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Chapter 25
Amino Acids, Peptides,
and Proteins
25.1
Classification of Amino Acids
Fundamentals
While their name implies that amino acids are
compounds that contain an —NH2 group and a
—CO2H group, these groups are actually
present as —NH3+ and —CO2– respectively.
They are classified as , , , etc. amino acids
according the carbon that bears the nitrogen.
Amino Acids
+
NH3

CO2–
+
–
H3NCH2CH2CO2

+
–
H3NCH2CH2CH2CO2

an -amino acid that is an
intermediate in the biosynthesis
of ethylene
a -amino acid that is one of
the structural units present in
coenzyme A
a -amino acid involved in
the transmission of nerve
impulses
The 20 Key Amino Acids
More than 700 amino acids occur naturally, but
20 of them are especially important.
These 20 amino acids are the building blocks of
proteins. All are -amino acids.
They differ in respect to the group attached to
the  carbon.
These 20 are listed in Table 25.1.
Table 25.1
H
+
H3N
C
O
C
O
–
R
The amino acids obtained by hydrolysis of
proteins differ in respect to R (the side chain).
The properties of the amino acid vary as the
structure of R varies.
Table 25.1
H
+
H3N
C
O
C
O
–
R
The major differences among the side chains
concern:
Size and shape
Electronic characteristics
Table 25.1
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Table 25.1
H
Glycine
(Gly or G)
+
H3N
C
O
C
O
–
H
Glycine is the simplest amino acid. It is the only
one in the table that is achiral.
In all of the other amino acids in the table the 
carbon is a chirality center.
Table 25.1
H
+
H3N
C
O
C
O
–
CH3
Alanine
(Ala or A)
Alanine, valine, leucine, and isoleucine have
alkyl groups as side chains, which are nonpolar
and hydrophobic.
Table 25.1
H
+
H3N
C
O
C
O
CH(CH3)2
Valine
(Val or V)
–
Table 25.1
H
+
H3N
C
O
C
O
–
CH2CH(CH3)2
Leucine
(Leu or L)
Table 25.1
H
+
H3N
C
O
C
O
–
CH3CHCH2CH3
Isoleucine
(Ile or I)
Table 25.1
H
+
H3N
C
O
C
O
–
CH3SCH2CH2
Methionine
(Met or M)
The side chain in methionine is nonpolar, but
the presence of sulfur makes it somewhat
polarizable.
Table 25.1
H
+
H2N
C
O
C
CH2
H2C
C
H2
O
–
Proline
(Pro or P)
Proline is the only amino acid that contains a
secondary amine function. Its side chain is
nonpolar and cyclic.
Table 25.1
H
+
H3N
C
CH2
O
C
O
–
Phenylalanine
(Phe or F)
The side chain in phenylalanine (a nonpolar
amino acid) is a benzyl group.
Table 25.1
H
+
H3N
C
O
C
O
–
Tryptophan
CH2
(Trp or W)
N
H
The side chain in
tryptophan (a nonpolar
amino acid) is larger
and more polarizable
than the benzyl group
of phenylalanine.
Table 25.1
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Table 25.1
H
+
H3N
C
O
C
O
–
CH2OH
Serine
(Ser or S)
The —CH2OH side chain in serine can be
involved in hydrogen bonding.
Table 25.1
H
+
H3N
C
O
C
O
–
CH3CHOH
Threonine
(Thr or T)
The side chain in threonine can be involved in
hydrogen bonding, but is somewhat more
crowded than in serine.
Table 25.1
H
+
H3N
C
O
C
O
–
CH2SH
Cysteine
(Cys or C)
The side chains of two remote cysteines can be
joined by forming a covalent S—S bond.
Table 25.1
H
+
H3N
C
O
C
O
–
Tyrosine
(Tyr or Y)
CH2
OH
The side chain of
tyrosine is similar to
that of phenylalanine
but can participate in
hydrogen bonding.
