Lecture 2

Question 1

Draw the ionization state of a generic AA at pH =1, 6, and 11

Answer 1

N/A

Question 2

Write the law of mass action equilibrium

Answer 2

Ka = [H+][A-]/[HA]

Question 3

What is the pKa for the following ionizable fxnal groups in AA’s and polypeptides? (for the free amino acid solubilized in water, not one buried residue within a protein):

1. Aspartate (carboxylate)
2. Glutamate (carboxylate)
3. Histidine (imidazole)
4. Cysteine (thiol, sulfhydryl)
5. Tyrosine (phenol group)
6. Lysine (ε-amino group)
7. Arginine (guanidinium group)
8. Serine (hydroxyl) 9. Threonine (hydroxyl)
9. α-carboxyl of free amino acid
10. α-amino of free amino acid
11. C-terminal carboxyl group of polypeptide
12. N-terminal amino group of polypeptide

Answer 3

1. Aspartate (carboxylate) = 4
2. Glutamate (carboxylate) = 4
3. Histidine (imidazole) = 6.5, 14.6
4. Cysteine (thiol, or sulfhydryl) = 8.5
5. Tyrosine (phenol group) = 10.5
6. Lysine (ε-amino group) = 10.5
7. Arginine (guanidinium group) = 12.5
8. Serine (hydroxyl) = 14
9. Threonine (hydroxyl) = 14
10. α-carboxyl of free amino acid = 2
11. α-amino of free amino acid = 9.5
12. C-terminal carboxyl group of polypeptide = 3
13. N-terminal amino group of polypeptide = 8

Question 4

What factors do pKa values depend on?

Answer 4

temperature, ionic strength, and most of all the microenvironment of the functional group.

Question 5

Draw the structure of aspartic acid, it’s pKa, and its depronated state and the name of its fxnal group before and after deprotonation

Answer 5

N/A

Question 6

Draw the structure of glutamic acid, it’s pKa, and its depronated state and the name of its fxnal group before and after deprotonation

Answer 6

N/A

Question 7

Draw the structure of histidine, it’s pKa, and its depronated state and the name of its fxnal group before and after deprotonation

Answer 7

N/A

Question 8

Draw the structure of cysteine, it’s pKa, and its depronated state and the name of its fxnal group before and after deprotonation

Answer 8

N/A

Question 9

Draw the structure of tyrosine, it’s pKa, and its depronated state and the name of its fxnal group before and after deprotonation

Answer 9

N/A

Question 10

Draw the structure of lysine, it’s pKa, and its depronated state and the name of its fxnal group before and after deprotonation

Answer 10

N/A

Question 11

Draw the structure of arginine, it’s pKa, and its depronated state and the name of its fxnal group before and after deprotonation

Answer 11

N/A

Question 12

Write the henderson-hasselbalch equation

Answer 12

pH = pKa + log [A-]/[HA]

Question 13

How does Ka relate to pH?

Answer 13

HA  A- + H+
Ka = [H+][A-]/[HA]
log10Ka = log10 ([H+][A-]/[HA])
logKa = log[H+] + log[A-]/[HA]
recall: p = -log
-pKa = -pH + log[A-]/[HA]
pH = pKa + log[A-][HA]

Question 14

Ratio vs. Fraction problem
Q: A protein has one cysteine residue on its surface (solvent exposed). What fraction of the protein will have the cysteine side chain in the deprotonated state when the protein is suspended in a buffer at the following pH’s: 6, 7, 8, 9, 10? Assume a pKa value of 8.0 for the cysteine side chain thiol group.

