Enzyme inhibitions

1. Enzyme InhibitionEnzyme Inhibition
ByBy
Prof. V.K. GuptaProf. V.K. Gupta
Department of BiochemistryDepartment of Biochemistry
Kurukshetra University, KurukshetraKurukshetra University, Kurukshetra
email: [email protected] email: [email protected]

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

3. Type of Enzyme Inhibitors Type of Enzyme Inhibitors
ReversibleReversible
IrreversibleIrreversible
Type of Type of
InhibitorsInhibitors
CompetitiveCompetitive
UncompetitiveUncompetitive
Non- CompetitiveNon- Competitive
Active Site Active Site
DirectedDirected
Suicide / kSuicide / k
catcat
InhibitorsInhibitors

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

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

6. Competitive Inhibition Competitive Inhibition
 A competitive I combines with the free enzyme to form an EI A competitive I combines with the free enzyme to form an EI
complex in a manner that prevents S binding complex in a manner that prevents S binding
Binding of S & I is mutually exclusiveBinding of S & I is mutually exclusive
Inhibition can be reversed by increasing the concentration of S at a Inhibition can be reversed by increasing the concentration of S at a
constant [I]constant [I]
Degree of inhibition will depend on the concentrations of S & I and Degree of inhibition will depend on the concentrations of S & I and
on the relative affinities of the enzyme for S & Ion the relative affinities of the enzyme for S & I

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

SS
II
EnzymeEnzyme

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

9. 4. The binding sites for S & I are distinct but 4. The binding sites for S & I are distinct but
overlapping.overlapping.
EnzymeEnzyme
II
SS

10. 5. Binding of I to a distinct inhibitor site causes a 5. Binding of I to a distinct inhibitor site causes a
conformational change in the enzyme that distorts conformational change in the enzyme that distorts
or masks the S binding site or vice versa.or masks the S binding site or vice versa.
EnzymeEnzyme
II SS
EnzymeEnzyme
II
SS
EnzymeEnzyme
II
SS

11. Examples for Competitive Inhibition

COO-
CH2
CH2
COO-
+ FAD
SDH HCCOO-
-OOCCH
+ FADH2
Succinate Fumarate
i)
ii)
Cometitive inhibition accounts for the antibacterial action of sulfanilamide Cometitive inhibition accounts for the antibacterial action of sulfanilamide
which is a structural analog of PABAwhich is a structural analog of PABA
Sulfanilamide inhibits the bacterial enzyme dihydropteroate synthetase Sulfanilamide inhibits the bacterial enzyme dihydropteroate synthetase
which catalyzes the incorporation of PABA into 7,8-dihydropteroic acid.which catalyzes the incorporation of PABA into 7,8-dihydropteroic acid.
H2N COOH
PABA
H2N S NH2
O
O
Sulfanilamide
Malonate is a competitive inhibitor of SDH.Malonate is a competitive inhibitor of SDH.

12. Derivation of velocity equationDerivation of velocity equation
Ki = Ki =
[E] [I][E] [I]
[EI][EI]
or [EI]or [EI] = =
[E] [I][E] [I]
KiKi
In the steady state assumptionIn the steady state assumption
[E] [S][E] [S]
[ES][ES]
kk
-1-1 + k + k
22
kk
11
== ==KmKm
E + S ES E+P
I
EI + S
+
No ReactionX
Ki
k1 k2
k-1
[ES][ES] = =
[E] [S][E] [S]
KmKm
v=kv=k
22[ES] [ES] ÞÞ Vmax = k Vmax = k
2 2 [E][E]
T T ÞÞ
Now [E]Now [E]
T T = [E] + [ES] + [EI]= [E] + [ES] + [EI]
X

13. kk
22 [ES] [ES]
kk
22 ( [E] + [ES] + [EI] ) ( [E] + [ES] + [EI] )
VV
max max = k= k
2 2 ( [E] + [ES] + [EI] )( [E] + [ES] + [EI] )
[ES] [ES]
[E] + [ES] + [EI] [E] + [ES] + [EI]
== ==
vv
VV
maxmax
Putting the value of [ES] and [EI}Putting the value of [ES] and [EI}
[E] [S][E] [S]
KmKm
[E] [E] [S] [E] [I][E] [E] [S] [E] [I]
Km KKm K
ii
++ ++
vv
VV
maxmax
==
[S][S]
KmKm
[S] [I][S] [I]
Km KKm K
ii
1 +1 + ++
==
vv
VV
maxmax

