Chapter 4 Amino Acids

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Chapter 4Amino Acids
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Outline

• What are the structures and properties of amino
  acids ?
• What are the acid-base properties of amino acids
  ?
• What reactions do amino acids undergo ?
• What are the optical and stereochemical
  properties of amino acids ?
• What are the spectroscopic properties of amino
  acids ?
• How are amino acid mixtures separated and
  analyzed ?

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4.1 What Are the Structures and Properties of
Amino Acids?

• The common amino acids contain a carbon atom
  alpha to a carboxyl carbon.
• This a-carbon has an amino group attached and an
  R group (R H in glycine).
• The are 20 common a-amino acids are used by the
  ribosomes to make proteins. These 20 have L
  chirality at the a-carbon.
• Amino acids join together via peptide bonds.
• Several modified amino acids occur only rarely in
  proteins.
• Some amino acids are not found in proteins.

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4.1 What Are the Structures and Properties of
Amino Acids?
Figure 4.1 Anatomy of an amino acid. Except for
proline and its derivatives, all of the amino
acids commonly found in proteins possess this
type of structure.
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4.1 What Are the Structures and Properties of
Amino Acids?
Figure 4.2 Two amino acids can react with loss
of a water molecule to form a covalent bond.
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The 20 Common Amino Acids

• You should know names, structures, approximate
  a-pKa values, 3-letter and 1-letter codes
• Classification based on sidechain structure
• Non-polar amino acids.
• Polar, uncharged amino acids.
• Acidic amino acids.
• Basic amino acids.
• Other sidechain structural classifications
• Aromatic, cyclic, hydroxyl, and thiol amino acids.

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The 20 Common Amino Acids
Figure 4.3 Some of the nonpolar (hydrophobic)
amino acids.
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The 20 Common Amino Acids
Figure 4.3 Some of the nonpolar (hydrophobic)
amino acids.
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The 20 Common Amino Acids
Figure 4.3 Some of the polar, uncharged amino
acids.
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The 20 Common Amino Acids
Figure 4.3 Some of the polar, uncharged amino
acids.
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The 20 Common Amino Acids
Figure 4.3 The acidic amino acids.
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The 20 Common Amino Acids
Figure 4.3 The basic amino acids.
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Several Amino Acids Occur Rarely in Proteins

• We'll see some of these in later chapters
• Selenocysteine in many organisms.
• Pyrrolysine in several archaeal species.
• Hydroxylysine, hydroxyproline collagen.
• Carboxyglutamate - blood-clotting proteins.
• Pyroglutamate in bacteriorhodopsin.
• GABA, epinephrine, histamine, serotonin act as
  neurotransmitters and hormones.
• Phosphorylated amino acids a signaling device.

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Several Amino Acids Occur Rarely in Proteins
Figure 4.4(a) Some amino acids are less common
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Several Amino Acids Occur Rarely in Proteins
Figure 4.4(b) Some amino acids are less common,
but nevertheless found in certain proteins.
Hydroxylysine and hydroxyproline are found in
connective-tissue proteins carboxy-glutamate is
found in blood-clotting proteins pyroglutamate
is found in bacteriorhodopsin (see Chapter 9).
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Several Amino Acids Occur Rarely in Proteins
Figure 4.4(c) Several amino acids that act as
neurotransmitters and hormones.
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4.2 What Are Acid-Base Properties of Amino Acids?

• Amino Acids are Weak Polyprotic Acids
• The degree of dissociation depends on the pH of
  the medium
• The carboxylic acid group always ionizes first.
• Then the side chain (if ionizable) or the a-amino
  group.
• The order of ionization always proceeds from most
  acidic to least acidic.

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4.2 What Are Acid-Base Properties of Amino Acids?

• The first dissociation is the carboxylic acid
  group (using glycine as an example)
• NH3CH2COOH ? NH3CH2COO- H
• (NH3CH2COO-)(H)
• Ka1 ---------------------------
• (NH3CH2COOH)

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4.2 What Are Acid-Base Properties of Amino Acids?

