PKa Lecture

8/12/2019 PKa Lecture

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Protonation States and pKa

Suggested Readings:

Markley, J. L. (1975). Observation of Histidine Residues in Proteins by Nuclear Magnetic

Resonance Spectroscopy.Acc. Chem. Res. 8, 70-80.

Cosgrove, M.S. et.al (2002), The Catalytic Mechanism of G-6-P Dehydrogenases,

Biochemistry, 41, 6939-6945.

Bartik, K. et al. (1994), Measurement of the Individual pKa Values of Acidic Residues of Hen

And Turkey Lysozymes by Two Dimensional 1H NMR. Biophysical J., 66, 1180-1184

Anderson, D.E. et al. (1990). pH induced denaturation of proteins: A single salt bridge

contributes 3-5 kCal/mol to the free energy of folding of T4 lysozyme. Biochemistry,29, 2403-

2408.

Smith, R. et al. (1996) Ionization states of the catalytic residues in HIV-1 protease. Nat. Struct.

Biol.,3, 946-950.

Dyson, H.J. et al. (1996) Direct Measurement of the Aspartic Acid 26 pKa for reduced E.coli

Thioredoxin by 13C NMR Biochemistry,35, 1-6

Pujato, M. (2006), The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in

intrinsically disordered proteins with only local interactions Biochimica Biophysica Acta,1227-

1233

Munia [email protected] 16

th, 2007


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Deprotonation reaction:

The pKaof a titrating site is defined as the pHfor which the site is 50%

occupied: The pHfor which the occupancy is 0.5.

HA + H2O

A-

+ H3O+

Ka = [A-] [H3O+]

[HA]

1[H3O

+] =1

Ka

[A-][HA]

Henderson-Hasselbach equation:

-log [H3O+] = -log Ka + log [A-]/[HA]

pH = pKa + log 1-

is degree of protonation or occupancy: Number of bound protons as a

function of pH

(1)

(2)

(3)

(4)


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( ) ( )HKaeH pp10ln1

1p +=

One state transition

0.4p =a

K

Definition of pKa

pH = pKa + log 1-

Titration curve


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Titration curves of amino acids

Since amino acids are (at least) diprotic their titration curves appear a little

different from a simple acid-each proton will have a pKa value and thus there are two or more stages in the

titration curve

Depending on where in the titration you are looking (i.e. at which pH) a

different form of the amino acid will be prevalent

Remember that pH is notation for proton concentration and that pKa is the

equilibrium constant for ionization

- thus pKa is a measure of the tendency for a group to give up a proton

-as the pKa increases by one unit the tendency to give up the protondecreases tenfold


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The positively charged amino group attached to the a-

carbon helps to push the departing proton of the carboxyl

group out more easily.

The inflection point pIis the point when removal of thefirst proton is complete and he second has just begun so

the amino acids prevalent form is as a dipolar ion

pH < pI: net positive charge

pH > pI: net negative charge


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pKa of ionizable side chains

pKa= pH for 50%

dissociation,

Note range


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pKaof some amino acids


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Factors that affect pKavalues

Ionizable residues encounter two differences inside folded proteinscompared to water

They are partly desolvated by the protein

This is especially unfavorable for the charged form (because its anion) but its also unfavorable for the neutral form (because its adipole.

They form new interactions with other residue.

These new interactions may be energetically favorable or unfavorable.

Usually the charged form is more affected than the neutral form dueto these interactions


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e.g. an aspartate with a low pKa

this aspartate is partly buried but itaccepts ~4 hydrogen bonds fromnearby residues

its also close to some positivelycharged residues

the charged form is very happyhere, so it becomes more difficult toadd a proton to it

so we have to increase [H+] (lowerpH) to add the proton

so the pKa of the residuedecreases from 4 to ~2


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Simple rules for guessing pKashifts

acidic residues (asp & glu)

if charged form is unhappy:

deprotonation is more

difficult so pKa shifts up

if charged form is happy:

deprotonation is easier

so pKa shifts down

basic residues

(arg, lys & his)

if charged form is unhappy:

deprotonation is easier

so pKa shifts down

if charged form is happy:

deprotonation is more

difficult so pKa shifts up

COOHCOOH COOCOO+ H+ H33OO++ NHNH33++NHNH22+ H+ H33OO++

remember: a pKais just the G for deprotonation


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Reasons for interest in pKas [1]

enzyme activity is pH dependent

many catalytic steps involve addition or removal of

protons

the rates of these steps will depend on the pH and the

pKas of the residues involved

enzymes have optimal pHs

(sometimes loss of activity at

non-optimal pHs is due to

unfolding of the enzyme)


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protein stability is pH dependent

if the pKaof a residue is different in the folded state from

its value in the unfolded state, the proteins stability will

depend on pH

For most proteins the folded state is

only 1-5 kCal/mol more favored

than the unfolded state. A typical

ionic interaction is around 2-5

kCal/mol. So a single ionicinteraction can determine whether

or not a protein will fold.

pHpH

GG

un

fold

un

fold

(Pace et al. Biochemistry 2: 2564 (1990)(Pace et al. Biochemistry 2: 2564 (1990)


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a protonation equilibrium can be thought of as a very simple

ligand-binding reaction (with the ligand being H+)

knowing the pKaof a protein residue and the proteins

structure...

we can start to determine the relative importance of different

factors, e.g.:

1. desolvation effects

2. charge-charge interactions

3. protein dielectric properties


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From the change in pKa, one can determine the free energy (G)

associated with the reaction:

The standard free energy of dissociation (HA H++ A-) is given by:

G= -RT ln([H+] [A-]/[HA]) = -RT ln Ka = 2.303 RT pKa (standard state) -------(1)

Actual free energy of ionization: Gioniz= G+ RT ln ([H+] [A-] / [HA]) ----(2)

Suppose the ionization reaction is coupled to some other interaction: e.g. binding of a

proton to A-changes the interaction of A-with some other group in the molecule.

