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Horton • Moran • Scrimgeour • Perry • Rawn
Principles of Biochemistry
Fourth Edition
Chapter 3
Amino Acids and the Primary
Structures of Proteins
Prentice Hall c2002
Chapter 3 Copyright © 2006 Pearson Prentice
1
Hall, Inc.
Chapter 3 - Amino Acids and the
Primary Stucture of Proteins
Important biological functions of proteins
1. Enzymes, the biochemical catalysts
2. Storage and transport of biochemical molecules
3. Physical cell support and shape (tubulin, actin,
collagen)
4. Mechanical movement (flagella, mitosis,
muscles)
(continued)
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Functions of proteins (continued)
5. Decoding cell information (translation,
regulation of gene expression)
6. Hormones or hormone receptors (regulation
of cellular processes)
7. Other specialized functions (antibodies,
toxins etc)
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3.1 General Structure of Amino Acids
• Twenty common a-amino acids have carboxyl
and amino groups bonded to the a-carbon atom
• A hydrogen atom and a side chain (R) are also
attached to the a-carbon atom
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Zwitterionic form of amino acids
• Under normal cellular conditions amino
acids are zwitterions (dipolar ions):
Amino group =
-NH3+
Carboxyl group =
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-COO-
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Fig 3.1 Two representations of an amino acid
at neutral pH
(a) Structure
(b) Ball-and stick model
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Stereochemistry
• Stereoisomers (立體異構物)- compounds that
have the same molecular formula but differ in the
arrangement of atoms in space
• Enantiomers - nonsuperimposable mirror images
• Chiral carbons - have four different groups
attached
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Stereochemistry of amino acids
• 19 of the 20 common amino acids have a
chiral a-carbon atom (Gly does not)
• Threonine and isoleucine have 2 chiral
carbons each (4 possible stereoisomers each)
• Mirror image pairs of amino acids are
designated L (levo) and D (dextro)
• Proteins are assembled from L-amino acids
(a few D-amino acids occur in nature)
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Fig 3.2 Mirror-image pairs of amino acids
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Assignment of configuration
by the RS system
(a) Assign a priority to each group attached to a
chiral carbon based upon atomic mass priority
(1 highest, 4 lowest)
• If two atoms are identical, move to the next atoms
• For double or triple bonds, count atom once for
each bond (-CHO higher priority than -CH2OH)
• Priorities (low to high): -H, -CH3, -C6H5, -CH2OH,
-CHO, -COOH, -NH2, -NHR, -OH, -OR, -SH
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RS system (continued)
(b) Orient the molecule with priority 4 pointing away
(behind the chiral carbon). Trace path from
highest priority to lowest priority (1, 2, 3, 4)
(c) Clockwise path: absolute configuration R
Counterclockwise path: absolute configuration S
NOTE: All of the 19 common chiral L-amino acids
except cysteine have the S configuration.
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Assignment of RS configuration
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3.2 Structures of the 20 Common Amino Acids
• Fischer projections - horizontal bonds from a
chiral center extend toward the viewer, vertical
bonds extend away from the viewer
• Abbreviations can be one letter or three letters
• Amino acids are grouped by the properties of
their side chains (R groups)
• Classes: Aliphatic, Aromatic, Sulfur-containing,
Alcohols, Bases, Acids and Amides
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A. Aliphatic R Groups
• Glycine (Gly, G) - the a-carbon is not chiral
since there are two H’s attached (R=H)
• Four amino acids have saturated side chains:
Alanine (Ala, A) Valine (Val, V)
Leucine (Leu, L) Isoleucine (Ile, I)
• Proline (Pro, P) 3-carbon chain connects
a-C and N
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Four aliphatic amino acid structures
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Fig 3.3 Stereoisomers of Isoleucine
• Ile has 2 chiral carbons, 4 possible stereoisomers
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Proline has a nitrogen in the
aliphatic ring system
• Proline (Pro, P) - has a three
carbon side chain bonded to
the a-amino nitrogen
• The heterocyclic pyrrolidine
ring restricts the geometry of
polypeptides
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B. Aromatic R Groups
• Side chains have aromatic groups
Phenylalanine (Phe, F) - benzene ring
Tyrosine (Tyr, Y) - phenol ring
Tryptophan (Trp, W) - bicyclic indole group
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Aromatic amino acid structures
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C. Sulfur-Containing R Groups
• Methionine (Met, M) - (-CH2CH2SCH3)