• The second dissociation is the amino group in the
  case of glycine
• NH3CH2COO- ? NH2CH2COO- H
• (NH2CH2COO-)(H)
• Ka2 ---------------------------
• (NH3CH2COO-)

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4.2 What Are Acid-Base Properties of Amino Acids?
Figure 4.5 The ionic forms of the amino acids,
shown without consideration of any ionizations on
the side chain.
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pKa Values of the Amino Acids

• You should know these numbers and know what they
  mean
• Alpha carboxyl group pKa 2
• Alpha amino group pKa 9
• These numbers are approximate, but entirely
  suitable for our purposes.

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Isoelectric Point, pI

• The isoelectric point is the pH at which there is
  zero net charge
• Using Gly again
• Charges in the first ionization 1 ? 0
• Charges in the second ionization 0 ? -1
• So, in the case of glycine, the pH at which there
  is most of the zero net charge form occurs half
  way between the first and second ionizations.
• pI (pKa1 pKa2)/2 5.95

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4.2 What Are Acid-Base Properties of Amino Acids?
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4.2 What Are Acid-Base Properties of Amino Acids?
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pKa Values of the Amino Acids

• You should know these numbers and know what they
  mean
• Arginine, Arg, R pKa(guanidino group) 12.5
• Aspartic Acid, Asp, D pKa 3.9
• Cysteine, Cys, C pKa 8.3
• Glutamic Acid, Glu, E pKa 4.3
• Histidine, His, H pKa 6.0
• Lysine, Lys, K pKa 10.5
• Tyrosine, Tyr, Y pKa 10.1

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Titrations of polyprotic amino acids
Figure 4.7 Titration of glutamic acid
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Titrations of polyprotic amino acids
Figure 4.7 Titration of lysine.
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A Sample Calculation

• What is the pH of a glutamic acid solution
• if the alpha carboxyl is 1/4 dissociated?
• pH 2.2 (-0.477)
• pH 1.723
• Note that, when the group is ¼ dissociated, ¼ are
  dissociated and ¾ are not thus the ratio in the
  log term is ¼ over ¾ or 1/3.

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Another Sample Calculation

• What is the pH of a lysine solution if the side
  chain amino group is 3/4 dissociated?
• pH 10.5 (0.477)
• pH 10.977 11.0
• Note that, when the group is ¾ dissociated, ¾ is
  dissociated and ¼ is not thus the ratio in the
  log term is ¾ over ¼ or 3/1.

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Amino Acids as Buffers
From Table 4.1 we can see that Histidine has pKa
values of 1.8, 6.0, and 9.2. It can provide
effective buffering at a pH equal to any one of
these three pKas. The titration curve has three
regions in which pH is relatively unaffected by
addition of acid or base. What is the zero net
charge form of His ? In which ionizations does it
appear ? What is the charge on His at pH 4?
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Reactions of Amino Acids

• Amino acid composition an amino acid analysis
  gives the number of each amino acid in the
  peptide or protein.
• Peptide bonds are cleaved by acid hydrolysis (6M
  HCl, 110oC, 18-72 hours). Trp is destroyed.
• Amino acid sequence order of the amino acids in
  a peptide or protein.
• Edmans reagent (phenylisothiocyanate) is the
  currently preferred reagent. It reacts with a
  free a-amino group of an amino acid or peptide to
  produce a phenylthiohydantoin (PTH) derivative.

