LECTURE 4: REACTION MECHANISM & INHIBITORS · Protein Synthesis Two general classes of inhibitors...
Transcript of LECTURE 4: REACTION MECHANISM & INHIBITORS · Protein Synthesis Two general classes of inhibitors...
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LECTURE 4:REACTION MECHANISM &
INHIBITORS
Chymotrypsin
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LECTURE OUTCOMES
After mastering the present lecture materials,students will be able to1. to explain reaction mechanisms of between enzyme
and substrate2. to explain the influence of irreversible inhibitors on
enzymatic reactions3. to explain the influence of reversible inhibitors on
enzymatic reactions4. to explain competitive, uncompetitive, noncompetitiive
inhibition of enzymatic reactions5. to calculate KM and Vmax of reactions catalyzed by
enzymes with the presence of inhibitors
LECTURE LAYOUT1. REACTION MECHANISMS1.1 Sequential Reactions1.2 Ping-Pong Reactions1.3 Kinetics of Bi-Bi Reactions
2. REACTON INHIBITION2.1 Irreversible Inhibition2.2 Reversible Inhibition2.2.1 Competitive inhibition,2.2.2 Uncompetitive inhibition.2.2.3 Noncompetitive inhibition2.3 Feedback Inhibition
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1. REACTION MECHANISMS Most biochemical reactions are not simple-,
single-substrate reactions, but typicallyinvolve two or three substrates that combineto release multiple products.
Yet, enzymatic reactions involving twosubstrates and yielding two productsaccount for ~60% of known biochemicalreactions.
Bi-Bi reactions, transferring groupscatalyzed by enzymes, fall under two majormechanistic classifications.
- Sequential reactions- Ping pong reactions
1.1 Sequential reactions Reactions in which all substrates combine
with the enzyme before a reaction can occurand products be released are known assequential reactions which is also calledsingle-displacement reactions.
Sequential reactions can be further classifiedinto 2 types :(a) Ordered Mechanism is that with a compulsory
order of substrate addition to the enzyme
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(b) Random mechanism is that with no preferencefor the order of substrate addition
A B P Q
EA EQ EEABEPQESequential Reactions: Ordered MechanismAll substrates must combine with enzyme beforereaction can occur
Bisubstrate reactions
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Sequential Reactions: Random Mechanism
1.2 Ping pong reactions Ping pong reactions are those in which one
or more products are released before allsubstrates have been added.
A P B Q
EAFP FBEQ EFE
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1.3 Kinetics of Bi-Bi Reactions tSteady state kinetic measurements can be
utilized to distinguish among the foregoingbisubstrate mechanisms. For doing so, onemust first derive their rate equations.
This can be done in much the same way asfor single-substrate-enzymes, i.e., solving aset of simultaneous linear equationsconsisting of an equation expressing thesteady state condition for each kineticallydistinct enzyme complex plus one equationrepresenting the conservation condition forthe enzyme.
The rate equations for the above-describedbisubstrate mechanisms in the absence ofproducts are given below in doublereciprocal form.For ordered Bi-Bi reactions:
Y = a + bX1 + cX2 + dX3Y = 1/V, a = 1/Vm, b = KM
A/Vm, c =KM
B/Vm, d = KSAKM
B/Vm, X1 = 1/[A], X2 =1/[B], and X3 = 1/([A][B])
]B][A[VmKK
]B[VmK
]A[VmK
VmV
BM
AS
BM
AM
11
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For random Bi Bi reactions :
Y = a + bX1 + cX2 + dX3Y = 1/V, a = 1/Vm, b = KS
AKMB/(VmKS
B), c =KM
B/Vm, d = KSAKM
B/Vm, X1 = 1/[A], X2 =1/[B], and X3 = 1/([A][B])
For ping pong Bi Bi reactions :
]B][A[VmKK
]B[VmK
]A[VmKKK
VmV
BM
AS
BM
BS
BM
AS
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]B[VmK
]A[VmK
VmV
BM
AM
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2. REACTION INHIBITION An important number of compounds have the
ability to combine with certain enzymes in eithera reversible or irreversible manner, and thereby1. block the enzyme, but do not usually destroy it2. reduce the rate of enzymic reactions3. work specifically in general, and at low concentrations
Such compounds are called INHIBITORS andinclude;- drugs,- antibiotics,- poisons,
- anti metabolites- products of enzymic
reactions.
