Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme...
Transcript of Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme...
![Page 1: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/1.jpg)
Allosteric regulation of enzyme activity
Allostery: Key PointBinding of a ligand at a site different from the active site
modulates the activity. This behavior extends well beyond the normal use of the
word “allostery” which is often used to discuss cooperative interactions.
The molecular basis for allostery provides insight into many regulatory mechanisms.
That which has been learned by studying allosterically regulated enzymes/proteins has profoundly influenced our understanding of cooperativity and enzyme regulation in general.
![Page 2: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/2.jpg)
• The terms allostery and cooperativity are confusing.
• Allostery strictly refers to influence of activity by a distant site.
• Cooperativity indicates that the occupancy of one site in a multisubunit enzyme influences the binding on the others. This is a form of allostery, but is only one manifestation of a general phenomena.
• Unfortunately allostery had become almost exclusively associated with the behavior of multi-subunit enzymes.
Allostery vs. cooperativity
![Page 3: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/3.jpg)
• Multisubunit enzymes that exhibit cooperativity show a sigmoidal initial velocity curve in contrast to the hyperbolic curve for independent subunits.
Kinetic Signature of Cooperativity in Enzymes
0
20
40
60
80
100
0 20 40 60 80 100
Vi
[ substrat ]
LDHhyperbole
PFK-1sigmoïde
0
0.05
0.1
0.15
0.2
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Data 1
1/v 11/v 4
1/v
1/s
Double reciprocal plot
![Page 4: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/4.jpg)
This is the traditional view of feed-back inhibition and regulation in “allosteric” enzymes.
Kinetic Consequences of Allosteric Effectors on Cooperative Enzymes
![Page 5: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/5.jpg)
• Homotrophic (or: homotropic) responses: This refers to allosteric modulation of enzyme activity by substrate molecules. This necessarily must occur in multisubunit enzymes.
• Heterotrophic (or heterotropic) responses: This refers to regulation by non-substrate molecules or combinations of non-substrate and substrate molecules.
• Allosteric regulation can be positive or negative.
Types of Regulation
![Page 6: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/6.jpg)
Allosteric regulation of enzyme activity
Based on genetic data obtained in the 1940
Negative feed-back: the product of a metabolic pathway inhibity the first step
A ---------� B ---------� C ---------� D ---------� ZE1 E2 E3 E4
![Page 7: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/7.jpg)
Allosteric regulation of enzyme activity: an example
Inhibitor
![Page 8: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/8.jpg)
Allosteric regulation of enzyme activity
a. Homotropic effect (POSITIVE or NEGATIVE COOPERATIVITY)Subunit interactions are essential
b. Heterotropic effect (allosteric effectors)Act on the cooperativity
2 type of systemsa. systems V (regulation of Vmax) very unusual!
b. systems K (régulation de l’affinité)
![Page 9: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/9.jpg)
E4 + S ���� E4S KD1
E4S + S ���� E4S2 KD2
E4S2 + S ���� E4S3 KD3
E4S3 + S ���� E4S4 KD4
Allosteric regulation of enzyme activity
KD1 = KD2 = KD3 = KD4 Equal affinity; no cooperativity
KD1 > KD2 > KD3 > KD4 Increased affinity; positive cooperativity
KD1 < KD2 < KD3 < KD4 Decreased affinity; negative cooperativity
0
20
40
60
80
100
0 20 40 60 80 100
N = 1N = 40.5
Vi
[ substrat ]
![Page 10: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/10.jpg)
Empirical Hill equation
Allosteric regulation of enzyme activity
]S[Km
]S[Vmaxv +=
0
20
40
60
80
100
0 20 40 60 80 100
N = 1N = 40.5
Vi
[ substrat ]
NN.
Nmax
]S[K
]S[Vv+
=50
Michaelis equation
![Page 11: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/11.jpg)
NN.
Nmax
]S[K
]S[Vv+
=50
v*K0.5N + v*[s]N = Vmax *[s]N
v*K0.5N = Vmax *[s]N - v*[s]N
v*K0.5N = [s]N * (Vmax – v)
v /(Vmax – v) = [s]N K0.5N
Log(v /(Vmax – v)) = [s]N K0.5N
log(v /(Vmax – v)) =Nlog [s] + Nlog K0.5
Plot log(v /(Vmax – v) in fonction of log[s]: slope N
Allosteric regulation of enzyme activityThe empirical Hill equation
Hill plot
![Page 12: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/12.jpg)
0
20
40
60
80
100
0 20 40 60 80 100
N = 1N = 40.5
Vi
[ substrat ]
Les enzymes allostériques comme switch (intérupteur)
How many times should increase [s] to have v increased from 10% to 90%VmaxN = 1 81 foldN = 4 3 foldN = 0.5 6500 fold NN
.