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Detecting Amino Acids
Ninhydrin is the classical reagent for detecting
amino acids. Reaction requires 2-5 min at
100oC and is sensitive at the nanomole level.
Ruhemanns Purple 570 nm
Note The product from Pro is Yellow and absorbs
at 440 nm.
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N-Terminal Reagents
DNFB - Sangers reagent (dinitrofluorobenzene
) DANSYL choride (dimethylaminonaphthalenesu
lfonyl chloride)
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Other Reagents
C-terminal analysis Hydrazine Disulfide
reduction Dithiothreitol - Clelands
Reagent Thiols reactions Iodoacetate 5,5-dit
hiobis-(2-nitrobenzoic acid) - Ellmans reagent
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Edmans Reaction
Figure 4.8(a) Edmans reagent reacts with the
N-terminal residue of a peptide or protein and
cleaves the peptide bond forming a cyclic
thiazoline derivative that reacts in weak aqueous
acid to form a PTH-amino acid. This reaction can
proceed down the chain cleaving successive
residues. Samples are used to identify each
residue.
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Oxidation of Cysteine to Cystine
Figure 4.8(b) Cysteine residues react with each
other to form disulfides. This will occur with
O2 in air.
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Stereochemistry of Amino Acids

• All common AA except glycine are chiral at the
  a-carbon atom.
• L-amino acids predominate in nature and are the
  only ones used in ribosomal protein synthesis.
• D,L-nomenclature is based on D- and
  L-glyceraldehyde.
• R,S-nomenclature system is more convenient, since
  amino acids like isoleucine and threonine (with
  two chiral centers) can be named unambiguously.

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Stereochemistry of Amino Acids
39
Discovery of Optically Active Molecules and
Determination of Absolute Configuration
Emil Fischer deduced the structure of glucose in
1891. Fischers proposed structure was confirmed
by J. M. Bijvoet in 1951 (by X-ray diffraction).
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Rules for Description of Chiral Centers in the
(R,S) System
Naming a chiral center in the (R,S) system is
accomplished by viewing the molecule from the
chiral center to the atom with the lowest
priority. The priorities of the functional groups
are SH gt OH gt NH2 gt COOH gt CHO gt CH2OH gt CH3
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Spectroscopic Properties

• All amino acids absorb in the infrared (bond
  vibrations).
• Only Phe, Tyr, and Trp absorb in the UV
  (electronic transitions between energy levels).
• Absorbance at 280 nm is a good method for
  determining protein concentration.
• NMR spectra are characteristic of each residue in
  a protein, and high resolution NMR measurements
  can be used to elucidate three-dimensional
  structures of proteins.

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Spectroscopic Properties
Figure 4.10 The UV spectra of the aromatic amino
acids at pH 6. Beers Law A ecl
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Spectroscopic Properties
Figure 4.11 Proton NMR spectra of several amino
acids.
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Spectroscopic Properties
Figure 4.12 A plot of C13 chemical shifts versus
pH for the carbons of lysine.
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Separation of Amino Acids

• Mikhail Tswett, a Russian botanist, first
  separated colorful plant pigments by
  chromatography.
• Many chromatographic methods exist for separation
  of amino acid mixtures
• Ion exchange chromatography.
• High-performance liquid chromatography.

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Separation of Amino Acids
Figure 4.13 Gradient separation of common
PTH-amino acids
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Peptides and Proteins the Peptide Bond
Figure 4.14 Peptide formation is the creation of
an amide bond between the carboxyl group of one
amino acid and the amino group of another amino
acid.
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4.7 What is the Fundamental Structural Pattern in
Proteins?

• Proteins are unbranched polymers of amino acids.
• Amino acids join head-to-tail through formation
  of covalent peptide bonds.
• Peptide bond formation results in release of
  water.
• The peptide backbone of a protein consists of the
  repeated sequence N-Ca-Co-.
• N is the amide nitrogen of the amino acid.
• Ca is the alpha-C of the amino acid.
• Co is the carbonyl carbon of the amino acid.

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The Peptide Bond

• Is the biochemical nomenclature for the amide
  bond between formed two amino acids.
• Is usually found in the trans conformation.
• Has partial (40) double bond character due to
  resonance.
• Is about 0.133 nm long - shorter than a typical
  single bond but longer than a double bond.
• Due to the double bond character, the six atoms
  of the peptide bond group are always coplanar.
• N partially positive O partially negative.

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The Peptide Bond
Figure 4.15 The trans conformation of the
peptide bond.
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4.7 What is the Fundamental Structural Pattern
in Proteins?