Gtotal= Gioniz+ Ginter= G+ Ginter+ RT ln ([H+] [A-] / [HA]) ------(3)

At equillibrium Gtotal= 0. The H+ concentration at which the acid is half ionized is:(H+)1/2= e

-(G+ Ginter

)/RT -----------(4)

The apparent pKa is: pKa = -log (H+

)1/2 = (G+ Ginter) / 2.303 RT ---------(5)

For a model system without coupling: pKa = G/ 2.303 RT ----------(6)

Therefore, from the difference in the two pKa values, the interaction energy can be

calculated as Ginter = 2.303 RT (pKa pKa) --------------(7)


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pKa analysis by NMR

The side chain1

H,13

C or15

N chemical shift changes with ionization. Usually thelargest change occurs closest to the site of protonation / deprotonation.

Monitor the chemical shift change as a function of pH. Fit to modified Hill Equation:

1

2 3

48.0-8.8 ppm

6.8-7.2 ppm

C2H proton appears at higher frequency thanmost other protons and is sensitive to theprotonation of the ring.

Ionization of Histidine

1+ 10pH-pKaobs=

HA+ A-x 10pH-pKa

HAis the chemical shift in the acidic pH limit

A- is the chemical shift in the basic pH limit


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C2HC4H CH CH

010

Raise pH

100ppm

C

H

COO-+H3N

C

C

NC

N

C

HH

H

+

1

2

3

4

H H

H

Shift measured with multiple 1Dspectra starting with pH 1.0 and

moving through to pH 9. The

chemical shift change of the proton

on C2 reflects the protonation state

of N1

pH1 3 5 7 9 11

Chemical

ShiftChange

(ppm)

0

1

pKa = 5.2

50% ofcomplete

change

Titration of the C2H of Histidine


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4 histidines which could

be monitored and have their

pKas measured.

Observation of Histidine Residues in Proteins by Means of Nuclear Magnetic

Resonance Spectroscopy.(Markley J., Acc Chem Res. 8, 1975, 70-80)

Chemical shift change of C2H

and C4H monitored as a

function of pH using 1D NMR


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H1 = His105

H2 = His119

H3 = His12

H4 = His48

MeasurepKaof each histidine

pKa

His105 6.7

His119 6.2

His12 5.8

His48 is more complex,

sudden discontinuity in the

curve.

C2H titration

C4H titration


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Found that 200mM Na+CH3COO-

helped to stabilize the protein.

Can then determine that the pKaof C2H is 6.31.

There is a conformational change affectingthis peak so that at some pHs two peaks wereobserved. H4a and H4b were acid and base

stable forms.


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His105

His12

His48

His119 His48 and His105 are unchanged

His12 and His119 curved are shifted

downfield.

Why downfield??

His119 changes from 6.2 to 8.0His 12 changes from 5.8 to 7.4

Both His12 and His119 are protonated in the enzyme-inhibitor complex. The proton is protected from exchange

by the presence of the inhibitor. Need to go to higher pHto remove it.

Repeat titrations in the presenceof an inhibitor.

in this case, cytidine-3-monophosphate (3-CMP) O

OPO3-

OH

NHOCH2

NH2

O


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O

OPO3-

OH

NHOCH 2

NH2

O


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pKa values of acidic residues of hen and turkey lysozymes by two

dimensional 1H NMR(Bar tik , K. et.al. B io ph ys J., 66, 1994, 1180-1184)

pH=

1.1

pH=5.9

Both enzymes have identical activity profile as a function

of pH as indicated by identical pKa values of the residues

in the active site.

2D DQFCOSY


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Protein is positively charged (pI = 11) between pH 1 to pH 7 (titration range). This

results in an overall decrease in the stability of the positively charged histidine

residues and increase in the stability of the negatively charged Asp and Glu

residues. Therefore, a decrease in the pKa values is observed for these residuesfrom their standard values.

pKa values of the conserved residues at the active site (Glu35 and Asp52) is higher

than rest of the residues due to the hydrophobic nature of the active site cleft and

interaction between Glu 35 and Asp52.


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Direct Measurement of the Aspartic Acid 26 pKa for Reduced E. Coli

Thioredoxin by 13C NMR(J. Dyson et al., B ioc hem ist ry , 35, 1996, 1-6.)

Two dimensional HCACO spectrum of thioredoxin at pH 8.52.

pKa determined using modified 2D HCACO experiment that detects coupling between13CO of a carboxyl group and the adjacent 13CH or 13CH.

O-O

CCNCC-

HCHH

HO

C

H

pH


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Plot of chemical shift as a function of pH

The carboxyl group of Asp 26 is buried in a

hydrophobic environment that elevates its pKa value

to 7.3-7.5 from a standard value of 4.0.

Ionization of Asp26 also affected by two Cysteine

thiol groups ionizing at the active site.

O-O

CCNCC-

HCHH

HO

C

H

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