Nmax
]S[K
]S[Vv+
=50
Power supply with feedback and current limiting
Allosteric regulation of enzyme activity
![Page 13: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/13.jpg)
• Concerted (conceptually simple and often effective)
• Sequential (probably correct but difficult to prove)
There are two Models for Allosteric Regulation
![Page 14: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/14.jpg)
The concerted mechanism
Hypothesis: conformationnal changes in proteinsEnzyme studied: PFK-1 of E. coli
Jacques Monod (1910-1976)
Jeffries Wyman (1901–1995 )
Jean-Pierre Changeux (1936- )
Genetist
Protein biochemist(thermodynamic coupling)
PhD student (at that time)
![Page 15: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/15.jpg)
• Allosteric enzymes are composed of identicalprotomers that occupy equivalent positions in the enzyme. Each protomer contains a binding site for each specific ligand.
• Each protomer can exist in only one of two states. The R (relaxed or high substrate affinity state) or T (taut or low substrate affinity state).
• All protomers in enzyme molecule must be in either the R or T state. The R and T states are in equilibrium with each other.
• The binding affinity of a specific ligand depends on the conformation of the enzyme (R or T) and not on the neighboring site occupancy.
The concerted mechanism
![Page 16: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/16.jpg)
• A general set of equilibrium rate equations can be derived from this model.
The concerted mechanism
![Page 17: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/17.jpg)
Simple Version of the Concerted Model
• This approximate model implies that the substrate does not bind to the inactive state. This must be a gross simplification but it explains the principle. Interestingly, it accounts for a lot of enzymatic behavior (it is the simplest model). It cannot explain negative cooperativity.
![Page 18: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/18.jpg)
Rate Equations for the Simplified Concerted Model
• Where: T state is inactive, kR, and L are the same for all species. n is the number of protomers, kR is the intrinsic enzyme-substrate dissociation const.
• This simple equation provides a simple kinetic model.• Allosteric regulators affect the value of “L”
L =
[T0 ][R0 ]
For the transition R T
Where L is the allosteric constant for the native enzyme v
V max= ([S]/ kR )(1+ [S]/ kR )n−1
L + (1+ [S] / kR )n
![Page 19: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/19.jpg)
Effect of Activator and Inhibitors on the Concerted Model
• Allosteric effectors modify the apparent equilibrium constant for the T to R transition. In this approximation it is assumed that the inhibitor binds to the T state whereas the activator binds exclusively to the R state.
Lapp = L(1+ [ I ]/ kTI )n
(1+ [A] / kRI )n
![Page 20: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/20.jpg)
Régulation allostérique de l’activité enzymatique
Modèle concerté ou symétrique MonodWymanChangeux (cooperativité positive)
Conformation R Conformation T
The concerted mechanism
![Page 21: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/21.jpg)
Sequential Model for Allosteric Regulation of Cooperative Enzymes
Daniel E. Koshland Jr. (1920-2007)
![Page 22: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/22.jpg)
Exemple: La phosphofructokinase, enzyme-clé de la glycolyse
fructose-6-phosphate + ATP => fructose-1,6-bis phosphate + ADPLa cinétique est coopérative pour le fructose-6-phosphate, mais pas pour l'ATP, à basses concentrations. A partir de 0.5 mM, l'ATP est un inhibiteur allostérique (agissant sur un autre site que le site catalytique où il est un substrat).Activateurs allostériques de la phosphofructokinase : ADP, AMP, cAMP, fructose-2,6-bisphosphate , etc (selon l'organisme). Ils se fixent tous au même site allostérique et l'empêchent l’ATP d'avoir son effet inhibiteur.
Régulation allostérique de l’activité enzymatique
![Page 23: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/23.jpg)
Régulation allostérique de l’activité enzymatique
How the allosteric effectors act?positive: stabilise conformation R, decrease the cooperativitynégative: stabilise conformation T, increase the cooperativity
ATP
ADP ADP
T T
T
R
R
Site actif
Site allostérique
![Page 24: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/24.jpg)
S07b Allostérie et coopérativitéThe BASIC concept of the concerted mechanism: the energetic coupling
K = [R]/[T]T R
A ligand binds to the T conformation, but not to the R conformation. How will it change the [R]/[T] equilibrium?