Table 25.1
H
+
H3N
C
H2NCCH2
O
C
O
–
Asparagine
(Asn or N)
O
The side chains of asparagine and glutamine
(next slide) terminate in amide functions that are
polar and can engage in hydrogen bonding.
Table 25.1
H
+
H3N
C
H2NCCH2CH2
O
O
C
O
–
Glutamine
(Gln or Q)
Table 25.1
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Table 25.1
H
+
H3N
–
C
OCCH2
O
C
O
–
Aspartic Acid
(Asp or D)
O
Aspartic acid and glutamic acid (next slide) exist
as their conjugate bases at biological pH. They
are negatively charged and can form ionic
bonds with positively charged species.
Table 25.1
H
+
H3N
–
C
OCCH2CH2
O
C
O
–
Glutamic Acid
(Glu or E)
O
Table 25.1
General categories of -amino acids
nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains
Table 25.1
H
Lysine
(Lys or K)
+
H3N
C
O
C
O
–
+
CH2CH2CH2CH2NH3
Lysine and arginine (next slide) exist as their
conjugate acids at biological pH. They are
positively charged and can form ionic bonds
with negatively charged species.
Table 25.1
H
Arginine
(Arg or R)
+
H3N
C
O
C
O
–
CH2CH2CH2NHCNH2
+ NH2
Table 25.1
H
Histidine
+
H3N
(His or H)
C
CH2
N
NH
O
C
O
–
Histidine is a basic
amino acid, but less
basic than lysine and
arginine. Histidine can
interact with metal ions
and can help move
protons from one site
to another.
25.2
Stereochemistry of Amino
Acids
Configuration of -Amino Acids
Glycine is achiral. All of the other amino acids
in proteins have the L-configuration at their
carbon.
–
CO2
+
H3N
H
R
25.3
Acid-Base Behavior of Amino
Acids
Recall
While their name implies that amino acids are
compounds that contain an —NH2 group and a
—CO2H group, these groups are actually
present as —NH3+ and —CO2– respectively.
How do we know this?
Properties of Glycine
The properties of glycine:
high melting point: (when heated to 233°C
it decomposes before it melts)
solubility: soluble in water; not soluble in
nonpolar solvent
more consistent with this
than this
•• O ••
+
H3NCH2C
•• O ••
•• •–
O•
••
••
H2NCH2C
••
OH
••
Properties of Glycine
The properties of glycine:
high melting point: (when heated to 233°C
it decomposes before it melts)
solubility: soluble in water; not soluble in
nonpolar solvent
more consistent with this
•• O ••
+
H3NCH2C
•• •–
O•
••
called a zwitterion or
dipolar ion
Acid-Base Properties of Glycine
The zwitterionic structure of glycine also follows
from considering its acid-base properties.
A good way to think about this is to start with the
structure of glycine in strongly acidic solution,
say pH = 1.
At pH = 1, glycine exists in its protonated form
(a monocation).
•• O ••
+
H3NCH2C
••
OH
••
Acid-Base Properties of Glycine
Now ask yourself "As the pH is raised, which is
the first proton to be removed? Is it the proton
attached to the positively charged nitrogen, or is
it the proton of the carboxyl group?"
You can choose between them by estimating
their respective pKas.
typical
ammonium
ion: pKa ~9
•• O ••
+
H3NCH2C
••
OH
••
typical
carboxylic
acid: pKa ~5
Acid-Base Properties of Glycine
The more acidic proton belongs to the CO2H
group. It is the first one removed as the pH is
raised.
•• O ••
+
H3NCH2C
••
OH
••
typical
carboxylic
acid: pKa ~5
Acid-Base Properties of Glycine
Therefore, the more stable neutral form of
glycine is the zwitterion.
•• O ••
+
H3NCH2C
•• •–
O•
••
•• O ••
+
H3NCH2C
••
OH
••
typical
carboxylic
acid: pKa ~5
Acid-Base Properties of Glycine
The measured pKa of glycine is 2.34.