14. [S][S]
[I] [I]
KiKi
KK
mm + [S] + + [S] +
KK
mm
==
vv
VV
maxmax
[S][S]
[I] [I]
KiKi
KK
mm (1+ ) (1+ )+ [S]+ [S]
==
vv
VV
maxmax
Multiplying by kMultiplying by k
m m both in the numerator and the both in the numerator and the
denominator denominator

15. In the presence of a competitive inhibitor KIn the presence of a competitive inhibitor K
m m increases increases
VV
max max unchanged unchanged
==
vv
VV
maxmax
[S][S]
KmKm
appapp + [S] + [S]
Where KmWhere Km
appapp
[I] [I]
KiKi
==
No inhibitorNo inhibitor
+ C Inhibitor+ C Inhibitor
VV
maxmax
½ V½ V
maxmax
KK
mmKmKm
appapp
[s][s]
vv
KK
mm (1+ ) (1+ )==

16. Lineweaver Burk plot Km
Vmax

=
1
v
[I]
2
[I]
1
1
Km
app
1
Km
( 1+ )
[I]
Ki
1
[S]
1
Vmax

+
Slope =
Km
Vm
ax
( 1+ )[I]
Ki

17. Calculation of KCalculation of K
ii
 From slope of the double reciprocal plot in the presence of a C. From slope of the double reciprocal plot in the presence of a C.
Inhibitor which is egual toInhibitor which is egual to

 From KmFrom Km
app app which is given by which is given by
[I] [I]
KiKi
KmKm
appappKK
mm (1+ ) (1+ )==
(1+ )(1+ )
[I] [I]
KiKi
KmKm
VV
maxmax
Slope =Slope =

18. KK
mm
VV
maxmax
KK
mm
VV
max max KK
ii
Slope =Slope =
- K- K
ii
[I][I]
 A graphical method is preferred to direct substitution of A graphical method is preferred to direct substitution of
numbers to allow errors in individual determination to be numbers to allow errors in individual determination to be
averaged out averaged out
KmKm
appapp
VV
maxmax
From the replot of slope vs. [I] From the replot of slope vs. [I]
Slope =Slope =
KK
mm
VV
max max
++
KK
mm
VV
maxmaxKiKi

[I][I]
Slope =Slope =

19. KK
mm
KK
mm
KK
ii
Slope =Slope =
- K- K
ii
[I][I]
KmKm
appapp
 From replot of KmFrom replot of Km
app app Vs. [I]Vs. [I]
KK
mm
KK
ii
[I][I]
++
KK
mm
KmKm
appapp = =

20. KK mm
VV m
ax
m
ax [S] K
[S] K ii
Slope =
Slope =
- K- K
ii
[I][I]
(1+ )(1+ )
Km Km
[S][S]
11
VV
maxmax
11
vv
11
VV
maxmax
Slope = 0Slope = 0
[S] = [S] = ¥¥
[S][S]
22
[S][S]
11
I
n
c
r
e
a
s
i
n
g

[
S
]

I
n
c
r
e
a
s
i
n
g

[
S
]

(1+ )(1+ )
[S] [S]
KmKm
 From Dixon’s plotFrom Dixon’s plot KmKm
Vmax[S] Ki Vmax[S] Ki

==
11
vv
11
Vmax Vmax

[I] [I]
++
(1+ )(1+ )
Km Km
[S][S]

21. Non-competitive Inhibition Non-competitive Inhibition
 An inhibitor that binds to an enzyme to form a dead end complex, 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 whether or not the active site is occupied by a substrate is termed as a
NC InhibitorNC Inhibitor
Can bind either to E or ES complexCan bind either to E or ES complex
Since I doesn't bear structural resemblance to the S, it must bind to the Since I doesn't bear structural resemblance to the S, it must bind to the
enzyme at a site distinct from the S binding siteenzyme at a site distinct from the S binding site
The presence of I does not affect S bonding but does interfere with the The presence of I does not affect S bonding but does interfere with the
catalytic functioning of the enzyme catalytic functioning of the enzyme
The binding of I often deforms the E so that it doesn’t form ES complex 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 at a normal rate and once formed, ES complex doesn’t decompose at
normal rate to yield productsnormal rate to yield products

22.  A NC I doesn’t affect the Km because the binding of I does not A NC I doesn’t affect the Km because the binding of I does not
block S binding or vice-versablock S binding or vice-versa
 I effectively lowers the concentration of active enzyme and I effectively lowers the concentration of active enzyme and
hence decreases the apparent Vhence decreases the apparent V
maxmax
 since there is no competition between S & I, the inhibition is not since there is no competition between S & I, the inhibition is not
reversed by increasing the [S]reversed by increasing the [S]
EnzymeEnzyme EnzymeEnzyme
EnzymeEnzyme
EnzymeEnzyme
SS
II
SS
II