T RK
X
X
Kx
Kx = [TX]/[T][X] � [TX]=Kx [T] [X]
K’ = -------------[R]
[T] + [TX]
K’ = ------------------[R]
[T] + Kx [T] [X]
K’ = ------------------ < K[R]
[T](1 + Kx [X] )
Binding to the T conformer willdecrease the R conformer concentration
![Page 25: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/25.jpg)
PFK de E. coli
ADP
ADP
ADP
ADP
FBP
FBP
PDB 1PFK
Active site
Allosteric siteBetween the subunits
Régulation allostérique de l’activité enzymatique
![Page 26: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/26.jpg)
Site actif
Site allostériqueEntre les sous-unités
Régulation allostérique de l’activité enzymatique
Message to take home: the schematic representation of the two conformation by circles and squares is a gross exageration!
PDB files1pfk ADP, ADP, FBP(conformation R)2pfk (conformation T)
![Page 27: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/27.jpg)
Rea
ctio
n ra
te
[ F-6-P ]
PFK at low and high [ATP]
(practicals in Bordeaux)
![Page 28: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/28.jpg)
Exemples d’enzymes Activateurs Inhibiteursallostériques allostériques allostériques
Hemoglobin 2,3-bisphopshoglycérate(enzyme honoris-causa) pH acide
PFK-1 (muscle) ADP, AMP ATP
Pyruvate kinase L (liver) F-1,6-BP , ATPPhenylalanine
F-1,6-BPase ATP AMP
Glutamate dehydrogenase ADP GTP
Ribonucléotide réductase ATP 2-dATP
Aspartate carbamoyltransferase ATP CTP
Régulation allostérique de l’activité enzymatique
![Page 29: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/29.jpg)
Class I RNR is activated by binding ATP or inactivate d by binding dATP to the activity site located on the RN R1 subunit. When the enzyme is activated, substrates are reduced i f the corresponding effectors bind to the allosteric substrate specificity site. A = when dATP or ATP is bound at th e allosteric site, the enzyme accepts UDP and CDP into the catalyticsite; B = when dGTP is bound, ADP enters the cataly tic site; C = when dTTP is bound, GDP enters the cat alytic site. The substrates (ribonucleotides UDP, CDP, ADP, and GDP) ar e converted to dNTPs by a mechanism involving the gen eration of a free radical.
Régulation allostérique de l’activité enzymatique
RibonucleotideReductase
NDP � 2’-dNDP
![Page 30: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/30.jpg)
• Feed-back inhibition is a common feature of complex biosynthetic pathways. It prevents the accumulation of unwanted intermediates and allows regulation of the level of important metabolites.
• Because the substrate and final product of the pathway are generally chemically different, this demands that the final product bind at a different site relative to the substrate of the allosteric enzyme.
Feed-Back Inhibition
![Page 31: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/31.jpg)
Example 1:Aspartate Transcarbamoylase
Cooperative Allosteric Regulation
This enzyme catalyzes the first committed step in pyrimidine biosynthetic pathway. It is a cooperative enzyme that is heterotropically activated by ATP and heterotropically inhibited by CTP
S07b Allostérie et coopérativité
![Page 32: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/32.jpg)
Synthèse des nucléotides: a. de novo (ATCase)b. voie de récupération
Régulation allostérique de l’activité enzymatique
![Page 33: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/33.jpg)
Etat de transition
Analogue de bi-substrat
Régulation allostérique de l’activité enzymatique
![Page 34: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/34.jpg)
Allosteric Regulation of ATCase
![Page 35: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/35.jpg)
ATCase Subunit Composition
![Page 36: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/36.jpg)
Structure of Active State top view(complex with PALA)
The D3 symmetery is preserved, but this is implicit from the crystal lattice(?).
The position and orientation of the catalytic and regulatory subunits change on transition from the T→R state.