Glycine is stronger than a typical carboxylic acid
because the positively charged N acts as an
electron-withdrawing, acid-strengthening
substituent on the  carbon.
•• O ••
+
H3NCH2C
••
OH
••
typical
carboxylic
acid: pKa ~5
Acid-Base Properties of Glycine
A proton attached to N in the zwitterionic form of
nitrogen can be removed as the pH is increased
further.
•• O ••
+
H3NCH2C
•• •–
O•
••
HO
–
•• O ••
••
H2NCH2C
•• •–
O•
••
The pKa for removal of this proton is 9.60.
This value is about the same as that for NH4+
(9.3).
Isoelectric Point (pI)
•• O ••
+
H3NCH2C
••
OH
••
pKa = 2.34
•• O ••
+
H3NCH2C
•• •–
O•
••
pKa = 9.60
•• O ••
••
H2NCH2C
•• •–
O•
••
The pH at which the
concentration of the
zwitterion is a
maximum is called the
isoelectric point. Its
numerical value is the
average of the two
pKas.
The pI of glycine is
5.97.
Acid-Base Properties of Amino Acids
One way in which amino acids differ is in
respect to their acid-base properties. This is the
basis for certain experimental methods for
separating and identifying them.
Just as important, the difference in acid-base
properties among various side chains affects
the properties of the proteins that contain them.
Table 25.2 gives pKa and pI values for amino
acids with neutral side chains.
Table 25.2
Amino Acids with Neutral Side Chains
H
Glycine
+
H3N
C
H
O
C
O
–
pKa1 = 2.34
pKa2 = 9.60
pI = 5.97
Table 25.2
Amino Acids with Neutral Side Chains
H
Alanine
+
H3N
C
CH3
O
C
O
–
pKa1 = 2.34
pKa2 = 9.69
pI = 6.00
Table 25.2
Amino Acids with Neutral Side Chains
H
Valine
+
H3N
C
O
C
O
CH(CH3)2
–
pKa1 = 2.32
pKa2 = 9.62
pI = 5.96
Table 25.2
Amino Acids with Neutral Side Chains
H
Leucine
+
H3N
C
O
C
O
–
CH2CH(CH3)2
pKa1 = 2.36
pKa2 = 9.60
pI = 5.98
Table 25.2
Amino Acids with Neutral Side Chains
H
Isoleucine
+
H3N
C
O
C
O
–
CH3CHCH2CH3
pKa1 = 2.36
pKa2 = 9.60
pI = 6.02
Table 25.2
Amino Acids with Neutral Side Chains
Methionine
+
H3N
H
O
C
C
CH3SCH2CH2
pKa1 = 2.28
–
pKa2 = 9.21
O
pI = 5.74
Table 25.2
Amino Acids with Neutral Side Chains
H
Proline
+
H2N
C
O
C
CH2
H2C
C
H2
O
–
pKa1 = 1.99
pKa2 = 10.60
pI = 6.30
Table 25.2
Amino Acids with Neutral Side Chains
H
Phenylalanine
+
H3N
C
CH2
O
C
O
–
pKa1 = 1.83
pKa2 = 9.13
pI = 5.48
Table 25.2
Amino Acids with Neutral Side Chains
H
Tryptophan
+
H3N
C
CH2
N
H
O
C
O
–
pKa1 = 2.83
pKa2 = 9.39
pI = 5.89
Table 25.2
Amino Acids with Neutral Side Chains
H
Asparagine
+
H3N
C
H2NCCH2
O
O
C
O
–
pKa1 = 2.02
pKa2 = 8.80
pI = 5.41
Table 25.2
Amino Acids with Neutral Side Chains
H
Glutamine
+
H3N
C
H2NCCH2CH2
O
O
C
O
–
pKa1 = 2.17
pKa2 = 9.13
pI = 5.65
Table 25.2
Amino Acids with Neutral Side Chains
H
Serine
+
H3N
C
O
C
CH2OH
O
–
pKa1 = 2.21
pKa2 = 9.15
pI = 5.68
Table 25.2
Amino Acids with Neutral Side Chains
H
Threonine
+
H3N
C
O
C
CH3CHOH
O
–
pKa1 = 2.09
pKa2 = 9.10
pI = 5.60
Table 25.2
Amino Acids with Neutral Side Chains
H
Tyrosine
+
H3N
C
CH2
OH
O
C
O
–
pKa1 = 2.20
pKa2 = 9.11
pI = 5.66
Table 25.3
Amino Acids with Ionizable Side Chains
H
Aspartic acid
+
H3N
–
C
OCCH2
O
C
O
–
pKa1 =
pKa2 =
pKa* =
pI =
1.88
9.60
3.65
2.77
O
For amino acids with acidic side chains, pI is the
average of pKa1 and pKa*.