• Cysteine (Cys, C) - (-CH2SH)
• Two cysteine side chains can be cross-linked by
forming a disulfide bridge (-CH2-S-S-CH2-)
• Disulfide bridges may stabilize the threedimensional structures of proteins
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Methionine and cysteine
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Fig 3.4 Formation of cystine
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D. Side Chains with Alcohol Groups
• Serine (Ser, S) and Threonine (Thr, T) have
uncharged polar side chains
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E. Basic R Groups
• Histidine (His, R) - imidazole
• Lysine (Lys, K) - alkylamino group
• Arginine (Arg, R) - guanidino group
• Side chains are nitrogenous bases which are
substantially positively charged at pH 7
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Structures of histidine, lysine and arginine
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F. Acidic R Groups and Amide Derivatives
• Aspartate (Asp, D) and Glutamate (Glu, E)
are dicarboxylic acids, and are negatively
charged at pH 7
• Asparagine (Asn, N) and Glutamine (Gln, Q)
are uncharged but highly polar
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Structures of aspartate, glutamate,
asparagine and glutamine
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G. The Hydrophobicity of
Amino Acid Side Chains
• Hydropathy: the relative hydrophobicity of each
amino acid
• The larger the hydropathy, the greater the
tendency of an amino acid to prefer a
hydrophobic environment
• Hydropathy affects protein folding:
hydrophobic side chains tend to be in the interior
hydrophilic residues tend to be on the surface
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Table 3.1
Amino
acid
Free-energy change
for transfer (kjmol-1)
• Hydropathy scale for
amino acid residues
(Free-energy change for
transfer of an amino acid
from interior of a lipid
bilayer to water)
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3.3 Other Amino Acids and
Amino Acid Derivatives
• Over 200 different amino acids are found in
nature
• Most are precursors to common amino acids
or chemically modified derivatives
• Some amino acids are chemically modified
after incorporation into a polypeptide
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Fig 3.5 Compounds derived from
common amino acids
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Selenocysteine
• Selenocysteine is
incorporated into a few
proteins
• Constitutes the 21st
amino acid
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3.4 Ionization of Amino Acids
• Ionizable groups in amino acids: (1) a-carboxyl,
(2) a-amino, (3) some side chains
• Each ionizable group has a specific pKa
AH
B + H+
• For a solution pH below the pKa, the protonated
form predominates (AH)
• For a solution pH above the pKa, the unprotonated
form predominates (B)
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Fig 3.6 Titration curve for alanine
• Titration curves
are used to
determine pKa
values
• pK1 = 2.4
• pK2 = 9.9
• pIAla = isoelectric
point
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Fig 3.7 Ionization of Histidine
(a) Titration curve
of histidine
pK1 = 1.8
pK2 = 6.0
pK3 = 9.3
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Fig 3.7 (b) Deprotonation of imidazolium ring
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Table 3.2
pKa values of
amino acid
ionizable groups
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Henderson-Hasselbach equation:
calculating group ionizations
[proton acceptor]
pH = pKa + log
[proton donor]
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Fig 3.8 (a) Ionization of the protonated
g-carboxyl of glutamate
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Fig 3.8 (b) Deprotonation of the
guanidinium group of Arg
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3.5 Peptide Bonds Link Amino Acids
in Proteins
• Peptide bond - linkage between amino acids
is a secondary amide bond
• Formed by condensation of the a-carboxyl of
one amino acid with the a-amino of another
amino acid (loss of H2O molecule)
• Primary structure - linear sequence of
amino acids in a polypeptide or protein
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Fig 3.9 Peptide bond between
two amino acids
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Polypeptide chain nomenclature
• Amino acid “residues” compose peptide chains
• Peptide chains are numbered from the N (amino)
terminus to the C (carboxyl) terminus
• Example: (N) Gly-Arg-Phe-Ala-Lys (C)
(or GRFAK)
• Formation of peptide bonds eliminates the
ionizable a-carboxyl and a-amino groups of the
free amino acids
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Fig 3.10 Aspartame, an artificial sweetener
• Aspartame is a
dipeptide methyl ester
(aspartylphenylalanine
methyl ester)
• About 200 times
sweeter than table
sugar
• Used in diet drinks
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3.6 Protein Purification Techniques
