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MBB 324 > Lecture 2 > Flashcards

Lecture 2 Flashcards

1
Q

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

A

N/A

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2
Q

Write the law of mass action equilibrium

A

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

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3
Q

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
A
  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
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4
Q

What factors do pKa values depend on?

A

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

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5
Q

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

A

N/A

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6
Q

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

A

N/A

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7
Q

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

A

N/A

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8
Q

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

A

N/A

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9
Q

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

A

N/A

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10
Q

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

A

N/A

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11
Q

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

A

N/A

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12
Q

Write the henderson-hasselbalch equation

A

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

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13
Q

How does Ka relate to pH?

A 
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]
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14
Q

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.

A 
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 =

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15
Q

Relate ratio to fraction

A 
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

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16
Q

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

A

50%

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17
Q

2 pH units below the pKa 99% will be _________

A

protonated

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18
Q

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

A

deprotonated

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19
Q

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

A

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 (-)

20
Q

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

A

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

21
Q

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

A
  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)
22
Q

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?

A

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

23
Q

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?

A

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

24
Q

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?

A

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

25
Q

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

A
  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.
26
Q

Q: Are all potential pKa values important?

A

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

27
Q

Draw the tautomers of histidine

A

N/A

28
Q

What is amphoteric

A

It can function as an acid

or a base.

29
Q

Which fxnal group is amphoteric

A

Imidazole

30
Q

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

A

pKa2 can shift lower, if the environment is right.

31
Q

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

A

Human copper-zinc superoxide dismutase (CuZn SOD)

32
Q

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

A

imidazolate

33
Q

Fxn of CuZn SOD

A

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

34
Q

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

A

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

35
Q

Draw arginine’s resonance forms

A

N/A

36
Q

What is the isoelectric point (pI)

A

Average of the pKa values surround the net neutral state.

37
Q

What is the eqn for the isoelectric point (pI)

A

pI = 1/2 x (pK1 + pK2)

38
Q

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

A

N/A

39
Q

Draw the titration graph of glycine (pKa1, pKa2)

A

N/A

40
Q

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

A

N/A

41
Q

Draw the titration graph of glutamate (pKa1, pKa2)

A

N/A

42
Q

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

A

N/A

43
Q

Draw the titration graph of histamine (pKa1, pKa2)

A

N/A

44
Q

What is the isoelectric point (pI) of proteins

A

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

45
Q

When does a protein have minimum solubility?

A

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.

46
Q

pH&raquo_space; pI gives a protein a ____ charge

A

Neg

47
Q

pH &laquo_space;pI gives a protein a ____ charge

A

Pos

MBB 324 (17 decks)

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