Enzyme Kinetics - Inhibition

Inhibitors
 Inhibitors are chemicals that reduce the rate of
enzymic reactions
 The are usually specific and they work at low
concentrations
 They block the enzyme but they do not
usually destroy it
 Many drugs and poisons are inhibitors of
enzymes in the nervous system
© 2008 Paul Billiet ODWS
2 jenis inhibitor enzim :
1. Irreversibel (bekerja secara tidak dapat balik)
2. Reversible (dapat balik)
Reversibel inhibitor
 Competitive
 Non-competitive
 Uncompetitive
Types of Inhibition
 Competitive Inhibition
 Noncompetitive Inhibition
 Uncompetitive Inhibition
 Irreversible Inhibition
Competitive Inhibition
Enzyme
S
I

In competitive inhibition,
the inhibitor competes
with the substrate for the
same binding site
 Reversible Inhibitors (Competitive Inhibition)

• A reversible inhibitor
goes on and off, allowing
the enzyme to regain
activity when the inhibitor
leaves
• A competitive inhibitor is
reversible and has a
structure like the substrate
- it competes with the
substrate for the active site
- its effect is reversed by
increasing substrate
concentration
 Example of a Competitive Inhibitor
• Malonate is a competitive inhibitor of succinate dehydrogenase
- it has a structure that is similar to succinate
- inhibition can be reversed by adding succinate
• Dehidrogenase suksinat adalah anggota golongan enzim yang
mengkatalisa siklus asam sitrat, lintas akhir metabolik bagi
degradasi oksidatif karbohidrat dan lemak di dalam
mitokondria.
• Enzim ini mengkatalisa pembebasan 2 atom H dari 2 gugus
metilen (-CH2-).
 Example - Competitive Inhibition
NH2
Sulfanilamide is a competitive
inhibitor of p-aminobenzoic
folic acid acid. Sulfanilamides (also
known as sulfa drugs,
COOH discovered in the 1930s)
p-aminobenzoic acid were the first effective
NH2 systemic antibacterial
agents.
Because we do not make folic
acid, sulfanilamides do not
affect human cells.
SO2 NH2
sulfanilamide
Asam Folat (bahasa Latin : “folium” berarti daun) pertama kali
diisolasi dari daun bayam.

Kekurangan asam folat (asam pteroilglutamat) menyebabkan

sejenis anemia dengan sel darah merah yang tidak cukup
matang sebagaimana mestinya.
 Competitive Inhibition
- Reaction Mechanism

E+S ES E+P
+
I
In competitive inhibition, the
inhibitor binds only to the
EI free enzyme, not to the ES
complex
General Michaelis-Menten Equation

Vmax,app [S]
v=
Km,app + [S]

This form of the Michaelis-Menten equation

can be used to understand how each type of
inhibitor affects the reaction rate curve
In competitive inhibition, only the apparent Km
is affected (Km,app> Km),

The Vmax remains unchanged by the presence

of the inhibitor.

Nilai Km mengekspresikan konsentrasi substrat yang

diperlukan oleh enzim untuk mencapai ½ Vmax
(afinitas substrat terhadap enzim).
.
Competitive inhibitors alter the
apparent Km, not the Vmax
- Inhibitor
Vmax
Reaction Rate

+ Inhibitor
Vmax
2
Vmax,app = Vmax
Km,app > Km

Km Km,app
[Substrate]
 The Lineweaver-Burk plot is
diagnostic for competitive inhibition
1 = Km,app 1
+ 1 Increasing [I]
v Vmax [S] Vmax

Km,app
1 Slope =
Vmax
v

1
Vmax

-1 1
Km,app
[S]
Relating the Michaelis-Menten equation, the v vs. [S]
plot, and the physical picture of competitive inhibition

Inhibitor
competes with
substrate,
decreasing its .

apparent affinity:
Km,app > Km Vmax
- Inhibitor

Reaction Rate
+ Inhibitor
Vmax
2
Km,app > Km
Formation
Formation ofofEIEI
Vmax,app = Vmax
complex shifts
complex reaction
shifts reaction
to the
to theleft:
left:KK
m,app > K
m,app > Km
m Km Km,app
[Substrate]
Noncompetitive Inhibition
.

