Peter Fajer

Biophysical Methods in Biology

Lecture 10

Ligand Binding
Consider ligand A binding to macromolecule P:

A + P AP
Equilibrium constant is defined as:

[1]

Kd =

[ P ][ A] [ PA]

[2]

(note that Kd is often referred wrongly as binding constant: Kb = 1/Kd) define fractional occupancy (moles of ligand mole of macromolecule) r:

r=

[ A] bound [ PA] = [ P] total [ P] + [ PA]

[!]

substitute [P][A]/Kd for [PA]:

r=

[ A] K d + [ A]

["]

this hyperbolic dependence is called binding isotherm or (Langmuir isotherm)#

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Peter Fajer

Biophysical Methods in Biology

Lecture 10

binding isotherm
1#2 1 $#& $#% $#" $#2 $ $ ' 1$ [A] 1' 2$ 2' (d)1 (d)' (d)' (d)1$

*ote that although the isotherm has the concentration for free ligand [A] as well as bound ligand ([+]bound in r ) you ha,e to measure only one (bound or free) because conser'ation o( mass the total ligand [A]total = [A]+[PA] (and total macromolecule [P]total = [P]+[PA])# -easure the concentrations of free or bound ligand by ,ariety of methods: chromatography. equilibrium dialysis. ultrafiltration. spectroscopy#

Multiple binding sites

-acromolecule can ha,e more than one site for binding the ligand# /hese sites can be independent : binding of one ligand does not influence binding of the ne0t. or cooperative (binding of one affects binding of another)#

Independent
A + P A1 P + A2 P +.......
identical sites: +11)+21 or different sites: +11+21 [']

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Peter Fajer

Biophysical Methods in Biology

Lecture 10

Identical sites
site 1

site 2 macromolecule

all sites are independent and identical so that total number of sites is n[P]total rather than [P]total :

rall _ sites = nrsin gle _ site = n

[ A] K d + [ A]

[%]

Scatchard plot
plot the ratio of r/[A] ,ersus r:

r n r = [ A] K d K d

[&]

2or identical binding this results in a linear plot# 3e,iation from linearity implies non4identical sites#

r/[A]

Di erent sites
to be ,ery general: each class of sites with different Kdi can ha,e ni identical sites

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"

Peter Fajer

Biophysical Methods in Biology

Lecture 10

site class 1

site class 2

macromolecule

/he binding isotherm is a sum of the single binding isotherms. each corresponding to different class of sites:

r = r1 + r2 +..... =

n1 [ A] n2 [ A] + +........ K d ,1 + [ A] K d ,2 + [ A]

[5]

!ooperati"it#
6inding to one site on a molecule modulates binding of another# 2or strong cooperati,ity. binding of one ligand triggers binding to n sites:

nA + P An P

[7]

Kd =

[ P ][ A] n [ An P ]
n [ PAn ] n[ A] =n = [ P] + [ PAn ] K d + [ A] n

[1$]

[ A] r = bound [ P] total

[11]

or measure the ratio of filled sites $ %$=r/n& to the unfilled sites (1'$):

Y r [ A] = = 1Y n r Kd
)*ill+s e,uation)

[12]

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Peter Fajer

Biophysical Methods in Biology

Lecture 10

(ill)s Plot
1lot log%$/%1'$& "* log[A]. the slope is n8 an intercept is 1/Kd#

positi"e cooperati"it# n=log[r (n4r)] no cooperati"it# n=1 negati"e cooperati"it# n=+*,

log [+]

Kinetics
/ime course of reaction. 9inetics gi,e information about the rates rather than equilibria# :f course rates and equilibria are related Keg = .+1/.'1. howe,er the ratio of rates tells us nothing about the ,alue of each rate i#e# is it fast or is it slow# /wo sorts of 9inetics are usually considered:

stead#'state: in which the forward flu0 ) bac9ward flu0 resulting in no change of concentration8

transient .inetics: non4equilibrium 9inetics when concentrations are changing#

steady-state (Michaelis-Menten)
E + S X E + P
k 1 k 2 k1 k2

[1!]

for a reaction with an intermediate / the rate of intermediate;s production is equal to that of its disappearance:

d[ X ] = ( k 1 + k 2 ) [ X ] k1 [ E ][ S ] k 2 [ E ][ P ] = 0 dt

[1"]

/he rates of substrate disappearance and the appearance of the product are identical:
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Peter Fajer

Biophysical Methods in Biology

Lecture 10

d [ S ] d [ P] = = k 1 [ E ][ S ] k 1 [ X ] dt dt

[1']

2rom mass conser,ation we ha,e:

[ E ] = [ E ] +[ X ] [ S ] = [ S ] + [ P]
o o

[1%]

+t the beginning of the reaction:

[ P] = 0

[15]

thus the initial ,elocity " can be sol,ed since we ha,e fi,e equations with " un9nowns (concentrations of E. <. =. 1)

v=

d[ S] Vmax [ S ] = dt [ S ] + K Michaelis

[1&]

(dirty shortcut: reali>e that initial ,elocity is a ma0imum ,elocity reduced by the partial en>yme occupancy. i#e#:

v = rVmax
then substitute " for r in the equation for Langmuir isotherm#) t0rno"er

[1&]

k cat =

Vmax Eo

[17]

non'stead# state
<teady4state appro0imation is not always applicable. for general reaction:

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Peter Fajer

Biophysical Methods in Biology

Lecture 10

E + S X1
k 1

k1

[2$]

the rate equations are:

d[ E ] d[ S ] d X 1 = = = k 1 [ E ][ S ] k 1 X 1 dt dt dt

[ ]

[ ]

[21]

with two mass con,ersation relationships:

[ E ] = [ E ] +[ X ] [ S ] = [ S] +[ X ]
o 1 o 1

[22]

/he differential (rate) equation can be sol,ed but it is a mess since the equation is non4linear (the rate depends on product of [E] and [<]). better appro0imate that < o ?? Eo so that concentration of substrate ne,er changes (pse0do' irst order appro1imation ):

d X1 dt

[ ] =k

( [ E ] [ X ] )[ S ] k [ X ]
o 1 o 1 1

[2!]

/his equation is a linear in /1 and it can be integrated to gi,e the concentration of /1 as function of time:

ln

[ E ] [ E
o

[ E ] [ E equilibrium

equilibrium

] = ( k [ S ] + k )t ]
1 o 1

[2"]

1resence of two intermediates complicates the affairs considerably:

E + S X1 X 2
k 1 k 2

k1

k2

[2']

conser,ation of mass gi,es:

[ E ] = [ E] +[ X ] +[ X ] [ S ] = [ S] +[ X ] +[ X ]
o 1 2 o 1 2

[2%]

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Peter Fajer

Biophysical Methods in Biology

Lecture 10

the rate of en>yme disappearance is:

d[ E ] = k 1 [ E ][ S ] k 1 X 1 dt

[ ]

[25]

the rate of product appearance is:

d X2 dt

[ ] =k

[ X ] k [ X ]
2 2 1

[2&]

+t this point you are ad,ised to gi,e up# @hat you ha,e is the set of simultaneous/ non0linear di((erential e,uations which e,en your grandma can;t sol,e# 6ill Aates to the rescue8 integrate the set using a 1C and any mathematical pac9age with n0merical integration routines e#g# -athcad. -athematica. -atlab#

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