Reading - Chapter 5
Practice problems - Chapter 5 - 1-5, 7, 8, 10 (For #10, use "generic" pK values for groups in peptides and proteins from THESE LECTURE NOTES, not values for free amino acids from text table; that of course means different answers from back of book!); Amino Acids and Peptides extra problems [PDF]

Acid dissociation reactions for functional groups of amino acid residues in proteins: [PDF]

• Both the a-amino group (amino group substituent on the aC) and the a-carboxyl group (carboxyl substituent on the aC) are ionizable.

• a-COOH group:  a weak acid, can DONATE its proton, with a pKa of about 2-3. What's the conjugate base form of the carboxyl group? Which form is charged, and is it a positive or a negative charge?
• a-NH₂ group:  a weak base (there's an unshared pair of electrons on the N; the neutral amino group can ACCEPT a proton). What's the conjugate acid form of the amino group? Which form is charged, and is it a positive or a negative charge?
• pK_as of a-amino and a-carboxyl groups are different for different amino acids, and also are altered if they're the terminal groups on a chain of amino acids, i.e., a peptide or protein.
  
  

• The nonionic form shown above does NOT occur in water. WHY NOT?

  Predominant form in H₂O is the zwitterion:   .  

• Perspective formulas show stereochemistry; projection formulas CAN be written "correctly", with convention that horizontal bonds project out of paper and vertical bonds behind plane of paper, but often biochemists use projection formulas casually (inaccurately), knowing that if it's in a protein, it's always an L-amino acid. (See also Fig. 5-3 in Lehninger Principles.)

• Fig. 5-4 (Nelson & Cox: Lehninger Principles of Biochemistry): Absolute configurations of D-glyceraldehyde as the reference compound for a-amino acids.  D- and L- apply only to the absolute configuration around the chiral a carbon; 2 of the 20 amino acids (threonine and isoleucine) have a second chiral center, requiring the RS system to describe their structures accurately, but we aren't going to worry about using the RS system here.
  

* Glx = either acid or amide (when it isn't known which it is)
**Asx = either acid or amide (when it isn't known which it is)

Properties of Amino Acid Side Chains

Side chains ("R groups") provide proteins with unique structural and functional properties.
Additional C atoms in R groups (after the a C) designated by successive Greek letters: b, g, d, e, as shown in the structure of the amino acid LYSINE (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed., p. 116):

• The side chains of the amino acids play an essential role in determining the properties of proteins. 
  There is a wide diversity in the chemical properties of amino acid side chains, but they can be grouped into classes, sometimes with overlapping "membership" (e.g., tyrosine is both aromatic and hydroxyl-containing). Other classifications are also possible (for example, the 5 classes in textbook, Fig. 5-5, discussed below).  You are expected to know all 20 amino acid structures and their R group properties, including ionization properties (see table below with "generic" pK_a values for groups in peptides and proteins and links to titration curves, and the PDF of proton dissociation reactions).

  Side Chain Class	Amino Acids
  Aliphatic	glycine, alanine, valine, leucine, isoleucine
  Cyclic	proline
  Aromatic	phenylalanine, tyrosine, tryptophan
  Hydroxyl-Containing	serine, threonine, tyrosine
  Sulfur-Containing	cysteine, methionine
  Basic	histidine, lysine, arginine
  Acidic and Their Amides	aspartic acid, glutamic acid, asparagine, glutamine

• Structures and classification below are from Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed., Fig. 5-5. States of ionization are the PREDOMINANT forms found at pH 7.
• Nonpolar, aliphatic R groups
  • Gly: quite water-soluble (as is Pro)
  • Ala, Val , Leu and Ile: increasing hydrophobicity with increasing number of C atoms in hydrocarbon chain
  • Pro: cyclic (--> unusual properties)
    • shares many properties with the aliphatic group
    • rigidity of ring plays critical role in protein structure (more about that later)
  • Met: methyl thioether (S-containing)
    • quite hydrophobic
    • Met's terminal methyl group important in metabolism

• Aromatic R groups
  • Phe: phenyl group (linked to b-CH₂, so Phe = alanine with a phenyl substituent on the methylene C)
    • VERY hydrophobic.
  • Trp: indole functional group on bC
    • electronegative atom in ring system
    • not as hydrophobic as Phe
    • hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)
  • Tyr: phenylalanine with aromatic OH group (phenolic OH) = p-hydroxyphenylalanine
    • ionizable (pK_a around 10; loss of proton gives phenolate anion)
    • hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)
    • Tyr R group is the least hydrophobic of the 3 aromatic amino acid side chains.

