Amino Acids, Peptides, and pKa/pI Review (Vocabulary Flashcards)

Overview

  • Topic: how pKa values of each ionizable group in amino acids determine the net charge of peptides and proteins, and how to determine the isoelectric point (pI).
  • Build-up: start from single amino acid building blocks in solution, move to dipeptides and polypeptides (peptide bonds), and then tackle charge states across pH values.
  • Terminology:
    • polypeptide: polymer of amino acids linked by peptide bonds.
    • peptide bond: bondage between the carboxyl carbon of one amino acid and the amine nitrogen of the next; formation is a condensation reaction releasing water; hydrolysis uses water to break the bond.
    • translation: the process by which a DNA sequence is used to assemble a protein sequence, attaching amino acids via peptide bonds (N-terminus to C-terminus direction).
  • Structural note: every amino acid (except glycine) is chiral at the alpha carbon; the general backbone is drawn with the peptide bond in the plane and side chains (R groups) alternating in and out of the plane.
  • Practical note from class: an app can generate structures and help with drawing; however, pKa values are not always included in such tools.

Peptide bond formation and the polypeptide backbone

  • Condensation reaction forms a dipeptide from two amino acids and releases water:
    ext{Amino
    m a2Acid}1 + ext{Amino m a2Acid}2
    ightarrow ext{Dipeptide} + ext{H}_2 ext{O}
  • Reverse reaction (hydrolysis) consumes water to break the peptide bond:
    ext{Dipeptide} + ext{H}2 ext{O} ightarrow ext{AminoAcid}1 + ext{AminoAcid}_2
  • Peptide bond exhibits resonance, giving partial double-bond character and planarity along the backbone; this resonance contributes to the backbone dipole moment along the chain.
  • The backbone can participate in hydrogen bonding (backbone amide N–H and carbonyl O). Side chains (R groups) can also participate in hydrogen bonding, but focus here is on the backbone.
  • The N-terminus (amino end) and C-terminus (carboxyl end) define the polarity and overall charge of the peptide.

Structural notes about amino acids in polypeptides

  • Because every amino acid starts from the same core (except the side chain), the molecule looks similar except for the side chain (R group).
  • Chirality: most amino acids are chiral at the alpha carbon; note: common teaching sometimes erroneously states that lysine is not chiral. In reality, lysine is chiral (like other standard amino acids). Glycine is the exception and is achiral.
  • In 2D drawings, the backbone is usually shown in a plane, with R groups projecting above or below the plane.

Key pKa values to know in context

  • Carboxyl terminus (C-terminus) pKa ≈ 2 (often listed as about 2; minor variations exist by environment).
  • Amino terminus (N-terminus) pKa ≈ 9.5 (often cited around 9–10).
  • Side chains with ionizable groups (examples from the transcript):
    • Aspartate (Asp, D): pKa ≈ 3.9
    • Glutamate (Glu, E): pKa ≈ 4.1–4.4
    • Histidine (His, H): pKa ≈ 6
    • Lysine (Lys, K): pKa ≈ 10.5
  • These values determine when groups are protonated (neutral for carboxyls, positive for amines) or deprotonated (negative for acids, neutral for bases) as pH changes.

Isolectric point (pI): concept and method

  • The isoelectric point is the pH at which the molecule carries net zero charge.
  • How to find pI in practice:
    • List all ionizable groups: N-terminus, C-terminus, and ionizable side chains present in the sequence.
    • Order their pKa values from low to high.
    • Start at very low pH where all ionizable groups are protonated (maximum positive charge) and track how the net charge changes as pH increases by passing each pKa.
    • The pI is the pH where the net charge crosses zero. If a region of pH has zero net charge (i.e., a range of pH values with net zero), the pI is the average of the two pKa values that bracket that zero-charge region:
      extpI≈pK<em>a,i+pK</em>a,i+12ext{pI} \approx \frac{pK<em>{a,i} + pK</em>{a,i+1}}{2}
    • In many peptides, there is a distinct pair of pKa values surrounding the zero-charge state, yielding a well-defined pI.
  • Important nuance: if two or more ionizable groups share the same pKa, their deprotonation/protonation events occur around the same pH, which can affect the exact location and shape of the net charge vs pH curve.

