1. Allosteric regulation of enzyme activity
Allostery: Key Point
Binding 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.

2. Allostery vs. cooperativity
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.

3. Kinetic Signature of Cooperativity in Enzymes
LDH
hyperbole
PFK-1
sigmoïde
Double reciprocal plot
Multisubunit enzymes that exhibit cooperativity show a sigmoidal initial velocity curve in contrast to the hyperbolic curve for independent subunits.

4. Kinetic Consequences of Allosteric Effectors on Cooperative Enzymes
This is the traditional view of feed-back inhibition and regulation in “allosteric” enzymes.

5. Allosteric regulation can be positive or negative.
Types of Regulation
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.

6. Allosteric regulation of enzyme activity
E E E E4
A  B  C  D  Z
Based on genetic data obtained
in the 1940
Negative feed-back: the product of a metabolic pathway inhibity the first step

7. Allosteric regulation of enzyme activity: an example
Inhibitor

8. Allosteric regulation of enzyme activity
Homotropic effect
(POSITIVE or NEGATIVE COOPERATIVITY)
Subunit interactions are essential
2 type of systems
a. systems V (regulation of Vmax) very unusual!
b. systems K (régulation de l’affinité)
b. Heterotropic effect (allosteric effectors)
Act on the cooperativity

9. Allosteric regulation of enzyme activity
E4 + S  E4S KD1
E4S + S  E4S2 KD2
E4S2 + S  E4S3 KD3
E4S3 + S  E4S4 KD4
KD1 = KD2 = KD3 = KD4
Equal affinity; no cooperativity
KD1 > KD2 > KD3 > KD4
Increased affinity; positive cooperativity
KD1 < KD2 < KD3 < KD4
Decreased affinity; negative cooperativity

10. Allosteric regulation of enzyme activity
Empirical Hill equation
Michaelis equation

11. Allosteric regulation of enzyme activity
The empirical Hill equation
Hill plot
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

12. Allosteric regulation of enzyme activity
Power supply with feedback and current limiting
Les enzymes allostériques comme switch (intérupteur)
How many times should increase [s] to have v increased from 10% to 90%Vmax
N = fold
N = fold
N = fold

13. There are two Models for Allosteric Regulation
Concerted (conceptually simple and often effective)
Sequential (probably correct but difficult to prove)

14. The concerted mechanism
Hypothesis: conformationnal changes in proteins
Enzyme studied: PFK-1 of E. coli
Jean-Pierre Changeux
( )
Jacques Monod ( )
Genetist
PhD student (at that time)
Jeffries Wyman (1901–1995 )
Protein biochemist
(thermodynamic coupling)

15. The concerted mechanism
Allosteric enzymes are composed of identical protomers 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.

16. The concerted mechanism
A general set of equilibrium rate equations can be derived from this model.

17. 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.

18. Rate Equations for the Simplified Concerted Model
For the transition R T
Where L is the allosteric constant for the native enzyme
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”

19. 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.

20. The concerted mechanism
Régulation allostérique de l’activité enzymatique
The concerted mechanism
Modèle concerté ou symétrique MonodWymanChangeux
(cooperativité positive)
Conformation R
Conformation T

21. Sequential Model for Allosteric Regulation of Cooperative Enzymes
Daniel E. Koshland Jr. ( )

22. fructose-6-phosphate + ATP => fructose-1,6-bisphosphate + ADP
Régulation allostérique de l’activité enzymatique
Exemple: La phosphofructokinase, enzyme-clé de la glycolyse
fructose-6-phosphate + ATP => fructose-1,6-bisphosphate + ADP
La 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.