• Polar, uncharged R groups
  • Ser and Thr: aliphatic OH groups, not ionizable in pH range 1-13
    • pKa values so high that under any biologically reasonable pH conditions they're polar but not ionizable.
    • hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)
  • Asn and Gln: amide functional groups
    • VERY polar, but NOT ionizable
    • hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)
  • Cys: thiol (also called a sulfhydryl group) -- not very polar, and IS ionizable
    • sulfur atom makes protonated -SH group more hydrophobic than an aliphatic OH group
    • thiol DOES lose its proton in physiologically relevant pH range (pK_a about 8.5)
    • generates -S^- (thiolate anion is quite hydrophilic due to the charge).

• Positively charged R groups (sometimes called "basic" R groups)
  • Arg: guanidino group
    • VERY high pK_a (~12+), so a very weak acid (stronger base)
    • carries + charge all across physiological pH range
    • resonance forms of guanidino group stabilize protonated form (charge is delocalized)
    • hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)
  • Lys: e-amino group (a primary amine)
    • pK_a about 10
    • protonated form (predominates at physiological pH) carries + charge
    • hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)
  • His: imidazole functional group (has 2 N atoms in 5-membered unsaturated ring)
    • pKa about 6-6.5
    • protonated form carries + charge, but at pH 7 predominant form is neutral (despite textbook's categorization as "positively charged")
    • very important player in catalytic activity of many enzymes
    • hydrogen bonding capability, and also proton donor/acceptor

• Negatively charged R groups (sometimes called "acidic" R groups)
  • Asp and Glu: side chain carboxyl groups
    • pK_a values around 4
    • predominant form at physiological pH = carboxylate anion
    • hydrogen bonding capability (donor? acceptor? how many hydrogen bonds?)

Relative hydrophobicity/hydrophilicity of amino acid R groups

• Table 12.2 (Berg, Timoczko and Stryer, Biochemistry, 5th ed.): Polarity scale for amino acid residues based on free energy changes for moving a residue from a hydrophobic environment (dielectric constant = 2) into H₂O.
• Similar trends for relative hydrophobicities in text Table 5-1 (diff. numerical scale, and not arranged in order of relative polarity)
• Depending on how transfer experiments are done, different absolute numbers can be obtained, but the general trends of relative polarity are clear
  • Phe, Met, Ile, Leu, Val are very hydrophobic
  • Arg, Asp, Lys, Glu, Asn, Gln, and His are quite hydrophilic
  • The rest are in between -- neither very polar nor very hydrophobic

• Fig. 5-7: Reversible oxidation of 2 cysteine side chain thiols to form cystine, or re-reduction to 2 thiols
  • disulfide bonds between 2 Cys residues in a (usually extracellular) protein
  • often a critical structural feature in extracellular proteins (stabilize folded structures, in interior of protein structure)
  • When found in intracellular proteins, usually have a functional role.
    

Ionization Properties of Amino Acid Functional Groups (in PEPTIDES AND PROTEINS)

• weak conjugate acid/base groups in peptides and proteins crucial to functions
  • only one a-amino and one a-carboxyl group on a peptide or proteins (at the termini of the chain) because the rest of the a-amino and a-carboxyl groups are tied up in amide bonds holding monomers together in polymer (more later)
  • side chain ionizable groups (only 7 of the 20 amino acids)
• PDF of the acid dissociation reactions for functional groups of amino acid residues in peptides and proteins
• ionization states of side chain weak acid groups control charges on protein
• Note: local environment in peptide or protein determines actual pKa of that specific group, so the ranges shown below (and the rather arbitrary "generic" values, rounded off for simplicity) are only the usual expected ranges for pK_a values for the functional groups in peptides and proteins; the pKa of a specific group in a specific protein can lie significantly outside the expected range if the local environment is unusual.
• links in table below are to titration curves for that amino acid or functional group

Isoelectric point (pI)

• pI = "isoelectric pH" = "isoelectric point" = pH at which the NET charge on a molecule is ZERO. 
  • If pH < pI, net charge is positive (more + than - charges)
  • If pH > pI, net charge is negative (more - than + charges)
• pI = the pH exactly halfway between the two pK_a values surrounding the zero net charge equivalence point on the titration curve (examples to be analyzed in class: Gly and His)

• Fig. 5-10. Titration curve of glycine (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.)

• Fig. 5-12b. Titration curve of histidine (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.)