• Common types of column chromatography:
Ion-exchange chromatography - separation
based upon the overall charge of molecules
Gel-filtration chromatography - separation
based upon molecular size
Affinity chromatography - separation by specific
binding interactions between column matrix and
target proteins
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Fig 3.11 Column Chromatography
(a) Separation of a
protein mixture
(b) Detection of
eluting protein
peaks
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Electrophoresis
• Polyacrylamide gel electrophoresis (PAGE)
Separates molecules on a polyacrylamide gel
matrix when an electric field is applied
• SDS-PAGE. Sodium dodecyl sulfate (SDS)
coats proteins with negative charges. Coated
polypeptide chains then separate by molecular
mass (method to determine molecular weight)
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Fig 3.12 (a) SDS-PAGE Electrophoresis
(b) Protein banding pattern after run
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Fig. 3.14
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3.7 Amino Acid Composition of Proteins
• Amino acid analysis - determination of the
amino acid composition of a protein
• Peptide bonds are cleaved by acid hydrolysis
(6M HCl, 110o, 16-72 hours)
• Amino acids are separated
chromatographically and quantitated
• Phenylisothiocyanate (PITC) used to derivatize
the amino acids prior to HPLC analysis
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Fig 3.15 Acid-catalyzed hydrolysis of a peptide
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Fig 3.16 Amino acid treated with PITC
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Fig 3.17 Chromatogram from HPLCseparated PTC-amino acids
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3.8 Determining the Sequence of Amino Acids
• Edman degradation procedure - Determining
one residue at a time from the N-terminus
(1) Treat peptide with PITC which reacts with the
N-terminus to form a PTC-peptide
(2) Treat with trifluoroacetic acid (TFA) to
selectively cleave the N-terminal peptide bond
(3) Separate N-terminal derivative from peptide
(4) Convert derivative to PTH-amino acid
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Fig 3.18 Edman degradation procedure
Phenylisothiocyanate
(Edman reagent)
pH = 9.0
Phenylthiocarbamoyl-peptide
F3CCOOH
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Edman degradation procedure (cont)
Polypeptide chain
with n-1 amino acids
Aqueous acid
Returned to alkaline conditions
for reaction with additional
phenylisothiocyanate in the
next cycle of Edman degradation
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Cleaving and blocking disulfide bonds
• Disulfide bonds in proteins must be cleaved:
(1) To permit isolation of the PTH-cysteine
during the Edman procedure
(2) To separate peptide chains
• Treatment with thiol compounds reduces the
(R-S-S-R) cystine bond to two cysteine
(R-SH) residues
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Fig 3.19 Cleaving, blocking disulfide bonds
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3.9 Protein Sequencing Strategies
• Proteins may be too large to be sequenced
completely by the Edman method
• Proteases (enzymes cleaving peptide bonds)
and chemical agents are used to selectively
cleave the protein into smaller fragments
• Cyanogen bromide (BrCN) cleaves
polypeptides at the C-terminus of Met residues
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Fig 3.20 Protein cleavage by BrCN
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Protease enzymes cleave
specific peptide bonds
• Chymotrypsin - carbonyl side of aromatic or
bulky noncharged aliphatic residues (e.g. Phe,
Tyr, Trp, Leu)
• Trypsin - carbonyl side, basic residues (Lys,Arg).
• Staphylococcus aureus V8 protease - carbonyl
side of negatively charged residues (Glu, Asp).
NOTE: in 50mM ammonium bicarbonate cleaves
only at Glu.
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Fig 3.21 Cleavage, sequencing an oligopeptide
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Fig 3.20 Sequences of DNA and protein
• Protein amino acid sequences can be
deduced from the sequence of nucleotides in
the corresponding gene
• A sequence of three nucleotides specifies one
amino acid (A,C,G,T are DNA residues )
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3.10 Comparisons of the Primary Structures
of Proteins Reveal Evolutionary Relationships
• Closely related species contain proteins with
very similar amino acid sequences
• Differences reflect evolutionary change from a
common ancestral protein sequence
• Cytochrome c protein sequences from various
species can be aligned to show their similarities
• Phylogenetic tree shows evolutionary
differences in amino acid sequences
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Fig. 3.23
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Fig 3.24
Phylogenetic tree for
cytochrome c
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