Presentation on theme: "Allosteric regulation of enzyme activity"— Presentation transcript:
1Allosteric 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.
2Allostery 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.
3Kinetic Signature of Cooperativity in Enzymes LDHhyperbolePFK-1sigmoïdeDouble reciprocal plotMultisubunit enzymes that exhibit cooperativity show a sigmoidal initial velocity curve in contrast to the hyperbolic curve for independent subunits.
4Kinetic Consequences of Allosteric Effectors on Cooperative Enzymes This is the traditional view of feed-back inhibition and regulation in “allosteric” enzymes.
5Allosteric regulation can be positive or negative. Types of RegulationHomotrophic (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.
6Allosteric regulation of enzyme activity E E E E4A B C D ZBased on genetic data obtainedin the 1940Negative feed-back: the product of a metabolic pathway inhibity the first step
7Allosteric regulation of enzyme activity: an example Inhibitor
8Allosteric regulation of enzyme activity Homotropic effect(POSITIVE or NEGATIVE COOPERATIVITY)Subunit interactions are essential2 type of systemsa. systems V (regulation of Vmax) very unusual!b. systems K (régulation de l’affinité)b. Heterotropic effect (allosteric effectors)Act on the cooperativity
9Allosteric regulation of enzyme activity E4 + S E4S KD1E4S + S E4S2 KD2E4S2 + S E4S3 KD3E4S3 + S E4S4 KD4KD1 = KD2 = KD3 = KD4Equal affinity; no cooperativityKD1 > KD2 > KD3 > KD4Increased affinity; positive cooperativityKD1 < KD2 < KD3 < KD4Decreased affinity; negative cooperativity
10Allosteric regulation of enzyme activity Empirical Hill equationMichaelis equation
11Allosteric regulation of enzyme activity The empirical Hill equationHill plotv*K0.5N + v*[s]N = Vmax *[s]Nv*K0.5N = Vmax *[s]N - v*[s]Nv*K0.5N = [s]N * (Vmax – v)v /(Vmax – v) = [s]N K0.5NLog(v /(Vmax – v)) = [s]N K0.5Nlog(v /(Vmax – v)) =Nlog [s] + Nlog K0.5Plot log(v /(Vmax – v) in fonction of log[s]: slope N
12Allosteric regulation of enzyme activity Power supply with feedback and current limitingLes enzymes allostériques comme switch (intérupteur)How many times should increase [s] to have v increased from 10% to 90%VmaxN = foldN = foldN = fold
13There are two Models for Allosteric Regulation Concerted (conceptually simple and often effective)Sequential (probably correct but difficult to prove)
14The concerted mechanism Hypothesis: conformationnal changes in proteinsEnzyme studied: PFK-1 of E. coliJean-Pierre Changeux( )Jacques Monod ( )GenetistPhD student (at that time)Jeffries Wyman (1901–1995 )Protein biochemist(thermodynamic coupling)
15The 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.
16The concerted mechanism A general set of equilibrium rate equations can be derived from this model.
17Simple 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.
18Rate Equations for the Simplified Concerted Model For the transition R TWhere L is the allosteric constant for the native enzymeWhere: 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”
19Effect 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.
20The concerted mechanism Régulation allostérique de l’activité enzymatiqueThe concerted mechanismModèle concerté ou symétrique MonodWymanChangeux(cooperativité positive)Conformation RConformation T
21Sequential Model for Allosteric Regulation of Cooperative Enzymes Daniel E. Koshland Jr. ( )
22fructose-6-phosphate + ATP => fructose-1,6-bisphosphate + ADP Régulation allostérique de l’activité enzymatiqueExemple: La phosphofructokinase, enzyme-clé de la glycolysefructose-6-phosphate + ATP => fructose-1,6-bisphosphate + 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.
23Régulation allostérique de l’activité enzymatique How the allosteric effectors act?positive: stabilise conformation R, decrease the cooperativitynégative: stabilise conformation T, increase the cooperativityTTRSite actifATPSite allostériqueADPADPTR
24The BASIC concept of the concerted mechanism: the energetic coupling S07b Allostérie et coopérativitéThe BASIC concept of the concerted mechanism: the energetic couplingTRK = [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]TRKK’ =[R][T] + [TX]XKxK’ =[R][T] + Kx [T] [X]XBinding to the T conformer willdecrease the R conformerconcentrationK’ = < K[R][T](1 + Kx [X] )
25Régulation allostérique de l’activité enzymatique Allosteric siteBetween the subunitsPFK de E. coliADPFBPPDB 1PFKActive site
26Régulation allostérique de l’activité enzymatique Site allostériqueEntre les sous-unitésMessage to take home: the schematic representation of the two conformation by circles and squares is a gross exageration!Site actifPDB files1pfk ADP, ADP, FBP(conformation R)2pfk(conformation T)
27PFK at low and high [ATP] (practicals in Bordeaux)
28Régulation allostérique de l’activité enzymatique Exemples d’enzymes Activateurs Inhibiteursallostériques allostériques allostériquesHemoglobin ,3-bisphopshoglycérate(enzyme honoris-causa) pH acidePFK-1 (muscle) ADP, AMP ATPPyruvate kinase L (liver) F-1,6-BP , ATPPhenylalanineF-1,6-BPase ATP AMPGlutamate dehydrogenase ADP GTPRibonucléotide réductase ATP 2-dATPAspartate carbamoyltransferase ATP CTP
29Régulation allostérique de l’activité enzymatique RibonucleotideReductaseNDP 2’-dNDPClass 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.
30Feed-Back InhibitionFeed-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.
31S07b Allostérie et coopérativité Example 1: Aspartate Transcarbamoylase Cooperative Allosteric RegulationThis 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
32Régulation allostérique de l’activité enzymatique Synthèse des nucléotides: a. de novo (ATCase)b. voie de récupération
33Régulation allostérique de l’activité enzymatique Etat de transitionAnalogue de bi-substrat
36Structure 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 TR state.
37Comparison 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
38Régulation allostérique de l’activité enzymatique Conformation TConformation Tstabilisée
39Steady-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.)
40Molecular Basis of TR Transition (cooperativity) The TR 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.
41ATCase 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.
42Régulation allostérique de l’activité enzymatique
43Régulation allostérique de l’activité enzymatique
44Régulation allostérique de l’activité enzymatique PALA (inhibiteur) est activateur à faible concentration(conversion T -> R)
45S07b Allostérie et coopérativité Types of RegulationHomotropic responses: This refers to allosteric modulation of enzyme activity by substrate molecules. This necessarily must occur in multisubunit enzymes. => coopérativitéHeterotropic responses: This refers to regulation by non-substrate molecules or combinations of non-substrate and substrate molecules.Allosteric regulation can be positive or negative.
46Allosteric Regulation of ATCase S07b Allostérie et coopérativitéAllosteric Regulation of ATCase
48Bisubstrate Analogs: Useful Tools S07b Allostérie et coopérativitéBisubstrate Analogs: Useful ToolsBisubstrate analogs are enormously useful for trapping enzymes in their active conformation.
49Models 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.concertedsequential