The document discusses different types of enzyme inhibition. It describes reversible and irreversible inhibition, and types of reversible inhibition including competitive, uncompetitive, and non-competitive inhibition. Competitive inhibitors bind to the active site of the enzyme and prevent substrate binding, while non-competitive inhibitors bind elsewhere and affect the enzyme's activity. The document provides examples and derivations of equations to describe these inhibition patterns and calculate inhibition constants.

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