I I
S
Enzyme S Enzyme

S
I I
S
Enzyme Enzyme

the inhibitor does not

interfere with
substrate binding
(and vice versa)
 Reversible Inhibitors (Noncompetitive Inhibition)
• A noncompetitive inhibitor
has a structure that is different
than that of the substrate
- it binds to an allosteric site
rather than to the active site
- it distorts the shape of the
enzyme, which alters the
shape of the active site and
prevents the binding of the
Istilah alosterik
substrate diturunkan dari bhs
• The effect can not be reversed Yunani “allo” yang
berarti lain &
by adding more substrate “stereos” yang
berarti ruang atau
sisi.
 Example of a noncompetitive Inhibitor
• Sistem enzim bakteri yang mengkatalisa pengubahan L-treonin
menjadi L-isoleusin. Enzim dehidratase treonin dihambat oleh
isoleusin.

Aktivitas dehidratase treonin bereaksi dengan

sangat cepat dan bersifat dapat balik terhadap
fluktuasi konsentrasi isoleusin di dalam sel.

Jika konsentrasi isoleusin menurun, kecepatan

aktivitas reaksi dehidratase treonin meningkat.
Example of noncompetitive inhibition: fructose 1,6-
bisphosphatase inhibition by AMP
Fructose 1,6-bisphosphatase is a key regulatory
enzyme in the gluconeogenesis pathway. High
amounts of AMP signal that ATP levels are low and
gluconeogenesis should be shut down while
glycolysis is turned on.
High AMP levels inhibit fructose 1,6-bisphosphatase
(shutting down gluconeogenesis) and activate
phosphofructokinase (turning on glycolysis).
Regulation of fructose 1,6-bisphosphatase and
phosphofructokinase by AMP prevents a futile cycle
in which glucose is simultaneously synthesized and
broken down.
 Noncompetitive Inhibition -
Reaction Mechanism
E+S ES E+P
+ + In noncompetitive
inhibition, the
I I inhibitor binds
enzyme irregardless
of whether the
substrate is bound

EI + S ESI
Noncompetitive inhibitors decrease
the Vmax,app, but don’t affect the Km

Vmax,app < Vmax

Km,app = Km
 Why does Km,app = Km for
noncompetitive inhibition?
E+S ES E+P
+ + The inhibitor binds
equally well to free
I I enzyme and the ES
complex, so it doesn’t
alter apparent affinity
of the enzyme for the
EI + S ESI substrate
The Lineweaver-Burk plot is diagnostic
for noncompetitive inhibition
1 = Km 1 1 Increasing [I]
+
v Vmax,app [S] Vmax,app

1 Slope =
Km
v Vmax,app

1
Vmax,app
-1
Km
1
[S]
Relating the Michaelis-Menten equation, the v vs. [S] plot, and the
physical picture of noncompetitive inhibition

I
.

I
S
Enzyme S Enzyme
Inhibitor doesn’t interfere
with substrate binding,
Km,app = Km

S .

I I
S Vmax - Inhibitor
Enzyme Enzyme

Reaction Rate
Vmax,app
1 + Inhibitor
Even at high V
2 max

substrate levels, 1
V Km,app > K< mVmax
Vmax,app
Formation inhibitor
of EI still binds, 2 max,app

Vmax,app
Km,app== V
Kmmax
complex shifts
[E]t < reaction
[ES]
Vmax,app < Vmax
to the left: Km,app > Km Km Km,app
[Substrate]
Noncompetitive inhibitors
decrease the apparent Vmax, but
do not alter the Km of the
reaction
 Uncompetitive Inhibition
Enzyme.

Enzyme

S
In uncompetitive
S
inhibition, the
I
Enzyme
inhibitor binds
I
only to the ES
complex
Enzyme

I S
 Uncompetitive Inhibition -
Reaction Mechanism
E+S ES E+P
+ In uncompetitive
I inhibition, the
inhibitor binds only
to the ES complex,
it does not bind to
ESI the free enzyme
Uncompetitive inhibitors decrease both the
Vmax,app and the Km,app

Vmax,app < Vmax

Km,app < Km
Notice that at low substrate
concentrations,
uncompetitive inhibitors
have little effect on the
reaction rate because the
lower Km,app of the enzyme
offsets the decreased Vmax,app
Uncompetitive inhibitors decrease both the
Vmax,app and the Km,app of the enzyme

E+S ES E+P
+ Notice that
uncompetitive inhibitors
I don’t bind to the free
enzyme, so there is no
EI complex in the
reaction mechanism

ESI
 The Lineweaver-Burk plot is
diagnostic for uncompetitive inhibition
1 = Km,app 1 1
+
v Vmax,app [S] Vmax,app 1 Increasing [I]

=
Km 1
+
1 v
Vmax [S] Vmax,app
Km
Slope =
Vmax

1
Vmax,app

-1
Km,app
1
[S]
 Relating the Michaelis-Menten equation, the v vs. [S]
plot, and the physical picture of uncompetitive inhibition
Enzyme.