Table 25.3
Amino Acids with Ionizable Side Chains
H
+
H3N
Glutamic acid
–
C
OCCH2CH2
O
O
C
O
–
pKa1 =
pKa2 =
pKa* =
pI =
2.19
9.67
4.25
3.22
Table 25.3
Amino Acids with Ionizable Side Chains
H
+
H3N
C
O
C
O
–
+
CH2CH2CH2CH2NH3
pKa1 =
pKa2 =
pKa* =
pI =
2.18
8.95
10.53
9.74
Lysine
For amino acids with basic side chains, pI is the
average of pKa2 and pKa*.
Table 25.3
Amino Acids with Ionizable Side Chains
H
+
H3N
C
O
C
O
–
CH2CH2CH2NHCNH2
+ NH2
Arginine
pKa1 =
pKa2 =
pKa* =
pI =
2.17
9.04
12.48
10.76
Table 25.3
Amino Acids with Ionizable Side Chains
H
Histidine
+
H3N
C
CH2
N
NH
O
C
O
–
pKa1 =
pKa2 =
pKa* =
pI =
1.82
6.00
9.17
7.59
25.4
Synthesis of Amino Acids
From -Halo Carboxylic Acids
O
CH3CHCOH + 2NH3
Br
H2O
O
–
CH3CHCO + NH4Br
+NH3
(65-70%)
Strecker Synthesis
O
CH3CH
NH4Cl
NaCN
CH3CHC
N
NH2
1. H2O, HCl, heat
2. HO–
O
–
CH3CHCO
+NH3
(52-60%)
Using Diethyl Acetamidomalonate
O
O
C
C
C
CH3CH2O
CH3CNH
H
OCH2CH3
O
Can be used in the same manner as diethyl
malonate (Section 20.11).
Example
O O
CH3CH2OCCCOCH2CH3
H
CH3CNH
O
1. NaOCH2CH3
2. C6H5CH2Cl
O O
CH3CH2OCCCOCH2CH3
CH3CNH
O
CH2C6H5
(90%)
O O
Example
HOCCCOH
–CO2
CH2C6H5
H3 N
+
O
HBr, H2O, heat
HCCOH
H3N
+
CH2C6H5
O O
(65%)
CH3CH2OCCCOCH2CH3
CH3CNH
O
CH2C6H5
25.5
Reactions of Amino Acids
Acylation of Amino Group
The amino nitrogen of an amino acid can be
converted to an amide with the customary
acylating agents.
O
O O
+
– +
H3NCH2CO
CH3COCCH3
O
O
CH3CNHCH2COH
(89-92%)
Esterification of Carboxyl Group
The carboxyl group of an amino acid can be
converted to an ester. The following illustrates
Fischer esterification of alanine.
O
+
– +
H3NCHCO
CH3CH2OH
CH3
HCl
O
Cl
–
+
H3NCHCOCH2CH3
CH3
(90-95%)
Ninhydrin Test
Amino acids are detected by the formation of a purple
color on treatment with ninhydrin.