• Molecular separations based on charge properties (paper electrophoresis of amino acids as an example)
• paper strip soaked in buffer, in contact with 2 reservoirs with electrodes connected to a power supply

^
origin (sample of an amino acid applied)

• When a voltage is applied, in which direction will the amino acid move? What do you need to know to answer that question?
  • ________________________
    
    
  • ________________________
    
    
• e.g., Histidine: (sample dissolved in buffer, applied at origin above, and voltage applied)

Ultraviolet absorbance of amino acid side chains

• Aromatic amino acids (Trp, Tyr, Phe) absorb light in the near ultraviolet region of the spectrum (250-300 nm). 
• Trp has highest molar absorptivity, followed by Tyr, with Phe making only a small contribution.
• Disulfide bonds (between Cys residues in proteins) also absorb in the uv range, but much less than the aromatics.
• Fig. 5-6 (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed.): Absorbance of ultraviolet light by aromatic amino acids

Posttranslational modifications of amino acid side chains

• chemical modifications AFTER biosynthesis of proteins
• occur for a few amino acid residues in some proteins
• Some examples (see also Fig. 5-8, Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.)):

O-Phosphoserine	4-Hydroxyproline	5-Hydroxylysine	g-carboxyglutamate

• reversible phosphorylation and dephosphorylation of Ser, Thr, and Tyr residues very important in covalent regulation of activity of some enzymes and many biosignalling proteins, including some hormone receptors and transcription factors
• 4-hydroxyproline & 5-hydroxylysine important in structure of collagen (fibrous protein in connective tissue)
• g-carboxyglutamate important in a number of proteins whose function involves Ca²⁺ binding, including several proteins involved in blood clotting

Chemical Reactions of Amino Acids

• All amino acids have at least two reactive groups: the a-amino and a-carboxyl groups and these groups can react with a variety of reagents. Here are two examples:

• A particularly interesting example is the green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria, which has generated intense interest as a marker for gene expression and localization of gene products.  The chromophore, which results from the spontaneous cyclization and oxidation of the sequence -Ser65-Tyr66-Gly67- , is unusual because it does not involve a non-protein chromophore, as is usually the case for colored proteins. The chromophore is buried in the interior of GFP.

The Peptide Bond

• Peptides and proteins:polymers of amino acids joined bypeptide bonds
• amide linkages from condensation of a-carboxyl group of one amino acid with a-amino group of another amino acid

• process repeated many times --> linear chain of amino acids, a polypeptide chain
• convention: sequence written from left to right starting with residue with free a-amino group (the N-terminal or amino terminal amino acid residue) and ending with the residue containing the free a-carboxyl group (the C-terminal or carboxyl terminal residue),
  e.g., NH₂-Glu-Gly-Ala-Lys-COOH = EGAK
• average residue mass ~110 (average M_r of the 20 amino acids minus Mr of H₂O)
• a polypeptide chain with 100 amino acid residues would have a M_r of about 11,000)
• small peptides (a "few" amino acid residues) = oligopeptides

Peptide bond formation endergonic (DG^o' ~21 kJ/mol)

• (How would a cell make the reaction go in the direction of condensation in an aqueous environment? no details needed here for biochemical mechanism -- that's covered in BIOC 411)
• peptide bonds metastable in aqueous environment -- equilibrium lies far in direction of hydrolysis, but RATE of hydrolysis very slow in absence of catalyst
• Enzymes that catalyze peptide bond hydrolysis = peptidases or proteases, e.g., (specific examples of proteases) your digestive proteases like trypsin and pepsin

Ionization properties of peptides

• analyzed the same way as for free amino acids
• one a-amino group (pK_a approx. 8) and one a-carboxyl group (pK_a approx. 3), plus any ionizable side chains on residues in the peptide
• To figure out approximate net charge of a peptide at a given pH:
  • make yourself notes on the sequence to keep track of what you're doing
  • add up charges on all the ionizable groups

Example: Fig. 5-14 (Nelson & Cox: Lehninger Principles of Biochemistry, 3rd ed.): pentapeptide SGYAL = Ser-Gly-Tyr-Ala-Leu
= Serylglycyltyrosylalanylleucine

Amino Acid Analysis

• Sequence of amino acids in a protein is dictated by the sequence of nucleotides in the gene encoding that protein:
  (from Berg, Tymoczko & Stryer, Biochemistry, 5th ed., p. 28)

• Each protein (unique sequence) has unique amino acid composition.
• Can chemically hydrolyze (hot 6N HCl) a pure protein to generate the free amino acids and determine its amino acid composition chromatographically
• Because side chains of the amino acids have different properties, can separate and quantitate all 20 amino acids using a variety of chromatographic techniques, as illustrated below.
  
  

   

• Due to the partial double bond character of the peptide bond, the O, C, N and H atoms are nearly planar and there is no rotation about the peptide bond (peptide).  As we shall see later, the planarity of the these elements has important consequences for the three dimensional structure of proteins. 

• Generally, the two C_a groups are in a trans configuration, which minimizes steric interaction (cis/trans).