suicide / kcat
Non- Competitive
Inhibitors

4. Reversible Inhibition
 Inhibitor binds to Enzyme reversibly through weak non-covelent
interactions
 An Equilibrium is established between the free inhibitor & EI Complex
and is defined by an equilibrium constant (Ki)
E + I E I
 The activity of Enzyme Is fully restored on removing the Inhibitor by
dialysis.
Reversible Inhibitors depending on concentration of E, S and I, show a
definite degree of inhibition which is reached fairly rapidly and remains
constant when initial velocity studies are carried out.

5. Irreversible Inhibition
 Inhibitor binds at or near the active site of the enzyme irreversibly, usually
by covalent bonds, so it can’t dissociate from the enzyme
 No equilibrium exits
E + I E I
 Enzyme activity is not regained on dialysis
 Effectiveness of I is expressed not by equilibrium constant but by a
velocity constant, which determines the fraction of the enzyme inhibited in
a given period of time by a certain concentration of the I

6. 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

7. Binding of S & I in different Situations
1. Classical Competitive Inhibition (S & I compete for the
same binding site)
S I
Enzyme

8. 2. S & I are mutually 3. S & I have a common
exclusive because of binding group on the
steric hindrance enzyme.
S I S
I
Enzyme Enzyme

9. 4. The binding sites for S & I are distinct but
overlapping.
I
S
Enzyme

10. 5. 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.
I S
Enzyme
S I
I S
Enzyme Enzyme

11. Examples for Competitive Inhibition
CO O-
CH2 SDH HC COO-
+ FAD + F AD H
2
-OO CCH
i) CH2
CO O-
S uc c i na t e F um a ra t e
Malonate is a competitive inhibitor of SDH.
ii) Cometitive inhibition accounts for the antibacterial action of sulfanilamide
which is a structural analog of PABA
O
H2 N COOH H 2N S NH2
O
PABA
S ul fan i l a mi d e
Sulfanilamide inhibits the bacterial enzyme dihydropteroate synthetase
which catalyzes the incorporation of PABA into 7,8-dihydropteroic acid.

12. Derivation of velocity equation
k1 k2
E+S ES E+P
+ k-1
[E] [I]
I Ki = [EI]
Ki
[E] [I]
or [EI] = Ki
EI + S X No Reaction
In the steady state assumption
[E] [S] k-1 + k2
= = Km
[ES] k1
[E] [S]
[ES] = Km
v=k2[ES] ⇒ Vmax = k2 [E]T ⇒ Now [E]T = [E] + [ES] + [EI]

13. Vmax = k2 ( [E] + [ES] + [EI] )
v k2 [ES] [ES]
= =
Vmax k2 ( [E] + [ES] + [EI] ) [E] + [ES] + [EI]
Putting the value of [ES] and [EI}
[E] [S]
v Km
Vmax =
[E] [E] [S] [E] [I]
+ +
Km Ki
[S]
v Km
=
Vmax [S] [I]
1 + Km + K
i

14. Multiplying by km both in the numerator and the
denominator
[S]
v
=
Vmax [I] Km
Km + [S] +
Ki
[S]
v
=
Vmax [I]
Km (1+ )+ [S]
+
Ki )

15. In the presence of a competitive inhibitor Km increases
Vmax unchanged
v [S]
=
Vmax Kmapp + [S]
[I]
Where Kmapp = Km (1+
Ki )
Vmax
v
No inhibitor
½ Vmax
+ C Inhibitor
Km Kmapp [s]

16. [I]
Lineweaver Burk plot 1 Km ( 1+ Ki ) 1 1
=
v Vmax [S] + Vmax
[I] ) [I]2
+ i
(1 K
Km x [I]1
e = Vma
Sl op
1
Km
1
Kmapp

17. Calculation of Ki
 From slope of the double reciprocal plot in the presence of a C.
Inhibitor which is egual to
Km (1+ [I] )
Slope = Ki
Vmax
 From Kmapp which is given by
[I]
Kmapp = Km (1+
Ki )

18.  A graphical method is preferred to direct substitution of
numbers to allow errors in individual determination to be
averaged out
From the replot of slope vs. [I]
Km Km
Slope = + [I]
Vmax VmaxKi
Kmapp
Slope =
Vmax
Km
Slope =
Vmax Ki
Km
- Ki
Vmax
[I]

19.  From replot of Kmapp Vs. [I]
Km + Km [I]
Kmapp =
Ki
Kmapp
Km
Slope =
Ki
- Ki Km
[I]

20.  From Dixon’s plot Km [I] 1 (1+ Km )
1 = + [S]
v Vmax[S] Ki Vmax
IInc
nc
re
rea
as
[S]1
siin
ngg[
[SS]
]
1
Km Ki
v
x
[S] [S]
e = Vm
a
1 Km p
2
(1+ ) Slo
Vmax [S]
1 [S] = ∞
Vmax Slope = 0
[I]
[S]
- Ki (1+ Km )