O
O
OH
+
+ H3NCHCO–
OH
R
O
O
O–
O
RCH + CO2 + H2O +
N
O
O
25.6
Some Biochemical Reactions
of Amino Acids
Biosynthesis of L-Glutamic Acid
O
HO2CCH2CH2CCO2H + NH3
enzymes and
reducing coenzymes
–
HO2CCH2CH2CHCO2
+ NH3
This reaction is the biochemical analog of reductive
amination (Section 21.10).
Transamination via L-Glutamic Acid
O
–
HO2CCH2CH2CHCO2
+
CH3CCO2H
+ NH3
L-Glutamic acid acts as a source of the amine
group in the biochemical conversion of -keto
acids to other amino acids. In the example to be
shown, pyruvic acid is converted to L-alanine.
Transamination via L-Glutamic Acid
O
–
HO2CCH2CH2CHCO2
+
CH3CCO2H
+ NH3
enzymes
O
HO2CCH2CH2CCO2H
–
+ CH3CHCO2
+ NH3
Mechanism
–
HO2CCH2CH2CHCO2
+
+ NH3
PLP
The first step is imine formation between the
amino group of L-glutamic acid and a coenzyme
called pyridoxal phosphate (PLP).
Mechanism
–
HO2CCH2CH2CHCO2
+
+ NH3
HO2CCH2CH2CHCO2–
Formation of the imine is followed by proton
removal at one carbon and protonation of
another carbon.
H
HO2CCH2CH2CCO2–
HO2CCH2CH2CCO2–
H
HO2CCH2CH2CCO2–
HO2CCH2CH2CCO2–
Hydrolysis of the imine function gives
-ketoglutarate and pyridoxamine phosphate.
HO2CCH2CH2CCO2–
H2O
–
HO2CCH2CH2CCO2
O
+
The pyridoxamine can do the same sequence of
steps in reverse with pyruvate to generate alanine
and regenerate PLP.
O
CH3CCO2H
+
–
CH3CHCO2
+ NH3
O
CH3CCO2H
+
Biosynthesis of L-Tyrosine
L-Tyrosine is biosynthesized from L-phenylalanine.
A key step is epoxidation of the aromatic ring to
give an arene oxide intermediate.
–
CH2CHCO2
+ NH3
Biosynthesis of L-Tyrosine
–
CH2CHCO2
O
+ NH3
O2, enzyme
–
CH2CHCO2
+ NH3
Biosynthesis of L-Tyrosine
–
CH2CHCO2
O
+ NH3
enzyme
HO
–
CH2CHCO2
+ NH3
Biosynthesis of L-Tyrosine
Conversion to L-tyrosine is one of the major
metabolic pathways of L-phenylalanine.
Individuals who lack the enzymes necessary to
convert L-phenylalanine to L-tyrosine can suffer
from PKU disease. In PKU disease, Lphenylalanine is diverted to a pathway leading
to phenylpyruvic acid, which is toxic.
Newborns are routinely tested for PKU disease.
Treatment consists of reducing their dietary
intake of phenylalanine-rich proteins.
Decarboxylation
Decarboxylation is a common reaction of amino acids. An example is the conversion of
L-histidine to histamine. Antihistamines act by
blocking the action of histamine.
N
–
CH2CHCO2
N
H
+ NH3
Decarboxylation
N
CH2CH2 NH2
N
H
–CO2, enzymes
N
–
CH2CHCO2
N
H
+ NH3
Neurotransmitters
–
The chemistry of the
brain and central
nervous system is
affected by
neurotransmitters.
Several important
neurotransmitters
are biosynthesized
from L-tyrosine.
+
H3N
H
CO2
H
H
OH
L-Tyrosine
Neurotransmitters
–
The common name
of this compound is
L-DOPA. It occurs
naturally in the
brain. It is widely
prescribed to reduce
the symptoms of
Parkinsonism.