23. Régulation allostérique de l’activité enzymatique
How the allosteric effectors act?
positive: stabilise conformation R, decrease the cooperativity
négative: stabilise conformation T, increase the cooperativity T T R
Site actif
ATP
Site allostérique
ADP
ADP T R

24. The BASIC concept of the concerted mechanism: the energetic coupling
S07b Allostérie et coopérativité
The BASIC concept of the concerted mechanism: the energetic coupling T R
K = [R]/[T]
A ligand binds to the T conformation, but not to the R conformation. How will it change the [R]/[T] equilibrium?
Kx = [TX]/[T][X]  [TX]=Kx [T] [X] T R K
K’ =
[R]
[T] + [TX] X Kx
K’ =
[R]
[T] + Kx [T] [X] X
Binding to the T conformer will
decrease the R conformer
concentration
K’ = < K
[R]
[T](1 + Kx [X] )

25. Régulation allostérique de l’activité enzymatique
Allosteric site
Between the subunits
PFK de E. coli
ADP
FBP
PDB 1PFK
Active site

26. Régulation allostérique de l’activité enzymatique
Site allostérique
Entre les sous-unités
Message to take home: the schematic representation of the two conformation by circles and squares is a gross exageration!
Site actif
PDB files
1pfk ADP, ADP, FBP
(conformation R)
2pfk
(conformation T)

27. PFK at low and high [ATP]
(practicals in Bordeaux)

28. Régulation allostérique de l’activité enzymatique
Exemples d’enzymes Activateurs Inhibiteurs
allostériques allostériques allostériques
Hemoglobin ,3-bisphopshoglycérate
(enzyme honoris-causa) pH acide
PFK-1 (muscle) ADP, AMP ATP
Pyruvate kinase L (liver) F-1,6-BP , ATP
Phenylalanine
F-1,6-BPase ATP AMP
Glutamate dehydrogenase ADP GTP
Ribonucléotide réductase ATP 2-dATP
Aspartate carbamoyltransferase ATP CTP

29. Régulation allostérique de l’activité enzymatique
Ribonucleotide
Reductase
NDP  2’-dNDP
Class I RNR is activated by binding ATP or inactivated by binding dATP to the activity site located on the RNR1 subunit. When the enzyme is activated, substrates are reduced if the corresponding effectors bind to the allosteric substrate specificity site. A = when dATP or ATP is bound at the allosteric site, the enzyme accepts UDP and CDP into the catalytic site; B = when dGTP is bound, ADP enters the catalytic site; C = when dTTP is bound, GDP enters the catalytic site. The substrates (ribonucleotides UDP, CDP, ADP, and GDP) are converted to dNTPs by a mechanism involving the generation of a free radical.

30. Feed-Back Inhibition
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.

31. S07b Allostérie et coopérativité
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

32. Régulation allostérique de l’activité enzymatique
Synthèse des nucléotides: a. de novo (ATCase)
b. voie de récupération

33. Régulation allostérique de l’activité enzymatique
Etat de transition
Analogue de bi-substrat

34. Allosteric Regulation of ATCase

35. ATCase Subunit Composition

36. 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.

37. 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

38. Régulation allostérique de l’activité enzymatique
Conformation T
Conformation T
stabilisée

39. Steady-state kinetic behavior of aspartate transcarbamoylase
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: , 1970; J. C. Gerhart and A. B. Pardee, Cold Spring Harbor Symp. Quant. Biol. 28: , 1963.)

40. 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 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.

41. 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.

42. Régulation allostérique de l’activité enzymatique

43. Régulation allostérique de l’activité enzymatique

44. Régulation allostérique de l’activité enzymatique
PALA (inhibiteur) est activateur à faible concentration
(conversion T -> R)

45. S07b Allostérie et coopérativité
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.

46. Allosteric Regulation of ATCase
S07b Allostérie et coopérativité
Allosteric Regulation of ATCase

47. S07b Allostérie et coopérativité

48. Bisubstrate Analogs: Useful Tools
S07b Allostérie et coopérativité
Bisubstrate Analogs: Useful Tools
Bisubstrate analogs are enormously useful for trapping enzymes in their active conformation.

49. Models for Allostery concerted sequential
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 second ligand more likely.
concerted
sequential

52. Pyruvate kinase

54. Glutamate dehydrogenase

56. GTP
NADH
Glutamate