21. Non-competitive Inhibition
 An inhibitor that binds to an enzyme to form a dead end complex,
whether or not the active site is occupied by a substrate is termed as a
NC Inhibitor
 Can bind either to E or ES complex
 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

22.  A NC 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 Vmax
 since there is no competition between S & I, the inhibition is not
reversed by increasing the [S]
S
Enzyme Enzyme
S
I I
Enzyme Enzyme

23. Examples for Non- Competitive
Inhibition
1. Enzymes requiring divalent metal ions (e.g. Mg2+ & Ca2+ etc) for their
activity are inhibited non-competitively by chelating agents like EDTA
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+

24. k2
E+S ES E+P [E] [S] [EI] [S]
Ks Ks = [ES] =
+ + [ES]
I I
Replacing Ks with Km
Ki
Ki [ES] =
[E] [S]
Km
EI + S ESI `
Ks
Vmax
Vmax i
v No inhibitor
½ Vmax
+ NC Inhibitor
½ Vmax i
Vmax = Decreases.
Km = Unchanged
Km [s]→

25.  Lineweaver – Burk Plot
1 Km 1 + 1
=
v Vmaxi [S] Vmaxi
[I]2
m
K
i
ax
m
V
e=
[I]1
op
1/v
Sl
1 No Inhibitor
Both slope & Intercept =
Intercept Vmaxi
Increased By
the factor Km
Slope =
(1+[ I ] ) Vmax
1
Ki
Km 1
Intercept =
Vmax
1/[s]→

26. Calculation of Ki
i) From the slope of the reciprocal plot
ii) from the intercept of the reciprocal
plot Km Km [I]
Slope = +
iii) from replot of slope of the reciprocal Vmax Vmax Ki
plot vs [ I ]
Slope
In partial NC inhibition
this plot is hyperbolic
Km
- Ki
Vmax
[I]

27. iv. Replot of intercept of the primary plot in the presence
of a NC I vs [I] is linear
1 1 [I]
Intercept = +
Vmax Vmax Ki
Intercept
In partial NC inhibition
this plot is hyperbolic
1
- Ki
Vmax
[I]

28. v. Dixon’s Plot
A plot of 1/v vs [I] will be linear at
fixed [E] and [S] for NC inhibition
[S]1
[S]2
1/v Km 1 1
Slope = ( Vmax [S] + Vmax
) Ki
Km 1
- Ki
(
Intercept = Vmax [S] + Vmax )
[I]

29. Uncompetitive Inhibition
 I doesn't bind to the free E rather it binds to the ES complex
 the binding of an UC I is presumed to cause structural distortion
of the active site making the enzyme catalytically inactive
 the binding of S could cause a conformational change in the E
thereby revealing an I binding site
 Inhibition can’t be reversed by increasing the [S] since I doesn't
compete with S for the same binding site
S
Enzyme
Enzyme
S
I
Enzyme

30.  UC Inhibition is rare in single-substrate reactions.
for e.g. Inhibition of intestinal alkaline phosphatase by L-
phenylalanine. It is common in multisubstrate reactions
E+S ES E+P
+
I
[E] [S]
[ES] = Km
[E] [S] [I]
ESI [ESI] =
Km Ki

31.  The equilibria show that at any [I] an infinitely high [S] will not
drive all the enzyme to ES form; some non productive ESI complex
will always be present. Consequently an UC I will decrease the V max
 An UC I will also decrease the Kmapp because the reaction
ES + I ESI removes some ES causing the reaction
E+S ES to proceed to the right

32. v [s]
=
Vmax Km [s]
[I] [I]
+
(1+ ) (1+ )
Ki Ki
The equation can also be written as
Vmax
v [s]
=
Vmaxi Kmapp +[s]
Vmax i
v ½ Vmax No inhibitor Vmax
+ UC Inhibitor Where Vmaxi = [I]
½ Vmax i (1+ )
Ki
Vmax = Decreases
Km = Decreases
Km
Kmapp=
[I]
Km [s]→ (1+ )
Kmapp Ki

33. Lineweaver Burk plot
[I]
1 Km 1 1 (1+ )
= + Ki
v Vmax [S] Vmax
Slope remains
Unchanged &
Intercept
Inc
Increases By
re
as
the factor
in
(1+[ I ] ) [I]2
g[
[I]1
I]
Ki 1/v
No I
1/Vmaxi
Incase of UC Inhibition Ki
Km is that concn of I which
Slope = halves the value of both
Vmax Vmax and Km
1/Vmax
1/[s]→
-1/Kmapp -1/Km

34. Calculation of Ki
i) From the slope of the reciprocal plot [I]
1 1 (1+ )
ii) From the Km app = Ki
Vmaxi Vmax
iii) From replot of 1/Vmaxi vs [ I ]
1 1 1 [I]
= +
Vmaxi Vmax VmaxKi
1/Vmaxi
1
Slope =
VmaxKi
1
- Ki
Vmax
[I]

