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April 16 th , 2007

Munia Mukherjee

munia@medusa.bioc.aecom.yu.edu

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

Deprotonation reaction:

HA + H 2 O

Deprotonation reaction: HA + H 2 O A - + H 3 O + Ka =

A - + H 3 O +

Ka = [A - ] [H 3 O + ]

[HA]

Henderson-Hasselbach equation:

1

[H

3 O + ]

=

1

[A-]

Ka

[HA]

-log [H 3 O + ] = -log Ka + log [A - ] / [HA]

pH = pKa + log 1- q

q

(1)

(2)

(3)

(4)

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

The pK a of a titrating site is defined as the pH for which the site is 50% occupied: The pH for which the occupancy q is 0.5.

Definition of pK a

pH = pKa + log 1- q

q

1 q ( p H ) = - ln10 p ( K - p H
1
q
(
p H
)
=
-
ln10 p
(
K
-
p
H )
1 + e
a
p K = 4.0 a
p
K = 4.0
a

Titration curve

One state transition

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 proton decreases tenfold

The positively charged amino group attached to the a- carbon helps to push the departing
The positively charged amino group attached to the a- carbon helps to push the departing

The positively charged amino group attached to the a- carbon helps to push the departing proton of the carboxyl group out more easily.

the departing proton of the carboxyl group out more easily. The inflection point pI is the

The inflection point pI is the point when removal of the first proton is complete and he second has just begun so the amino acid’s prevalent form is as a dipolar ion

pH < pI: net positive charge pH > pI: net negative charge

pK a of ionizable side chains

pK a of ionizable side chains pK a = pH for 50% dissociation, Note range

pK a = pH for 50% dissociation, Note range

pK a of some amino acids

pK a of some amino acids

Factors that affect pK a values

Ionizable residues encounter two differences inside folded proteins compared to water

They are partly desolvated by the protein

This is especially unfavorable for the charged form (because it’s an ion) but it’s also unfavorable for the neutral form (because it’s a dipole.

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 due to these interactions

e.g. an aspartate with a low pK a

e.g. an aspartate with a low pK a this aspartate is partly buried but it accepts

this aspartate is partly buried but it accepts ~4 hydrogen bonds from nearby residues

it’s also close to some positively charged residues

the charged form is very happy here, so it becomes more difficult to add a proton to it

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

so the pKa of the residue decreases from 4 to ~2

e.g. a glutamate with a high pK a

e.g. a glutamate with a high pK a this glutamate is partly buried & forms no

this glutamate is partly buried & forms no favorable interactions with other residues

this is very unfavorable for the charged form (compared with being in water)

this shifts the equilibrium in favor of the neutral form

so it will be protonated even at low

[H + ] (i.e. high pH) or does not want

to be deprotonated

so the pKa of the residue increases from 4.4 to ~6

Simple rules for guessing pK a shifts

remember: a pK a is just the DG for deprotonation

acidic residues (asp & glu)

COOHCOOH «« COOCOO ++ HH 33 OO ++ 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)

NHNH 33 ++ «« NHNH 22 ++ HH 33 OO ++

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

Reasons for interest in pK a s

[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 pK a s of the residues involved

depend on the pH and the pK a s of the residues involved enzymes have optimal

enzymes have optimal pHs (sometimes loss of activity at non-optimal pHs is due to unfolding of the enzyme)

(Pace(Pace etet al.al. BiochemistryBiochemistry 2:2: 25642564 (1990)(1990)

Reasons for interest in pK a s

[2]

protein stability is pH dependent if the pK a of a residue is different in the folded state from its value in the unfolded state, the protein’s stability will depend on pH

DGDG unfoldunfold
DGDG unfoldunfold

pHpH

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 ionic interaction can determine whether or not a protein will fold.

Reasons for interest in pK a s

[3]

a protonation equilibrium can be thought of as a very simple ligand-binding reaction (with the ligand being H + )

knowing the pK a of a protein residue and the protein’s 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

From the change in pKa, one can determine the free energy (DG) associated with the reaction:

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

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

Actual free energy of ionization: DG ioniz = DG + 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.

DG total = DG ioniz + DG inter =

DG + DG inter + RT ln ([H + ] [A - ] / [HA]) ------(3)

At equillibrium DG total = 0. The H+ concentration at which the acid is half ionized is:

(H + ) 1/2 = e -(DG + DG inter )/RT

-----------(4)

The apparent pKa’ is: pKa’ = -log (H + ) 1/2 = (DG + DG inter ) / 2.303 RT ---------(5)

For a model system without coupling: pKa = DG / 2.303 RT

----------(6)

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

calculated as DG inter = 2.303 RT (pKa’ – pKa)

--------------(7)

pKa analysis by NMR

The side chain 1 H, 13 C or 15 N chemical shift changes with ionization. Usually the largest change occurs closest to the site of protonation / deprotonation.