+
H3N
H
CO2
H
H
HO
OH
L-3,4-Dihydroxyphenylalanine
Neurotransmitters
Dopamine is formed
by decarboxylation
of L-DOPA.
H
H2N
H
H
H
HO
OH
Dopamine
Neurotransmitters
H
H2N
H
H
OH
HO
OH
Norepinephrine
Neurotransmitters
H
CH3NH
H
H
OH
HO
OH
Epinephrine
25.7
Peptides
Peptides
Peptides are compounds in which an amide
bond links the amino group of one -amino acid
and the carboxyl group of another.
An amide bond of this type is often referred to
as a peptide bond.
Alanine and Glycine
H
+
H3N
C
CH3
H
O
C
–
O
+
H3N
C
H
O
C
–
O
Alanylglycine
H
+
H3N
C
CH3
H
O
C
N
C
H
H
O
C
–
O
Two -amino acids are joined by a peptide bond
in alanylglycine. It is a dipeptide.
Alanylglycine
H
+
H3N
N-terminus
C
CH3
H
O
C
N
C
H
H
Ala—Gly
AG
O
C
–
O
C-terminus
Alanylglycine and glycylalanine are
constitutional isomers
H
+
H3N
C
C
CH3
H
+
H3N
C
H
H
O
N
C
H
H
H
O
C
N
C
H
CH3
O
C
–
O
Alanylglycine
Ala—Gly
AG
–
O
Glycylalanine
Gly—Ala
GA
O
C
Alanylglycine
H
+
H3N
C
CH3
H
O
C
N
C
H
H
O
C
–
O
The peptide bond is
characterized by a
planar geometry.
Higher Peptides
Peptides are classified according to the number
of amino acids linked together.
dipeptides, tripeptides, tetrapeptides, etc.
Leucine enkephalin is an example of a
pentapeptide.
Leucine Enkephalin
Tyr—Gly—Gly—Phe—Leu
YGGFL
Oxytocin
3
2
4
5
Ile—Gln—Asn
Tyr
1
Cys
N-terminus
C-terminus
Cys—Pro—Leu—GlyNH2
6
S
7
8
9
S
Oxytocin is a cyclic nonapeptide.
Instead of having its amino acids linked in an
extended chain, two cysteine residues are
joined by an S—S bond.
Oxytocin
S—S bond
An S—S bond between two cysteines is
often referred to as a disulfide bridge.
25.8
Introduction to Peptide
Structure Determination
Primary Structure
The primary structure is the amino acid
sequence plus any disulfide links.
Classical Strategy (Sanger)
1. Determine what amino acids are present and
their molar ratios.
2. Cleave the peptide into smaller fragments,
and determine the amino acid composition of
these smaller fragments.
3. Identify the N-terminus and C-terminus in the
parent peptide and in each fragment.
4. Organize the information so that the
sequences of small fragments can be
overlapped to reveal the full sequence.
25.9
Amino Acid Analysis
Amino Acid Analysis
Acid-hydrolysis of the peptide (6 M HCl, 24 hr)
gives a mixture of amino acids.
The mixture is separated by ion-exchange
chromatography, which depends on the
differences in pI among the various amino
acids.
Amino acids are detected using ninhydrin.
Automated method; requires only 10-5 to 10-7 g
of peptide.
25.10
Partial Hydrolysis of Peptides
Partial Hydrolysis of Peptides and Proteins
Acid-hydrolysis of the peptide cleaves all of the
peptide bonds.
Cleaving some, but not all, of the peptide bonds
gives smaller fragments.
These smaller fragments are then separated
and the amino acids present in each fragment
determined.
Enzyme-catalyzed cleavage is the preferred
method for partial hydrolysis.
Partial Hydrolysis of Peptides and Proteins
The enzymes that catalyze the hydrolysis of
peptide bonds are called peptidases, proteases,
or proteolytic enzymes.