23. Examples for Non- Competitive Examples for Non- Competitive
InhibitionInhibition
1.1.Enzymes requiring divalent metal ions (e.g. MgEnzymes requiring divalent metal ions (e.g. Mg
2+ 2+
& Ca& Ca
2+ 2+
etc) for their etc) for their
activity are inhibited non-competitively by chelating agents like EDTA activity are inhibited non-competitively by chelating agents like EDTA
which removes metal ions from the enzymewhich removes metal ions from the enzyme
2.2.Enzymes with -SH groups that participate in the maintenance of the three Enzymes with -SH groups that participate in the maintenance of the three
dimensional conformation of the molecule are non-competitively inhibited dimensional conformation of the molecule are non-competitively inhibited
by heavy metal ions.by heavy metal ions.

EE SH + HgSH + Hg
2+2+
EE S HgS Hg
+ +
+ H+ H
++

24. E + S ES E+P
I
EI + S
+
Ki
k2
Ks
+
I
ESI
Ks
Ki
Ks = Ks =
[E] [S][E] [S]
[ES][ES]
[EI] [S][EI] [S]
[ES][ES]
==

Replacing Ks with KmReplacing Ks with Km
[ES] = [ES] =
[E] [S][E] [S]
KmKm
``
No inhibitorNo inhibitor
+ NC Inhibitor+ NC Inhibitor
VV
maxmax
½ V½ V
maxmax
KK
mm [s][s]®®
vv
½ V½ V
max imax i
VV
max imax i
Vmax = Decreases.Vmax = Decreases.
Km = UnchangedKm = Unchanged

25.  Lineweaver – Burk PlotLineweaver – Burk Plot
KmKm
Vmaxi Vmaxi

==
11
vv
11
[S] [S]
11
Vmaxi Vmaxi
++
[I][I]
22
[I][I]
11
No InhibitorNo Inhibitor
KK
mm
VV
maxmax
Slope =Slope =
Intercept = Intercept =
11
VV
maxmax
Intercept = Intercept =
11
VV
maximaxi
11
KmKm
1/[s]1/[s]®®
KK
mm
VV
m
a
x
i
m
a
x
i
S
lo
p
e =
S
lo
p
e =
Both slope & Both slope &
Intercept Intercept
Increased By Increased By
the factorthe factor
(1+[ I ] )(1+[ I ] )
KiKi
1/v1/v

26. KK
mm
VmaxVmax
- K- K
ii
[I][I]
SlopeSlope
[I][I]
++
KK
mm
VmaxVmax
Slope =Slope =
Calculation of KCalculation of K
ii
i)i)From the slope of the reciprocal plotFrom the slope of the reciprocal plot
ii)ii) from the intercept of the reciprocal from the intercept of the reciprocal
plotplot
iii)iii) from replot of slope of the reciprocal from replot of slope of the reciprocal
plot vs [ I ]plot vs [ I ]
KK
mm
Vmax KiVmax Ki
In partial NC inhibition In partial NC inhibition
this plot is hyperbolicthis plot is hyperbolic

27. iv.iv. Replot of intercept of the primary plot in the presence Replot of intercept of the primary plot in the presence
of a NC I vs [I] is linearof a NC I vs [I] is linear
11
VmaxVmax
- K- K
ii
[I][I]
InterceptIntercept
In partial NC inhibition In partial NC inhibition
this plot is hyperbolicthis plot is hyperbolic
[I][I]
++
11
VmaxVmax
Intercept =Intercept =
11
Vmax KiVmax Ki

28. v.v. Dixon’s PlotDixon’s Plot
KmKm
Vmax [S]Vmax [S]
- K- K
ii
[I][I]
1/v1/v
[S][S]
11
[S][S]
22
Intercept = Intercept = (( + + ) )

11
VmaxVmax
KmKm
Vmax [S]Vmax [S]
Slope = Slope = (( + + ) )

11
VmaxVmax
11
KiKi
A plot of 1/v vs [I] will be linear at A plot of 1/v vs [I] will be linear at
fixed [E] and [S] for NC inhibitionfixed [E] and [S] for NC inhibition