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30S1 32 GTP
1 2 3 GTP
Initiation Factors
mRNA
3
12 GTP
30SInitiationComplex
f-met-tRNA
Spectinomycin
Aminoglycosides
12
GDP + Pi 50S
70SInitiationComplex
AP
Inhibition ofProtein Synthesis
Two general classes of inhibitors arerecognized ;1. Irreversible2. Reversible
2.1 Irreversible An irreversible inhibitor forms a covalent bond with
a specific function, usually an amino acid residue,which may, in some manner, be associated withthe catalytic activity of the enzyme.- Many examples of enzyme inhibitors covalently bind
not at the active site, but physically block the activesite
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- The inhibitor cannot be released by dilution ordialysis; kinetically, the concentration and hence thevelocity of active enzyme is lowered in proportion tothe concentration of the inhibitor and thus the effectis that of noncompetitive inhibition.
k1E + S ES E + Pk2
k3
k4+I
EIkI
IRREVERSIBLE INHIBITORS
Examples of irreversible inhibitors include;- DIFP (diisopropyl fluorophosphate) which reacts
irreversibly with serine proteases, chymotrypsin- Iodoacetate which reacts with essential sulfhydryl
group of an enzyme such as triose phosphatedehydrogenase:E-SH+ICH2COOH E-SCH2COOH+HI
HH3C C CH3
O
O
H3C C CH3
H
F P OCH3OH +
Enz
HH3C C CH3
O
O
H3C C CH3
H
P OCH3 O
Enz
+ HF
DIPF
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2.2 Reversible Inhibition As the term implies, this type of inhibition
involves equilibrium between the enzyme andthe inhibitor, the equilibrium constant (Ki) beinga measure of the affinity of the inhibitor for theenzyme.
Three distinct types of reversible inhibition areknown;- Competitive inhibition,- Uncompetitive inhibition- Noncompetitive inhibition
2.2.1 Competitive Inhibition Compounds that may or may not be structurally
related to the natural substrate combinereversibly with the enzyme at or near the activesite
The inhibitor andthe substratetherefore competefor the same siteaccording to thereaction as shownon the right side.
ES and EI complexes are formed, butEIS complexes are never produced.
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● One can conclude that high concentrations ofsubstrate will overcome the inhibition by causing thereaction sequence to swing to the right. The velocityof reaction can be calculated by the followingequation
Competitive
]S[K
]I[1K
]S[VV
IM
max
The rate of reaction can be calculated from thefollowing equation.
1/[S]
1/V
-1/Vmax-1/KM
-1/{KM(1+[I]/KI}
-I
+I
Among other enzymes that may undergo competitiveinhibition is succinic dehydrogenase, which readilyoxidizes succinic acid to fumaric acid.
If increasing concentrations of malonic acid, whichclosely resembles succinic acid in structure, areadded, however, succinic dehydrogenase activity fallsmarkedly
Succinate Glutarate Malonate Oxalate
Succinate Dehydrogenase
Substrate Competitive Inhibitor
Product
C-OO-
C-HC-HC-OO-
C-OO-
H-C-HH-C-H
C-OO-
C-OO-
H-C-HH-C-HH-C-H
C-OO-
C-OO-
C-OO-
C-OO-
H-C-HC-OO-
This inhibition cannow be reversed byincreasing in turnthe concentration ofthe substratesuccinic acid.
Competitive Inhibition
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2.2.2 Uncompetitive Inhibition Compounds that combine only with the ES
complex, not with the free enzyme, are calleduncompetitive inhibitors. The inhibition is notovercome by high substrate concentrations.
k1E + S ES E + Pk2
k3
+
ESIkI
I
HIV protease in a complexwith the protease inhibitorritonavir
The structure of theprotease is shown by thered, blue and yellowribbons. The inhibitor isshown as the smaller ball-and-stick structure near thecentre. Created from PDB
Human immunodeficiency virusScanning electron micrograph of HIV-1 (in green) budding from culturedlymphocyte. Multiple round bumps on cell surface represent sites of assemblyand budding of virions
Peptide-based proteaseinhibitor ritonavir
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KM value is consistently smaller than the KM valueof the uninhibited reaction which implies that S ismore effectively bound to the enzyme in thepresence of the inhibitor.
The equation used to calculate the velocity of thenoncompetitive inhibition is as follows
IM
max
K]I[
1]S[K
]S[VV
1/[S]
1/V
-1/Vmax-1/KM
(1+[I]/KI)/Vmax
-(1+[I]/KI)/KMx
-I
+I
Uncompetitive
2.2.3 Noncompetitive Inhibition Compounds that reversibly bind with either the
enzyme or the enzyme substrate complex aredesignated as noncompetitive inhibitors
Noncompetitive inhibition therefore differs fromcompetitive inhibition in that the inhibitor cancombine with ES, and S can combine with EI to formin both instances EIS.
This type of inhibition is not completely reversed byhigh substrate concentration since the closedsequence will occur regardless of the substrateconcentration
Since the inhibitor binding site is not identical to nordoes it modify the active site directly, the KM is notaltered.