Trypsin
Trypsin is selective for cleaving the peptide bond
to the carboxyl group of lysine or arginine.
O
O
O
NHCHC
NHCHC
NHCHC
R
R'
R"
lysine or arginine
Chymotrypsin
Chymotrypsin is selective for cleaving the peptide
bond to the carboxyl group of amino acids with
an aromatic side chain.
O
O
O
NHCHC
NHCHC
NHCHC
R
R'
R"
phenylalanine, tyrosine, tryptophan
Carboxypeptidase
Carboxypeptidase is selective for cleaving
the peptide bond to the C-terminal amino acid.
O
O
+
H3NCHC
R
protein
C
O
–
NHCHCO
R
25.11
End Group Analysis
End Group Analysis
Amino sequence is ambiguous unless we know
whether to read it left-to-right or right-to-left.
We need to know what the N-terminal and Cterminal amino acids are.
The C-terminal amino acid can be determined
by carboxypeptidase-catalyzed hydrolysis.
Several chemical methods have been
developed for identifying the N-terminus. They
depend on the fact that the amino N at the
terminus is more nucleophilic than any of the
amide nitrogens.
Sanger's Method
The key reagent in Sanger's method for
identifying the N-terminus is 1-fluoro-2,4dinitrobenzene.
1-Fluoro-2,4-dinitrobenzene is very reactive
toward nucleophilic aromatic substitution
(Chapter 12).
NO2
O2N
F
Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the
amino nitrogen of the N-terminal amino acid.
NO2
O2N
O
O
F + H2NCHC
NHCHC
NHCH2C
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
–
NHCHCO
CH3
CH(CH3)2 CH2C6H5
NO2
O
O
O
–
NHCHCO
CH3
Sanger's Method
Acid hydrolysis cleaves all of the peptide bonds
leaving a mixture of amino acids, only one of
which (the N-terminus) bears a 2,4-DNP group.
NO2
O
O
O
O
+
+
+
NHCHCOH + H3NCHCO– + H3NCH2CO– + H3NCHCO–
O2N
CH(CH3)2
CH3
CH2C6H5
H3O+
NO2
O2N
O
NHCHC
O
NHCHC
O
NHCH2C
CH(CH3)2 CH2C6H5
O
–
NHCHCO
CH3
25.12
Insulin
Insulin
Insulin is a polypeptide with 51 amino acids.
It has two chains, called the A chain (21 amino
acids) and the B chain (30 amino acids).
The following describes how the amino acid
sequence of the B chain was determined.
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
The B Chain of Bovine Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Overlaps between the above peptide sequences were
found in four additional peptides:
SHLV
LVGA
ALT
TLVC
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
YTPKA
The B Chain of Bovine Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Overlaps between the above peptide sequences were
found in four additional peptides:
SHLV
LVGA
ALT
TLVC
Trypsin-catalyzed hydrolysis gave GFFYTPK which
completes the sequence.
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
GFFYTPK
YTPKA
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALY
YLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALYLVCGERGFFYTPKA
Insulin
The sequence of the A chain was determined
using the same strategy.
Establishing the disulfide links between cysteine
residues completed the primary structure.
Primary Structure of Bovine Insulin
N terminus
of A chain
S
S
C terminus
of A chain
15
5
E Q C
V
C S L Y Q L
I
F
E N
20
C
V
YC
A S
S
10
N
S
S
H
L
N Q
V
S
C
F
G S H L V G A L Y L V
5
C
15
G 20
10
N terminus
E
of B chain
G R
F
F
Y
K P T
A
C terminus
25
30
of B chain
25.13
The Edman Degradation and
Automated Sequencing of
Peptides
Edman Degradation
1. Method for determining N-terminal amino
acid.
2. Can be done sequentially one residue at a
time on the same sample. Usually one can
determine the first 20 or so amino acids from
the N-terminus by this method.
3. 10-10 g of sample is sufficient.
