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Allosteric regulation of enzyme activity Allostery: Key Point Binding of a ligand at a site different from the active site modulates the activity. This.

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Presentation on theme: "Allosteric regulation of enzyme activity Allostery: Key Point Binding of a ligand at a site different from the active site modulates the activity. This."— Presentation transcript:

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 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

3 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 LDH hyperbole PFK-1 sigmoïde Double reciprocal plot

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

5 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

6 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 Z E1 E2 E3 E4

7 Allosteric regulation of enzyme activity: an example Inhibitor

8 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 systems a. systems V (regulation of Vmax ) very unusual! b. systems K (régulation de laffinité)

9 E 4 + S E 4 SK D1 E 4 S + S E 4 S 2 K D2 E 4 S 2 + S E 4 S 3 K D3 E 4 S 3 + S E 4 S 4 K D4 Allosteric regulation of enzyme activity K D1 = K D2 = K D3 = K D4 Equal affinity; no cooperativity K D1 > K D2 > K D3 > K D4 Increased affinity; positive cooperativity K D1 < K D2 < K D3 < K D4 Decreased affinity; negative cooperativity

10 Empirical Hill equation Allosteric regulation of enzyme activity Michaelis equation

11 v*K 0.5 N + v*[s] N = Vmax *[s] N v*K 0.5 N = Vmax *[s] N - v*[s] N v*K 0.5 N = [s] N * (Vmax – v) v /(Vmax – v) = [s] N K 0.5 N Log(v /(Vmax – v)) = [s] N K 0.5 N log(v /(Vmax – v)) =Nlog [s] + Nlog K 0.5 Plot log(v /(Vmax – v) in fonction of log[s]: slope N Allosteric regulation of enzyme activity The empirical Hill equation Hill plot

12 Les enzymes allostériques comme switch (intérupteur) How many times should increase [s] to have v increased from 10% to 90%Vmax N = 1 81 fold N = 43 fold N = fold Power supply with feedback and current limiting Allosteric regulation of enzyme activity

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

14 The concerted mechanism Hypothesis: conformationnal changes in proteins Enzyme studied: PFK-1 of E. coli Jacques Monod ( ) Jeffries Wyman (1901–1995 ) Jean-Pierre Changeux (1936- ) Genetist Protein biochemist (thermodynamic coupling) PhD student (at that time)

15 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. The concerted mechanism

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

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 Where: T state is inactive, k R, and L are the same for all species. n is the number of protomers, k R is the intrinsic enzyme-substrate dissociation const. This simple equation provides a simple kinetic model. Allosteric regulators affect the value of L For the transition R T Where L is the allosteric constant for the native enzyme

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 Régulation allostérique de lactivité enzymatique Modèle concerté ou symétrique MonodWymanChangeux (cooperativité positive) Conformation RConformation T The concerted mechanism

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

22 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 lATP d'avoir son effet inhibiteur. Régulation allostérique de lactivité enzymatique

23 How the allosteric effectors act? positive: stabilise conformation R, decrease the cooperativity négative: stabilise conformation T, increase the cooperativity ATP ADP T T T R R Site actif Site allostérique

24 S07b Allostérie et coopérativité The 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 R K X X KxKx 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 will decrease the R conformer concentration

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

26 Site actif Site allostérique Entre les sous-unités Régulation allostérique de lactivité enzymatique Message to take home: the schematic representation of the two conformation by circles and squares is a gross exageration! PDB files 1pfk ADP, ADP, FBP (conformation R) 2pfk (conformation T)

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

28 Exemples denzymes ActivateursInhibiteurs allostériquesallostériques allostériques Hemoglobin 2,3-bisphopshoglycérate (enzyme honoris-causa) pH acide PFK-1 (muscle)ADP, AMPATP Pyruvate kinase L (liver)F-1,6-BP, ATP Phenylalanine F-1,6-BPaseATPAMP Glutamate dehydrogenaseADPGTP Ribonucléotide réductase ATP2-dATP Aspartate carbamoyltransferase ATPCTP Régulation allostérique de lactivité enzymatique

29 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. Régulation allostérique de lactivité enzymatique Ribonucleotide Reductase NDP 2-dNDP

30 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

31 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

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

33 Etat de transition Analogue de bi-substrat Régulation allostérique de lactivité enzymatique

34 Allosteric Regulation of ATCase

35 ATCase Subunit Composition

36 Structure of Active State top view (complex with PALA) The D 3 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 Conformation T stabilisée Conformation T Régulation allostérique de lactivité enzymatique

39 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 lactivité enzymatique


44 PALA (inhibiteur) est activateur à faible concentration (conversion T -> R) Régulation allostérique de lactivité enzymatique

45 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

46 Allosteric Regulation of ATCase S07b Allostérie et coopérativité


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

49 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 second ligand more likely. concertedsequential



52 Pyruvate kinase


54 Glutamate dehydrogenase


56 GTP NADH Glutamate

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