Example exercise: sequence Lysine, Alanine, Threonine, Isoleucine, Glutamate

  • Given sequence (one-letter codes): K A T I E
  • Ionizable groups present in this sequence:
    • N-terminus: pKa ≈ 9.5 (protonated at physiological pH; contributes +1 when protonated)
    • C-terminus: pKa ≈ 2 (deprotonated above this pH; contributes -1 when deprotonated)
    • Lysine side chain (K): pKa ≈ 10.5 (protonated at pH < 10.5; contributes +1 when protonated)
    • Glutamate side chain (E): pKa ≈ 4.1 (deprotonated above this pH; contributes -1 when deprotonated)
    • Alanine (A), Threonine (T), Isoleucine (I): non-ionizable in normal biological conditions
  • Determine isoelectric point (pI):
    • At very low pH: all ionizable groups are protonated; charges: N-term +1, Lys side +1, C-term neutral, Glu side chain neutral → net +2.
    • As pH increases past pKa of C-terminus (~2): C-term deprotonates to -1; net becomes +1 (N-term) +1 (Lys) -1 (C-term) = +1.
    • Past pKa of Glutamate side chain (~4.1): Glu side chain deprotonates to -1; net becomes +1 (N-term) +1 (Lys) -1 (C-term) -1 (Glu) = 0.
    • Between pKa ≈ 4.1 and pKa ≈ 9.5, no further ionizable group changes occur (no other pKa in this range for this sequence), so net charge remains 0.
    • Past pKa of N-terminus (~9.5): N-term deprotonates to 0; net becomes -1 (Lys) -1 (Glu) -1 (C-term) = -1.
    • Past pKa of Lysine (~10.5): Lys side chain deprotonates to 0; net becomes -2.
  • Therefore, the net zero charge region spans from about pH 4.1 to 9.5, so the isoelectric point is approximately:
    extpI≈pK<em>a,extGlu+pK</em>a,extN−term2≈4.1+9.52≈6.8.ext{pI} \approx \frac{pK<em>{a, ext{Glu}} + pK</em>{a, ext{N-term}}}{2} \approx \frac{4.1 + 9.5}{2} \approx 6.8.
  • Charge at neutral pH (≈7.4):
    • N-terminus: +1 (still protonated at pH 7.4)
    • Lysine side chain: +1 (still protonated at pH 7.4)
    • Glutamate side chain: -1 (deprotonated at pH 7.4)
    • C-terminus: -1 (deprotonated at pH 7.4)
    • Total charge: +1+(+1)+(−1)+(−1)=0.+1 + (+1) + (-1) + (-1) = 0. So the peptide is isoelectric around neutral pH.
  • Charge at pH 12:
    • N-terminus: 0 (deprotonated above 9.5)
    • Lysine side chain: 0 (deprotonated above 10.5)
    • Glutamate side chain: -1 (still deprotonated)
    • C-terminus: -1 (deprotonated)
    • Total charge: 0+0+(−1)+(−1)=−2.0 + 0 + (-1) + (-1) = -2. (Note: exact counts depend on the number of acidic/basic residues in the specific sequence; this example uses the specified residues.)

Case study: c-peptide (insulin precursor fragment)