35. iv. From replot of 1/Km appvs [I]
[I]
1 1 (1+ )
= Ki
Km app Km
1 1 1 [I]
= +
Kmapp Km KmKi
1/Kmapp
1
Slope =
KmKi
1
- Ki
Km
[I]

36. The equation for Dixon’s plot is
iv. Dixon’s Plot
Km
1 Km [I] 1 (1+ )
= + [S]
v Vmax Ki Vmax
In c
1 i
xK
rea
= ma
sin
1/v o pe V
Sl
g[
1 Km
(1+ )
S]
Vmaxi [S]
∞
]=
[S
1/Vmax
[I]→
Km -Ki
-Ki (1+ )
[S]

37. Irreversible Inhibition
An irreversible Inhibitor binds at or near the active site of the
enzyme irreversibly, usually by covalent bonds, so that it can’t
subsequently dissociate from the enzyme
The I destroys as essential functional group on the enzyme that
participates in normal S binding or catalytic action. As a result the
enzyme is rendered permanently inactive
Compounds which irreversibly denature the enzyme protein or
cause non-specific inactivation of the active site are not usually
regarded as irreversible inhibitors.

38. Examples:
Organophosphorus compounds (such as DFP) irreversibly react with the
–OH group of essential serine residue of some enzymes
DFP (Diisopropylphosphofluoridate) is a nerve poison since it inactivates
acetylcholinesterase that plays an important role in the transmission of
nerve impulses.
OCH(CH3)2 OCH(CH3)2
E CH2-OH + F—P=O E CH2-O- F—P=O + HF
OCH(CH3)2 OCH(CH3)2
DFP Catalytically inactive
enzyme

39. To distinguish between irreversible & NC Inhibition
t or
ib i
In h
r
to
t or
bi
no
hi
i bi
In
o l)
In h
Vmax
C
nt r
N
+
ble
(Co
rsi
ve
Ir re
[E]i
[E]T→

40. Types of Irreversible Inhibitors
Irreversible inhibitors
Active site directed Suicide Inhibitors
irreversible Inhibitors (Mechanism-based Inhibitors)
or or
(Affinity labels) (kcat Inhibitors)

41. Affinity labels
An affinity label is a chemically reactive compound that
is designed to resemble the substrate of an enzyme so
that it binds at the active site and forms a stable
covalent bond with a susceptible group of the nearby
residue in the enzyme protein.
Affinity labels are very useful for identifying catalytically
important residues

42. Examples:
TPCK acts as an affinity label for Chymotrypsin; even at very low concn
TPCK quantitatively inactivates chymotrypsin; TPCK is identical in
structure to a substrate of this enzyme i.e. tosyl-L-phenylalanyl methyl
ester, except that the carboxylic ester is replaced by the chloromethyl
group.
O OCH3
O CH2Cl
O
O
S
S N
N O H
O H
TPCK tosyl-L-phenylalanine methyl ester
(Affinity label) (Substrate)

43. CH2 His 57
N H
N
CH3
O CH2 Cl
O TPCK is attacked in a nucleophilic reaction by the
S N atom of the imidazole side chain of His57. the
N binding of TPCK to the Enz Brings the reactive –Cl
O H group in close proximity to the His57 residue and
facilitates the formation of a covelent bond
between the I & imidazole side chain
CH2 His 57
Cl- + H+
HO
CH3 N H
O CH2 N
O O
S
phenylpropionate N
O H Alkylated derivative of
Excess concn of this His 57
prevent the inactivation
by TPCK (inactive Enzyme)

44. Suicide Inhibitors
A suicide inhibitor is a relatively inert molecule that is transformed by an
enzyme at its active site into a reactive compound that irreversibly
inactivates the enzyme
They are substrate analogs designed so that via normal catalytic action of
the enzyme, a very reactive group is generated.
The latter forms a covalent bond with a nearby functional group within the
active site of the enzyme causing irreversible inhibition.
Such inhibitors are called suicide inhibitors because the enzyme appears
to commit suicide.
e.g. FdUMP is a suicide inhibitor of thymidylate synthase.

45. During thymidylate synthesis, N5,N10- methyleneTHF is
converted to 7,8-dihydrofolate; methyleneTHF is regenerated
in two steps

46. Conversion of dUMP to dTMP and its inhibition by FdUMP

47. Importance of Enzyme
Inhibition
 For understanding the regulation of enzyme activity within the
living cells
 To elucidate the kinetic mechanism of an enzyme catalyzing a
multisubstrate reaction
 Useful in elucidating the cellular metabolic pathways by causing
accumulation of intermediates
 Indentifiction of the catalytic groups at the active site
 Provide information about substrate specificity of the enzyme

48.  Form the basis of drug designing. The whole area of selective
toxicity , including the use of antibiotic, toxin, insecticides etc is
based on the exploitation of species differences in the
susceptibility to enzyme inhibitors.
 Competitive inhibitors are useful in x-rays crystallographic
studies to pin point the active site in crystal structure and thus
revealing how the surrounding amino acid residues interact with
the bound molecule.
 To treat methanol poisoning