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

d obs =

d HA + d A-

x 10 pH-pKa

1+ 10 pH-pKa

d HA is the chemical shift in the acidic pH limit

d A- is the chemical shift in the basic pH limit

3 2 4 6.8-7.2 ppm 1
3
2
4
6.8-7.2 ppm
1

8.0-8.8 ppm

Ionization of Histidine

3 2 4 6.8-7.2 ppm 1 8.0-8.8 ppm Ionization of Histidine C2H proton appears at higher

C2H proton appears at higher frequency than most other protons and is sensitive to the protonation of the ring.

H

+H 3 N

+H 3 N C C

C

+H 3 N C C

C

COO -

H

H

4 H C C + 3 N N H H 1 C 2
4
H
C C
+
3
N
N
H
H
1
C
2

H

C2HC4H

CaH

CbH

4 H C C + 3 N N H H 1 C 2 H C2HC4H C

10

H C C + 3 N N H H 1 C 2 H C2HC4H C a

Raise pH

0

3 N N H H 1 C 2 H C2HC4H C a H C b H

10

ppm

0

Titration of the C2H of Histidine

Shift measured with multiple 1D spectra 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

1

of the proton on C2 reflects the protonation state of N1 1 0 50% of complete

0

50% of complete change pKa = 5.2 1 3 5 7 9 11pH
50% of
complete
change
pKa = 5.2
1
3
5
7
9
11pH

Chemical

Shift

Change

Dd (ppm)

Observation of Histidine Residues in Proteins by Means of Nuclear Magnetic

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

Spectroscopy. ( Markley J., Acc Chem Res. 8, 1975, 70-80 ) 4 histidines which could be

4 histidines which could be monitored and have their pKa’s measured.

Chemical shift change of C2H and C4H monitored as a function of pH using 1D NMR

C2H titration C4H titration H1 = His105 H2 = His119 H3 = His12 H4 =

C2H titration

C4H titration

H1 = His105 H2 = His119 H3 = His12 H4 = His48

Measure pK a of each histidine

 

pKa

His105

6.7

His119

6.2

His12

5.8

His48 is more complex, sudden discontinuity in the curve.

There is a conformational change affecting this peak so that at some pHs two peaks

There is a conformational change affecting this peak so that at some pHs two peaks were observed. H4a and H4b were acid and base stable forms.

Found that 200mM Na + CH 3 COO - helped to stabilize the protein.

Can then determine that the pKa of C2H is 6.31.

His105 His12 His119 His48
His105
His12
His119
His48

Repeat titrations in the presence of an inhibitor.

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

O

NH 2 N
NH 2
N
HOCH 2 O OPO 3 - OH
HOCH 2
O
OPO 3 -
OH

His48 and His105 are unchanged

His12 and His119 curved are shifted downfield. His119 changes from 6.2 to 8.0 His 12 changes from 5.8 to 7.4

Why downfield??

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 pH to remove it.

NH 2 O N HOCH 2 O OPO 3 - OH
NH 2
O
N
HOCH 2
O
OPO 3 -
OH

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

dimensional 1 H NMR (Bartik, K. et.al. Biophys J., 66, 1994, 1180-1184)

pH=

1.1

pH=

5.9

Biophys J., 66, 1994, 1180-1184) pH= 1.1 pH= 5.9 2D DQFCOSY Both enzymes have identical activity

2D DQFCOSY

66, 1994, 1180-1184) pH= 1.1 pH= 5.9 2D DQFCOSY Both enzymes have identical activity profile as

Both enzymes have identical activity profile as a function of pH as indicated by identical pKa values of the residues in the active site.

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

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 residues from 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.

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

Thioredoxin by 13 C NMR (J. Dyson et al., Biochemistry, 35, 1996, 1-6.)

3 C NMR (J. Dyson et al., Biochemistry, 35, 1996, 1-6.) pH O H H H

pH

O

C NMR (J. Dyson et al., Biochemistry, 35, 1996, 1-6.) pH O H H H C—C—N—C—C-
H H
H
H
H
H

C—C—N—C—C-

Biochemistry, 35, 1996, 1-6.) pH O H H H C—C—N—C—C- H—C—H C O - O Two
H—C—H C O - O
H—C—H
C
O
-
O

Two dimensional HCACO spectrum of thioredoxin at pH 8.52.

pKa determined using modified 2D HCACO experiment that detects coupling between 13 CO of a carboxyl group and the adjacent 13 C b H or 13 C g H.

Plot of chemical shift as a function of pH O H H C—C—N—C—C- H H—C—H

Plot of chemical shift as a function of pH

O H H C—C—N—C—C-
O
H
H
C—C—N—C—C-
H H—C—H C O - O
H
H—C—H
C
O
-
O

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.