THE BINDING OF LIGAND TO PROTEIN

THE BINDING OF LIGAND TO PROTEIN: 

THE BINDING OF LIGAND TO PROTEIN PRESENTED BY : KALPESH GHEVARIYA

INTRODUCTION: 

INTRODUCTION Ligands are binds to monomeric and oligomeric proteins. Anything which binds to an enzyme or other protein is a ligand, whether it is not a substrate. We will considering binding processes where no subsequent reaction is taking place e.g. the binding to a protein of a non-substrate, or of a substrate for a two substrate reaction in the absence of a second substrate.

THE BINDING OF A LIGAND TO A PROTEIN HAVING A SINGLE LIGAND BINDING SITE: 

THE BINDING OF A LIGAND TO A PROTEIN HAVING A SINGLE LIGAND BINDING SITE Consider the binding of a ligand(S) to a protein(E), in the simplest possible system E + S ES The binding constant k b is defined by the relationship : k b = [ES]/([E][S]) (note that kb=1/ks) So, [ES] = kb [E][S]

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The fractional saturation (Y) of the protein is given by : Y=[ES]/[Eo] = [ES]/([E]+[ES]) = kb[E][S]/{[E]+(Kb[E][S])} = Kb[E][S]/[E] { 1+ (Kb[S])} So, Y = Kb[S] / 1+Kb[S]

COOPERATIVITY: 

COOPERATIVITY If more than one ligand binding site is present on a protein , there is a possibility of interaction between the binding sites during the binding process. This is termed as a cooperativity. There are many types of cooperativity.

Positive cooperativity: 

Positive cooperativity Positive cooperativity is said to occur when the binding of one molecule of a substrate of ligand increases the affinity of the protein for other molecules of the same or different substrate or ligand.

Negative cooperativity: 

Negative cooperativity Negative cooperativity occurs when the binding of one molecule of a substrate of ligand decreases the affinity of the protein for other molecules of the same or different substrate or ligand.

Homotropic cooperativity : 

Homotropic cooperativity Homotropic cooperativity occurs when the binding of one molecule of a substrate or ligand affects the binding to the protein of subsequent molecules of the same substrate or ligand (i.e. the binding of one molecule of a A affects the binding of further molecules of A)

Heterotropic cooperativity: 

Heterotropic cooperativity Heterotropic cooperativity occurs when the binding of one molecule of a substrate or ligand affects the binding to the protein of molecules of a different substrate or ligand. (i.e. the binding of one molecule of A affects the binding of B)

Cooperativity effects may be : 

Cooperativity effects may be Positive and homotropic Positive and heterotropic Negative and homotropic Negative and heterotropic e.g . Allosteric inhibition is an example of negative heterotropic cooperativity Allosteric activation is an example of positive heterotropic cooperativity

Positive homotropic cooperativity and the HILL EQUATION : 

Positive homotropic cooperativity and the HILL EQUATION Let us consider the simplest case of positive homotropic cooperativity in a dimeric protein. There are two identical binding sites, and when the ligand binds to one, it increases the affinity of the protein for the ligand at the other site, so the reaction sequence is : M2 + S → M2S (slow) M2S + S → M2S2 (rapid) Where M is monomeric subunit, termed a protomer , M2 is dimeric protein.

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If the increase in affinity is sufficiently large, and M2S will react with S almost immediately it is formed. Under these conditions ,[M2S2]>>[M2S] and Y = [M2S2]/[(M2)o] where [(M2)o] is the total concentration of dimer present.

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For complete cooperativity, where each protein molecule must be either free of ligand or completely saturated, the reaction may be written M2 + 2S M2S2 Binding constant Kb = [M2S2] / [M2] [S]² FROM WHICH , Y = Kb [S]² /1+Kb[S]² Alternatively , taking logs, log Kb + 2log[S] = log {[M2S2]/[M2]} = log {[M2S2]/{[(M2)o]}-[M2S2]} If n identical binding sites are there , logKb + nlog [S] =log { [ MnSn ] / {[( Mn )o]-[ MnSn ]} } =log (Y/1-Y) this is called the HILL EQUATION DERIVER: ARCHIBALD HILL

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If the HILL EQUATION IS obeyed, a graph of Log(Y/(1-Y)) against log[S] will be linear with slope = n and intercept = logKb. Such graph is called HILL PLOT, and its experimentally determined slope is known as the HILL COEFFICIENT and given the general symbol h.

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At values of Y below 0.1 and above 0.9, the slopes of Hill plots tend to a value of 1, indicating an absence of cooperativity….. The hill coefficient is therefore taken to be the slope of the linear, central portion of the graph, where cooperativity is at greatest point. Where cooperativity is complete , the hill coefficient (h) is equal to the no. of binding sites.

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In the case where S is a substrate and a reaction proceeds to yield products in such a way that the M.M.equation assumption is valid, then initial velocity is proportional to the conc. Of E-bound substrate,i.e Vo∞[MS], and Vo/Vmax=[MS]/[Mo]=Y Where,[MS] is the no. of substrate bound subunits present per unit volume , and [Mo] is total no. of sub units per unit volume, [Mo]=[M]+[MS] Under these conditions, Y/1-Y = Vo/(Vmax-Vo ) against log [So]

SCATCHARD PLOT: 

SCATCHARD PLOT For systems where a single ligand (S) binds to an oligomeric protein (E or Mn) having n identical and non-interacting binding sites for that ligand, Y= [MS]/[MS]+[M] = Kb[S]/1+Kb[S] So,[MS]+[MS]Kb[S] = Kb[S][MS]+Kb[S][M] Kb = [MS]/[M][S] = [MS]/([Mo]-[MS]) [S] SO, [MS]/[S]=Kb[Mo] - Kb[MS] GEORGE SCATCHARD plot a graph of [MS]/[S] against [MS] will be linear. Cooperativity will lead to non linearity.

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