Figure 4.16(a) The peptide bond has partial
double bond character. One of the postulated
resonance forms is shown here.
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4.7 What is the Fundamental Structural Pattern
in Proteins?
Figure 4.16(b) The peptide bond has partial
double bond character. One of the postulated
resonance forms is shown here.
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4.7 What is the Fundamental Structural Pattern
in Proteins?
Figure 4.16(c) The peptide bond is best
described as a resonance hybrid of the forms
shown on the two previous slides.
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4.7 What is the Fundamental Structural Pattern
in Proteins?
The coplanar relationship of the atoms in the
amide group is highlighted here by an imaginary
shaded plane lying between adjacent a-carbons.
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Charge on a Peptide

• Given the peptide Asp-Met-His-Ala
• Determine the charge on this peptide at pHs 2, 7
  and 10.
• Use pKas as given in the Table.
• Determine the pI of this peptide.
• First draw the structure.

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Charge on a Peptide

• Given the peptide Asp-Met-His-Ala
• Determine the charge on the peptide below at pHs
  2, 7 and 10. Use pKas as given in the Table.
• 2. Determine the pI of this peptide.

How many ionizable groups ? List the pKa values.
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Charge on a Peptide

• 1. Determine the charge on the peptide below at
  pHs 2, 7 and 10. Use pKas as given in the
  Table.
• 2. Determine the pI of this peptide Asp-Met-His-A
  la

pKa's 8.0(N-term) 3.9 6.0
3.1(C-term)
Analyze each ionizable group.
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Charge on a Peptide

• Analysis of charges
• at pH Charge on each group net charge
• 2 0 0 2
• 7 - 0 - -1
• 10 0 - 0 - -2
• N-Term Asp His C-Term
• pKa 8 3.9 6 3.1

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Isoelectric point of the Peptide

• Ionizations and charges at each pKa
• pKa conj. acid conj. Base
• 3.1 0 0 lt?gt 0
  -
• 3.9 0 - ltgt
  - -
• 6.0 - - ltgt
  - 0 -
• 8.0 - 0 - ltgt 0
  - 0 -
• Calc. pI (3.9 6.0)/2 4.95

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Peptides

• These are short polymers of amino acids.
• Each unit is called a residue.
• 2 residues dipeptide.
• 3 residues tripeptide.
• 12-20 residues oligopeptide.
• Many residues polypeptide.

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Protein

• One or more polypeptide chains
• One polypeptide chain - a monomeric protein.
• More than one - multimeric protein.
• Homomultimer - one kind of chain.
• Heteromultimer - two or more different chains.
• Hemoglobin, for example, is a heterotetramer.
• It has two alpha chains and two beta chains.

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Proteins - Large and Small

• Example proteins
• Insulin Has an A chain of 21 residues and a B
  chain of 30 residues - total mol. wt. of 5,733.
• Glutamine synthetase - 12 subunits of 468
  residues each - total mol. wt. of 600,000.
• Connectin proteins
• alpha - MW 2.8 million.
• beta - MW of 2.1 million, with a length of 1000
  nm -it can stretch to 3000 nm.

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Proteins - Large and Small
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Proteins - Large and Small
From Table 4.2 Size of protein
molecules. Molecular weights Insulin, 5,733
Cytochrome c, 12,500 Ribonuclease,
12,640 Lysozyme, 13,930 Myoglobin, 16,980.
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Proteins - Large and Small
From Table 4.2 Size of Protein
Molecules. Molecular weights Hemoglobin,
64,500 Immunoglobulin, 149,900 Glutamine
synthetase, 600,000.
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The Sequence of Amino Acids in a Protein

• Is unique for every protein.
• Predicts the folding arrangement (3o structure).
• Is encoded by the nucleotide sequence of DNA.
• So, is a form of genetic information.
• Is read from the amino terminus (1) to the
  carboxyl terminus (n).
• Is written from the amino terminus (left) to the
  carboxyl terminus (right).

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End Chapter 4Amino Acids