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IM
max
K]I[
1]S[K
]S[VV
1/[S]
1/V
-1/Vmax-1/KM
(1+[I]/KI)/Vmax
-I
+I
Mix inhibition
Noncompetitive
k1E + S ES E + Pk2
k3
kI
+
ESI
I+
EI
I
+ SkI
2.3 Feedback Inhibition (Allosteric Effectors) The activity of some enzymes is controlled by
certain molecules binding to a specific regulatory(or allosteric) site on the enzyme, distinct fromthe active site.
Different molecules can either inhibit or activatethe enzyme, allowing sophisticated control of therate. Only a few enzymes can do this, and theyare often at the start of a long biochemicalpathway.
They are generally activated by the substrate ofthe pathway and inhibited by the product of thepathway, thus only turning the pathway onwhen it is needed.
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The switch: Allosteric inhibition
Allosteric means “other site”
E
Active site
Allostericsite
© 2008 Paul Billiet ODWS
Switching off These enzymes
have two receptorsites
One site fits thesubstrate like otherenzymes
The other site fitsan inhibitormolecule
Inhibitor fits intoallosteric site
Substratecannot fit intothe active site
Inhibitormolecule
© 2008 Paul Billiet ODWS
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This process is known as feedbackinhibition.
Feedback Inhibition
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HOW TO SOLVE THE EQUATIONS
1. Competitive inhibitor
y =1/V; x = 1/[s] a = 1/Vmax b = KM(1+[I]/KI)/Vmax
]S[K
]I[1K
]S[VV
IM
max
maxV1
]S[1
V
K]I[
1K
V1
max
IM
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2. Uncompetitive
y =1/V; x = 1/[s] a = (1+[I]/KI)/Vmax b = KM/Vmax
IM
max
K]I[
1]S[K
]S[VV
maxV
K]I[
1
]S[1
maxVK
V1 IM
3. Noncompetitive Inhibition
y =1/V; x = 1/[s] a = (1+[I]/KI)/Vmax b = KM(1+[I]/KI)/Vmax
IM
max
K]I[
1]S[K
]S[VV
maxV
K]I[
1
]S[1
maxV
K]I[
1K
V1 II
M
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QUIZ (10 min)1. How is enzyme specificity achieved ?2. Calculate Vmax & KM from the following data, and does the
reaction obey Michaelis-Menten kinetics ?
[DNA]mol total
nucleotides/L
Free nucleotides in solution,V (pmol/L)
0 min 10 min
1.0 x 10-5 0.05 5.1
1.0 x 10-6 0.04 4.5
1.0 x 10-7 0.06 3.2
1.0 x 10-8 0.04 1.4
1.0 x 10-9 0.04 0.23
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ANSWERS1. The enzyme specificity is achieved
through the characteristic of active site2. Vmax = 4.36695
KM = 2.2E-08R2 = 0.999864, so the reaction obeysMichaelis-Menten kinetics
SOAL
Diketahui suatureaksi enzimatistanpa dan denganinhibitor dengan [I] =2,2.104M.
Hitunglah KM danVmax tanpa dandengan I serta KI
[S] V(-I) V(+I)
1*10-4 28 17
1.5*10-4 36 23
2.0*10-4 43 29
5*10-4 65 50
7.5*10-4 74 61
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Km
I II Competitive Non-competitive Uncompetitive
Dire
ct P
lots
Dou
ble
Rec
ipro
cal
Vmax Vmax
Km Km’ [S], mM
vo
[S], mM
vo
I I
Km [S], mM
Vmax
I
Km’
Vmax’Vmax’
Vmax unchangedKm increased
Vmax decreasedKm unchanged Both Vmax & Km decreased
I
1/[S]1/Km
1/vo
1/ Vmax
ITwo parallellines
I
Intersectat X axis
1/vo
1/ Vmax
1/[S]1/Km 1/[S]1/Km
1/ Vmax
1/vo
Intersectat Y axis
= Km’
Juang RH (2004) BCbasics
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Example of a suicide inhibitor
9/28/05
Diisopropylfluorophosphate(DIFP) forms a covalentbond with an active -siteresidue (Ser) of the enzymechymotrypsin.
Every molecule that reactsis inactivated irreversibly.
Here, a key active site Seris bound irreversibly to theinhibitor, preventing it fromdoing its "normal" job.
12Irreversible inhibitor
The effect of enzymeinhibition
Irreversible inhibitors: Combine with thefunctional groups of the amino acids in theactive site, irreversibly
Examples: nerve gases and pesticides,containing organophosphorus, combinewith serine residues in the enzymeacetylcholine esterase
© 2008 Paul Billiet ODWS