  • Context: c-peptide is a 31-residue peptide released with insulin; a table lists the amino acid composition and counts.
  • Goal: determine ionizable groups and net charge at a given pH (example: pH ≈ 7 and pH ≈ 12).
  • Ionizable residues to consider (as discussed in the lecture):
    • C-terminus: pKa ≈ 2 (carboxylate; deprotonates above 2 → -1)
    • N-terminus: pKa ≈ 9.5 (amine; protonated +1 below 9.5, neutral above)
    • Aspartate (Asp, D): pKa ≈ 3.9 (carboxylate; -1 when deprotonated)
    • Glutamate (Glu, E): pKa ≈ 4.1 (carboxylate; -1 when deprotonated)
    • Lysine (Lys, K): pKa ≈ 10.5 (amino side chain; +1 when protonated below 10.5, neutral above)
  • Ionizable residues count: a mix of acidic residues (Asp/Glu) and a basic residue (Lys), plus the termini.
  • Charge at pH ≈ 7:
    • N-terminus: +1 (protonated at pH 7)
    • Lys side chain: +1 (protonated at pH 7)
    • C-terminus: -1 (deprotonated)
    • Asp side chain: -1 (deprotonated if present at pH 7)
    • Glutamate side chain: -1 (deprotonated at pH 7)
    • Net charge (sum): +1+(+1)+(−1)+(−1)+(−1)=−1.+1 + (+1) + (-1) + (-1) + (-1) = -1. So the c-peptide is negatively charged at pH 7 in this composition.
  • Charge at pH ≈ 12:
    • N-terminus: 0 (deprotonated)
    • Lys side chain: 0 (deprotonated)
    • C-terminus: -1
    • Asp side chain: -1
    • Glutamate side chain: -1
    • Net charge: 0+0+(−1)+(−1)+(−1)=−3.0 + 0 + (-1) + (-1) + (-1) = -3. The peptide becomes more negatively charged at high pH.
  • Take-home: the exact net charge at a given pH depends on which residues are ionizable and their counts; the procedure above mirrors the reasoning shown in the lecture and aligns with the table of residues discussed.

Practical implications and common questions

  • Q: When drawing a peptide at a specific pH (e.g., pH 7.4), should you ionize the R-group side chains according to their pKa values? A: Yes. The protonation state of ionizable groups depends on how the ambient pH compares to each pKa. The termini follow the same rule.
  • Q: How do you handle cases where multiple residues have the same pKa or when the pH is between two pKa values? A: The net charge changes in steps at each pKa. If a zero-charge state exists over a narrow pH interval, pick the average of the surrounding pKa values to estimate pI.
  • Note on common errors: ensure you correctly assign the charges of the termini and side chains; mistakes about chirality (e.g., Lysine) can lead to confusion, but Lysine is indeed chiral; glycine is the only achiral amino acid.
  • Notation recap:
    • Charge contributions can be summarized for any peptide as the sum of the charges on all ionizable groups:
      Q<em>extnet(pH)=∑</em>iqi(pH)Q<em>{ ext{net}}(pH) = \sum</em>{i} q_i(pH)
    • The isoelectric point is the pH where Qextnet(pH)=0.Q_{ ext{net}}(pH) = 0.
  • Real-world relevance: understanding pI is important for protein purification (isoelectric focusing), formulation stability, and interpreting protein behavior in different pH environments.

Summary of key takeaways

  • Peptide bonds form via condensation, releasing water; they can be hydrolyzed by adding water.
  • Polypeptide properties (planarity, backbone dipole, hydrogen bonding) arise from peptide-bond resonance.
  • The ionization state of a peptide depends on the pH and the pKa of its ionizable groups (N-terminus, C-terminus, and side chains).
  • The pI is the pH where the net charge is zero; it often lies between two pKa values surrounding the zero-charge state.
  • For a given sequence, you can predict the charge at any pH by counting the protonated/deprotonated states of the ionizable groups.
  • Case studies (e.g., a five-residue peptide K-A-T-I-E or the 31-residue c-peptide) illustrate how to apply these rules to real sequences.

Quick reference table (selected pKa values)

  • C-terminus: pKa≈2pK_a \approx 2
  • N-terminus: pKa≈9.5pK_a \approx 9.5
  • Aspartate (D): pKa≈3.9pK_a \approx 3.9
  • Glutamate (E): pKa≈4.1pK_a \approx 4.1
  • Histidine (H): pKa≈6pK_a \approx 6
  • Lysine (K): pKa≈10.5pK_a \approx 10.5

Notes on references from the lecture

  • The workflow for determining the pI was demonstrated with cysteine as an example; the same logic applies to peptides with increasing complexity (polymerization and multiple ionizable groups).
  • A dipeptide drawing exercise was used to practice identifying N- and C- termini and R-group environments; an app was mentioned as a useful drawing tool, though not a substitute for understanding pKa values.
  • The lecture emphasized that, at a given pH, the ionization state of termini and side chains determines the net charge and thus the behavior of the peptide in solution.