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

30.  UC Inhibition is rare in single-substrate reactions. UC Inhibition is rare in single-substrate reactions.
for e.g. Inhibition of intestinal alkaline phosphatase by L- for e.g. Inhibition of intestinal alkaline phosphatase by L-
phenylalanine. It is common in multisubstrate reactionsphenylalanine. It is common in multisubstrate reactions
E + S E S E + PE + S E S E + P
++
II
ESIESI
[ES] = [ES] =
[E] [S][E] [S]
KmKm
[ESI] = [ESI] =
[E] [S] [I][E] [S] [I]
Km KiKm 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 Km
app
because the reaction
ES + I ESI removes some ES causing the reaction
E + S ES to proceed to the right

32. No inhibitor
+ UC Inhibitor
V
max
½ V
max
K
m
[s]®
½ V
max i
V
max i
Vmax = Decreases
Km = Decreases
Km
app
v
v
Vmax
(1+ )
=
[I]
Ki
[s]
Km
(1+ )
[I]
Ki
+
[s]
v
Vmaxi
=
[s]
Km
app
+[s]
The equation can also be written asThe equation can also be written as
Where Where Vmaxi =
Vmax
(1+ )
[I]
Ki
Km
app
=
Km
(1+ )
[I]
Ki

33. [I]
2
[I]
1
No I
-1/Km
app
-1/Km
K
m
Vmax
Slope =
1/Vmax
1/Vmaxi
1/v
1/[s]®
I
n
c
r
e
a
s
i
n
g

[
I
]

Lineweaver Burk plot
1
v
Km 1 1
Vmax [S] Vmax
= +
(1+ )
[I]
Ki
Slope remains
Unchanged &
Intercept
Increases By
the factor
(1+[ I ] )
Ki
Incase of UC Inhibition Ki
is that conc
n
of I which
halves the value of both
Vmax and Km

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

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

36. iv.iv. Dixon’s PlotDixon’s Plot
11
vv
Km [I] 1Km [I] 1
Vmax Ki VmaxVmax Ki Vmax
== ++
(1+ )(1+ )
KmKm
[S][S]
The equation for Dixon’s plot is The equation for Dixon’s plot is
-Ki
-Ki
1/Vmax
1
Vmaxi
1/v
[I]®
(1+ )
Km
[S]
(1+ )
Km
[S]
[S
] = ∞
1
V
m
axK
i
S
lo
p
e =
I
n
c
r
e
a
s
i
n
g

[
S
]

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

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

EE CHCH
22-O- F—P=O + HF -O- F—P=O + HF

OCH(CHOCH(CH
33))
22
OCH(CHOCH(CH
33))
22
OCH(CHOCH(CH
33))
22
OCH(CHOCH(CH
33))
22
DFPDFP
Catalytically inactive Catalytically inactive
enzymeenzyme

39. (C
o
n
tr
o
l) n
o
In
h
ib
ito
r
(C
o
n
tr
o
l) n
o
In
h
ib
ito
r
+
N
C
I n
h
i b
i t o
r
+
N
C
I n
h
i b
i t o
r
Ir
r
e
v
e
r
s
ib
le
In
h
ib
ito
r
Ir
r
e
v
e
r
s
ib
le
In
h
ib
ito
r
[E][E]
ii
[E][E]
TT®®
VmaxVmax
To distinguish between irreversible & NC InhibitionTo distinguish between irreversible & NC Inhibition

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

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

42. Examples:
TPCK acts as an affinity label for Chymotrypsin; even at very low conc
n

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.
N
H
O CH
2Cl
S
O
O
N
H
O OCH
3
S
O
O
tosyl-L-phenylalanine methyl ester
TPCK
(Affinity label) (Substrate)

43. N
H
O CH
2
S
O
O
Cl
N
NH
CH
2His 57
N
H
O CH
2
S
O
O
N
NH
CH
2His 57
Cl
-
+ H
+
Alkylated derivative of
His 57
(inactive Enzyme)
O
HO
phenylpropionate
Excess conc
n
of this
prevent the inactivation
by TPCK
TPCK is attacked in a nucleophilic reaction by the
N atom of the imidazole side chain of His57. the
binding of TPCK to the Enz Brings the reactive –Cl
group in close proximity to the His57 residue and
facilitates the formation of a covelent bond
between the I & imidazole side chain
CH
3
CH
3

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

45. During thymidylate synthesis, NDuring thymidylate synthesis, N
55
,N,N
1010
- methyleneTHF is - methyleneTHF is
converted to 7,8-dihydrofolate; methyleneTHF is regenerated converted to 7,8-dihydrofolate; methyleneTHF is regenerated
in two stepsin two steps

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

47.  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
Importance of Enzyme
Inhibition

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