![Page 37: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/37.jpg)
Comparison of Inactive and Active State of ATCase
•The transition is mediated by conformational changes in the interfaces between domains. This is a common (universal?) theme in allosteric enzymes
![Page 38: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/38.jpg)
Conformation Tstabilisée
Conformation T
Régulation allostérique de l’activité enzymatique
![Page 39: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/39.jpg)
Steady-state kinetic behavior of aspartate transcarbamoylase. The velocity of the enzyme-catalyzed reaction is measured by the rate at which the product carbamylaspartate (CAA) is produced. A: The sigmoidal dependence of enzyme velocity V on the concentration of aspartate, at a fixed concentration of the other substrate, carbamyl-P (3.6 mM). CTP lowers the apparent affinity for aspartate and increases the cooperativity, whereas ATP has the opposite effect. B: The kinetic behavior of native and of mercuric ion-treated enzyme. The treated enzyme is dissociated into catalytic trimers and regulatory dimers. The kinetic response of the treated enzyme to aspartate concentration is hyperbolic (i.e., normal Michaelis-Menten), and the value of Vmax is increased. C: The effect of the inhibitor ma-leate, which competes with aspartate, on the enzymatic activity of native and heat-dissociated aspartate transcarbamylase. With the dissociated enzyme, maleate acts as a normal competitive inhibitor, but it activates the native enzyme at low concentrations of both maleate and aspartate. The inhibitory effect of maleate binding at one or a few of the six active sites on the native enzyme must be more than compensated by an allosteric activating effect on the remaining active sites, increasing their affinity for aspartate. (From J. C. Ger-hart, Curr. Top. Cell Reg. 2:275-325, 1970; J. C. Gerhart and A. B. Pardee, Cold Spring Harbor Symp. Quant. Biol. 28:491 -496, 1963.)
![Page 40: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/40.jpg)
Molecular Basis of T→R Transition(cooperativity)
• The T→R transition is mediated through conformational changes at the interface between domains. In the T-state the active site is closed. The active site is not configured for substrate binding in this state.
• The largest change occurs in the loop that extends from 230-250 that lies in the interface between catalytic trimers. This loop contributes to the stability of the closed state. Binding of substrate requires movement of the domains and rearrangement of the hydrogen bonds (to an alternative set).
• The energetics of this transformation are small. This demands that disruption of one set of interactions is compensated by generation of another.
• In many cases these changes involve a order-disorder transition.
![Page 41: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/41.jpg)
ATCase Can Be Described by the Cooperative Model.
• All structures determined to date are consistent with a simple cooperative model for allosteric regulation.
• The hydrogen bonding pattern observed suggests that it is an all-or-nothing type of rearrangement, but this interpretation is biased by the crystallographic symmetry.
• Certainly conversion of one active site demands changes in all others to accommodate the new interactions.
• Suggests that the molecule switches between different but complementary arrangements of hydrogen bonding and non-polar interactions that occur in both the T and R states. This is a common theme.
![Page 42: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/42.jpg)
Régulation allostérique de l’activité enzymatique
![Page 43: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/43.jpg)
Régulation allostérique de l’activité enzymatique
![Page 44: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/44.jpg)
PALA (inhibiteur) est activateur à faible concentration(conversion T -> R)
Régulation allostérique de l’activité enzymatique
![Page 45: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/45.jpg)
Types of Regulation
• Homotropic responses: This refers to allosteric modulation of enzyme activity by substrate molecules. This necessarily must occur in multisubunit enzymes. => coopérativité
• Heterotropic responses: This refers to regulation by non-substrate molecules or combinations of non-substrate and substrate molecules.
• Allosteric regulation can be positive or negative.
S07b Allostérie et coopérativité
![Page 46: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/46.jpg)
Allosteric Regulation of ATCase
S07b Allostérie et coopérativité
![Page 47: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/47.jpg)
S07b Allostérie et coopérativité
![Page 48: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/48.jpg)
Bisubstrate Analogs: Useful Tools
Bisubstrate analogs are enormously useful for trapping enzymes in their active conformation.
S07b Allostérie et coopérativité
![Page 49: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/49.jpg)
Models for Allostery
• Two models for the cooperative binding of ligands to proteins with multiple binding sites have been advanced.
• The MWC (Monod, Wyman, and Changeux) model–which is designated the “concerted” model–assumes that each subunit is identical and can exist in two different conformations or states. The two states have different affinities for the ligand; however, all subunits within one protein can exist in only one of the two states. The binding of ligand to a subunit in the low affinity state, results in a conformational change that places it in the high affinity state. All other subunits, even though they do not have a bound ligand, must follow suit.
• In the Koshland model, which is the sequential model, ligand binding can induce a conformational change in just one subunit. This will then make a similar change in an adjacent subunit, making the binding of a secondligand more likely.
concerted sequential
![Page 50: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/50.jpg)
![Page 51: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/51.jpg)
![Page 52: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/52.jpg)
Pyruvate kinase
![Page 53: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/53.jpg)
![Page 54: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/54.jpg)
Glutamate dehydrogenasehttp://www.danforthcenter.org/smith/gdh.asp
![Page 55: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/55.jpg)
![Page 56: Allostery: Key Pointlascu.free.fr/enzymology/13b Allosterie.pdf · Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from](https://reader033.fdocuments.in/reader033/viewer/2022042210/5eae2417cbecb62c69110a34/html5/thumbnails/56.jpg)
GTP
NADHGlutamate