Enzyme

Vmax - Inhibitor

Reaction Rate
S
S
Enzyme
Vmax,app
I I 1
V
2 max
+ Inhibitor
Inhibitor 1
V Vmax,app < Vmax
increases Enzyme
2 max,app

the amount of Km,app< Km

enzyme bound
to substrate I S Km,app Km
.

[Substrate]
Km,app < Km

Even at high
Formation of EI levels,
substrate
complex shiftsinhibitor binds,
reaction
[E]t < [ES]
to the left: KVm,app > Km
max,app < Vmax
Uncompetitive inhibitors
decrease the apparent Km of the
enzyme and decrease the Vmax of
the reaction
Example of uncompetitive inhibition: alkaline
phosphatase inhibition by phenylalanine
.

Alkaline Alkaline Alkaline

phosphatase phosphatase phosphatase

O O
O O O P O- -
O P O-
P O- O-
O- O
-

Phe Phe

Alakaline
Phosphatase

O
O P O-
Phe O-
At alkaline pH, alkaline phosphatase catalyzes
the release of inorganic phosphate from
phosphate esters. It is found in a number of
tissues, including liver, bile ducts, intestine,
bone, kidney, placenta, and leukocytes.
Alkaline phosphatase plays a role in the
deposition of hydroxyapetite in osteoid cells
during bone formation. The function of
alkaline phosphatase in other tissues is not
known. Serum alkaline phosphatase levels are
important diagnostic markers for bone and
liver disease.
 Irreversible Inhibition
In irreversible
Enzyme inhibition, the
inhibitor binds to the
S enzyme irreversibly
O I through formation of
a covalent bond with
the enzyme ,
permanently
inactivating the
enzyme
Irreversible inhibitor
 Penghambat tak dapat balik adalah golongan yang
bereaksi dengan, atau merusak suatu gugus fungsional
pada molekul enzim yg penting bagi aktivitas
katalitiknya.
 Contoh : senyawa diisopropilfluorofosfat (DFP)
menghambat enzim asetilkolinesterase yang penting
dalam transmisi impuls syaraf.
Asetikolinesterase mengkatalisa hidrolisis asetilkolin,
suatu senyawa neurotransmitter yg berfungsi di dalam
bagian tertentu sistem syaraf (menyebabkan sel
menggandakan impuls syaraf).
Penghambatan irreversible Asetilkolinesterase oleh diisopropilfluorofosfat (DFP)

(a) Reaksi dikatalisa oleh asetilkolinesterase (b) Reaksi DFP dengan gugus hidroksil serin
Penghambatan irreversible suatu enzim yang
mengandung –SH oleh iodoasetamida
 Examples of Irreversible Inhibitors
• diisopropylphosphofluoridate
– prototype for the nerve gas sarin
– permanently inactivates serine proteases by
forming a covalent bond with the active site
serine
 Penicillin is a suicide inhibitor
R
O Penicillin
C
S CH3
H
H N

HC CH3

N COO-
C
H
O Strained
peptide bond R
O
glycopeptide C
glycopeptide H S CH3
transpeptidase transpeptidase
H N

HC CH3

N COO-
Ser OH Ser O C
H
H
O

Glycopeptide transpeptidase catalyzes the formation of cross-links between D-

amino acids in the cell walls of bacteria. This enzyme also catalyzes the
reverse reaction, the hydrolysis of peptide bonds. During the course of
hydrolyzing the strained peptide bond in penicillin, the enzyme activates the
inhibitor (penicillin), which then covalently modifies an active site serine in
the enzyme. In effect, the enzyme “commits suicide” by hydrolyzing the
strained peptide bond in penicillin.
Suicide inhibitors work by
“tricking” the enzyme into
activating the inhibitor, which
then forms a covalent bond with
the enzyme, leading to its
permanent inactivation.
 Irreversible Inhibitors
• An irreversible inhibitor destroys enzyme activity, usually
by bonding with side-chain groups in the active site.
Irreversible Inhibition - Reaction
Mechanism
E+S ES E+P
+ In irreversible inhibition,
I the inhibitor permanently
inactivates the enzyme.
The net effect is to remove
enzyme from the reaction.
EI Vmax decreases
No effect on Km
.
The Michaelis-Menten plot for an irreversible
inhibitor looks like noncompetitive inhibition

Vmax - Inhibitor
Reaction Rate

Vmax,app
1
V
+ Inhibitor
2 max
1
V
2 max,app Vmax,app < Vmax
Km,app = Km
Km [Substrate]
Km,app
 Irreversible inhibition is distinguished from
noncompetitive inhibition by plotting Vmax vs [E]t