Answer 14

Ratio (R) = [A-][HA]
pH = pKa + log[A-][HA]
Let [A-]/[HA] = R
pH = pKa + logR
pH - pKa = logR
10^pH-pKa = R

Fraction (F) = [A-]/[AT] = [A-]/ [HA] + [A-]
recall: Ratio R = [A-]/[HA]
recall: R[HA] = A-
Fraction (F) = [A-]/[AT] = [A-]/ [HA] + R[HA]
Fraction (F) = R / 1 + R

1) pH 6
R = 10^pH-pKa 
R = 10^6-8
R = 10^-2
R = 0.01

F = R / 1 + R
F = 0.01 / 1 + 0.01
F =

2) pH 7
R = 10^pH - pKa
R = 10^7 - 8
R = 10^-1
R = 0.1

F = R / 1 + R
F = 0.1 / 1 + 0.1
F =

3) pH 8
R = 10^pH-pKa
R = 10^8-8
R = 10^0
R= 

F = R / 1 + R
F =
F =

4) pH 9
R = 10^pH-pKa
R = 10^9-8
R = 10^1
R = 10

F = R / 1 + R
F = 10 / 1 + 10
F =

5) pH 10
R = 10^pH-pKa
R = 10^10-8
R = 10^2
R = 100

F = R / 1 + R
F = 100 / 1 + 100
F =

Question 15

Relate ratio to fraction

Answer 15

Ratio (R) = [A-][HA]
pH = pKa + log[A-][HA]
Let [A-]/[HA] = R
pH = pKa + logR
pH - pKa = logR
10^pH-pKa = R

Fraction (F) = [A-]/[AT] = [A-]/ [HA] + [A-]
recall: Ratio R = [A-]/[HA]
recall: R[HA] = A-
Fraction (F) = [A-]/[AT] = [A-]/ [HA] + R[HA]
Fraction (F) = R / 1 + R

Question 16

At the pKa of a fxnal group; ____% will be deprotonated

Answer 16

50%

Question 17

2 pH units below the pKa 99% will be _________

Answer 17

protonated

Question 18

2 pH units above the pKa, 99% will be ________

Answer 18

deprotonated

Question 19

For the following ionizable residue, a) pKa, b) Charge at pH 7.4 (physiological pH), c) Charge when pH is below pKa, d) Charge when pH is above pKa

1) Asp
2) Glu
3) His
4) Cys
5) Lys
6) Tyr
7) Arg
8) Ser
9) Thr

Answer 19

1) Asp
a) pKa: 4
b) Charge at pH 7.4 (physiological pH): Neg (-)
c) Charge when pH is below pKa: Neutral (0)
d) Charge when pH is above pKa: Neg (-)

2) Glu
a) pKa: 4
b) Charge at pH 7.4 (physiological pH): Neg (-)
c) Charge when pH is below pKa: Neutral (0)
d) Charge when pH is above pKa: Neg (-)

3) His
a) pKa: 6.5
b) Charge at pH 7.4 (physiological pH): Neutral (0)
c) Charge when pH is below pKa: Pos (+)
d) Charge when pH is above pKa: Neutral (0)

4) Cys
a) pKa: 8
b) Charge at pH 7.4 (physiological pH): Neutral (0)
c) Charge when pH is below pKa: Neutral (0)
d) Charge when pH is above pKa: Neg (-)

5) Lys
a) pKa: 10.5
b) Charge at pH 7.4 (physiological pH): Pos (+)
c) Charge when pH is below pKa: Pos (+)
d) Charge when pH is above pKa: Neutral (0)

6) Tyr
a) pKa: 10.5
b) Charge at pH 7.4 (physiological pH): Neutral (0)
c) Charge when pH is below pKa: Neutral (0)
d) Charge when pH is above pKa: Neg (-)

7) Arg
a) pKa: 12.5
b) Charge at pH 7.4 (physiological pH): Pos (+)
c) Charge when pH is below pKa: Pos (+)
d) Charge when pH is above pKa: Neutral (0)

8) Ser
a) pKa: 14
b) Charge at pH 7.4 (physiological pH): Neutral (0)
c) Charge when pH is below pKa: Neutral (0)
d) Charge when pH is above pKa: Neg (-)

9) Thr
a) pKa: 14
b) Charge at pH 7.4 (physiological pH): Neutral (0)
c) Charge when pH is below pKa: Neutral (0)
d) Charge when pH is above pKa: Neg (-)

Question 20

Q: At pH 7, what is the net charge on the peptide: DYGDKMICAWRYAE?

Answer 20

List the pKa value for each residue with a titratable functional group, then list the charge on that functional group at the given pH.
Answer: -1

Question 21

Q: What protein environments affect the pKa of a titratible functional group?