Enzyme is
inactivated
until all of the
irreversible
inhibitor is
used up
Irreversible inhibitors decrease
Vmax,app, but leave the apparent
Km unchanged. Irreversible
inhibitors differ from other types
of inhibitors because they
covalently modify the enzyme.
This results in the permanent
inhibition of the enzyme activity.
 Summary-Enzyme Inhibition
• Competitive Inhibitor
– Binds to substrate binding site
– Competes with substrate
– The affinity of the substrate appears to be decreased
when inhibitor is present (Km,app > Km)
• Noncompetitive inhibitor
– Binds to allosteric site
– Does not compete with the substrate for binding to the
enzyme
– The maximum velocity appears to be decreased in the
presence of the inhibitor (Vmax,app < Vmax)
• Uncompetitive Inhibitor
– Binds to the enzyme only after the substrate has
bound
– The affinity of the substrate appears to be increased
and the maximum velocity appears to be decreased
when inhibitor is present (Km,app <Km,
Vmax,app <Vmax),
• Irreversible Inhibitor
– Covalently modifies and permanently inactivates the
enzyme
 Latihan Soal
• Penanggulangan keracunan metanol
Metanol adalah pelarut komersil yang pernah digunakan sebagai
antibeku otomotif. Senyawa ini amat beracun dan dapat menyebabkan
kematian, jika seseorang menelan dalam jumlah 30 mL saja. Toksisitas
yang demikian tinggi ini, disebabkan bukan hanya oleh metanolnya
sendiri, tetapi oleh produk metaboliknya formaldehida. Metanol dengan
cepat dioksidasi menjadi formaldehida oleh kerja enzim dehidrogenase
alkohol pada hati :

Sebagian usaha medis penanggulangan keracunan metanol adalah

dengan memberikan si pasien etanol melalui mulut atau secara
intravenous dalam jumlah yang akan meracuni orang dalam keadaan
normal. Jelaskann mengapa perlakuan ini akan efektif !
 Enzyme Activators
• Chemicals that help the enzyme work.
• Activators increase the enzyme reaction rate.

Active
Site
Activator

X
Binding Substrate
Site
• Alosterik inhibitor
Penghambat yang dapat mempengaruhi enzim alosterik.
- Enzim alosterik adalah enzim yang mempunyai dua bagian
aktif, yaitu bagian aktif yang menangkap substrat dan bagian
yang menangkap penghambat.
- Apabila ada senyawa yang dapat memasuki bagian yang
menangkap penghambat maka enzim menjadi tidak aktif,
senyawa penghambat tersebut merupakan penghambat
alosterik.
- Struktur senyawa penghambat alosterik tidak mirip dengan
struktur substrat.
- Apabila enzim menangkap substrat maka penghambat tidak
dapat terikat pada enzim, sehingga enzim dapat aktif
mereaksikan substrat menjadi produk.
 The switch: Allosteric inhibition
Allosteric means “other site”

Active site

E
Allosteric
site

© 2008 Paul Billiet ODWS

 Switching off
 These enzymes
have two receptor
sites
 One site fits the Inhibitor
substrate like other Substrate molecule
enzymes cannot fit
into the
 The other site fits active site Inhibitor fits
an inhibitor into allosteric
molecule site

© 2008 Paul Billiet ODWS

 The allosteric site the enzyme “on-
off” switch
Active
site
Allosteric
Substrate E
site empty Conformational E
fits into change Inhibitor
the active molecule is
site Substrate
cannot fit present
The inhibitor into the
molecule is active site Inhibitor fits
absent into allosteric
site
A change in shape
 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
Negative feedback is achieved
 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
 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 molecules
© 2008 Paul Billiet ODWS
 Phosphofructokinase
 The 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

 Istilah
 Untuk aktivitasnya enzim sering
memerlukan adanya gugus non-protein yaitu
kofaktor, atau mungkin juga suatu molekul
organik kompleks yang disebut koenzim.
Istilah gugus prostetik digunakan untuk
kofaktor yang terikat kuat oleh ikatan
kovalen terhadap protein enzim. Bagian
protein dari enzim dinamakan apoenzim,
sedangkan enzim yang lengkap dinamakan
holoenzim.
 Protein Enzim protein
sederhana

Enzim Konjugasi
Enzim

Protein +
Bukan Protein

Bukan protein =
Protein = apoenzim Gugus prostetik

Organik = Anorganik = kofaktor

Koenzim
Contoh koenzim
Enzyme Cofactors
 Coenzyme - non protein organic, maybe a vitamin.
Enzymes - Cofactors
 Coenzymes (contain a vitamin) or metal ion