Answer 21

1. A hydrophobic environment
   - If in charged hydrophobic environment, its not energetically optimal. Enzymes shift the pKa by burying that fxnal group.
2. Local charged fxnal group (s)
   - E.g. bringing a neg charge close to another neg charge fxnal group will cause repulsion and elevation of the group’s pKa (less willing to give up proton)

Question 22

Q: A lysine residue side chain is buried within the hydrophobic core of a protein. What affect will this environment have on the pKa of the ε-amino group of the lysine?

Answer 22

There will be a decrease in the pKa of the lysine to prevent the energetically unfavourable situation of an un-neutralized buried charge

Question 23

Q: A glutamate residue side chain is buried within the core of a protein. What affect will this environment have on the pKa of the side chain carboxylate group?

Answer 23

There will be an increase in the pKa of the Glu to prevent the energetically unfavourable situation of an un-neutralized buried charge.

Question 24

Q: The ε-amino group of a lysine residue is within close proximity (within vdW distance) to a guanidinium group of an arginine. What effect will this arrangement have on the pKa of the lysine ε-amino group?

Answer 24

There will be a decrease in the pKa of one or both residues to prevent the energetically unfavourable situation of two like-charges (+,+) interacting

Question 25

Q: Why is knowing the pKa of a functional group within a protein important?

Answer 25

1. pKa values are important for calculating the net charge on a protein or peptide, this can be helpful for purifying a protein 2. It can give you clues to details about enzyme catalytic mechanism.

Question 26

Q: Are all potential pKa values important?

Answer 26

Yes. Some pKa values appear too non-physiological. But always remember that environmental effect can shift pKa values

Question 27

Draw the tautomers of histidine

Answer 27

N/A

Question 28

What is amphoteric

Answer 28

It can function as an acid | or a base.

Question 29

Which fxnal group is amphoteric

Answer 29

Imidazole

Question 30

Even though the pKa2 is usually non-physiological, an example of this state does show up in the enzyme:

Answer 30

pKa2 can shift lower, if the environment is right.

Question 31

Give an extreme example of the effect of environment on pKa values

Answer 31

Human copper-zinc superoxide dismutase (CuZn SOD)

Question 32

His63 is believed (based on structural evidence) to be in the ____ ionic state.

Answer 32

imidazolate

Question 33

Fxn of CuZn SOD

Answer 33

CuZn SOD is an important part of the cellular defense against reactive oxygen species such as superoxide radical anion O2 ^. -

Question 34

Mutations within the gene for the CuZn SOD enzyme can lead to what disease

Answer 34

Mutations within the gene for this enzyme can lead to the disease amyotrophic lateral sclerosis (ALS, AKA Lou Gehrig's disease).

Question 35

Draw arginine's resonance forms

Answer 35

N/A

Question 36

What is the isoelectric point (pI)

Answer 36

Average of the pKa values surround the net neutral state.

Question 37

What is the eqn for the isoelectric point (pI)

Answer 37

pI = 1/2 x (pK1 + pK2)

Question 38

Draw the 3 different states of glycine (pKa1, pKa2)

Answer 38

N/A

Question 39

Draw the titration graph of glycine (pKa1, pKa2)

Answer 39

N/A

Question 40

Draw the 4 different states of glutamate (pKa1, pKa2)

Answer 40

N/A

Question 41

Draw the titration graph of glutamate (pKa1, pKa2)

Answer 41

N/A

Question 42

Draw the 4 different states of histamine (pKa1, pKa2)

Answer 42

N/A

Question 43

Draw the titration graph of histamine (pKa1, pKa2)

Answer 43

N/A

Question 44

What is the isoelectric point (pI) of proteins

Answer 44

pH at which the net charge on the protein = 0 (The molecule carries no electrical charge or the negative and positive charges are equal)

Question 45

When does a protein have minimum solubility?

Answer 45

At its pI (isoelectric point). Here, its net charge is 0, so water (which is polar) is less attracted to it. Thus, it can aggregate and precipitate out of sln.

Question 46

pH >> pI gives a protein a ____ charge

Answer 46

Neg

Question 47

pH << pI gives a protein a ____ charge

Answer 47

Pos