4. Has been automated.
Edman Degradation
The key reagent in the Edman degradation is
phenyl isothiocyanate.
N
C
S
Edman Degradation
Phenyl isothiocyanate reacts with the amino
nitrogen of the N-terminal amino acid.
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
O
C6H5N
C
S
+
+ H3NCHC
R
NH
peptide
Edman Degradation
S
O
C6H5NHCNHCHC
NH
peptide
R
The product is a phenylthiocarbamoyl (PTC)
derivative.
The PTC derivative is then treated with HCl in
an anhydrous solvent. The N-terminal amino
acid is cleaved from the remainder of the
peptide.
Edman Degradation
S
O
C6H5NHCNHCHC
peptide
NH
R
HCl
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide
Edman Degradation
The product is a thiazolone. Under the
conditions of its formation, the thiazolone
rearranges to a phenylthiohydantoin (PTH)
derivative.
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide
Edman Degradation
C6H5
S
N
C
C
The PTH derivative is
isolated and identified.
The remainder of the
peptide is subjected to
a second Edman
degradation.
O
CH
HN
R
S
C6H5NH
C
C
N
CH
R
O
+
+
H3N
peptide
25.14
The Strategy of Peptide Synthesis
General Considerations
Making peptide bonds between amino acids is
not difficult.
The challenge is connecting amino acids in the
correct sequence.
Random peptide bond formation in a mixture of
phenylalanine and glycine, for example, will give
four dipeptides.
Phe—Phe
Gly—Gly
Phe—Gly
Gly—Phe
General Strategy
1. Limit the number of possibilities by
"protecting" the nitrogen of one amino acid
and the carboxyl group of the other.
N-Protected
phenylalanine
O
X
NHCHCOH
CH2C6H5
C-Protected
glycine
O
H2NCH2C
Y
General Strategy
2. Couple the two protected amino acids.
X
O
O
NHCHC
NHCH2C
Y
CH2C6H5
O
X
NHCHCOH
CH2C6H5
O
H2NCH2C
Y
General Strategy
3. Deprotect the amino group at the N-terminus
and the carboxyl group at the C-terminus.
X
O
O
NHCHC
NHCH2C
Y
CH2C6H5
O
+
H3NCHC
O
–
NHCH2CO
CH2C6H5
Phe-Gly
25.15
Amino Group Protection
Protect Amino Groups as Amides
Amino groups are normally protected by
converting them to amides.
Benzyloxycarbonyl (C6H5CH2O—) is a common
protecting group. It is abbreviated as Z.
Z-protection is carried out by treating an amino
acid with benzyloxycarbonyl chloride.
Protect Amino Groups as Amides
O
O
CH2OCCl
+
+
–
H3NCHCO
CH2C6H5
1. NaOH, H2O
2. H+
O
CH2OC
O
NHCHCOH
CH2C6H5
(82-87%)
Protect Amino Groups as Amides
O
CH2OC
O
NHCHCOH
CH2C6H5
is abbreviated as:
O
ZNHCHCOH
CH2C6H5
or Z-Phe
Removing Z-Protection
An advantage of the benzyloxycarbonyl
protecting group is that it is easily removed by:
a) hydrogenolysis
b) cleavage with HBr in acetic acid
Hydrogenolysis of Z-Protecting Group
O
CH2OC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
H2, Pd
O
CH3
CO2
H2NCHCNHCH2CO2CH2CH3
CH2C6H5
(100%)
HBr Cleavage of Z-Protecting Group
O
CH2OC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
CH2Br
CO2
+
H3NCHCNHCH2CO2CH2CH3
–
CH2C6H5 Br
(82%)
The tert-Butoxycarbonyl Protecting Group
O
(CH3)3COC
O
NHCHCOH
CH2C6H5
is abbreviated as:
O
BocNHCHCOH
CH2C6H5
or Boc-Phe
HBr Cleavage of Boc-Protecting Group
O
(CH3)3COC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
H3C
C
H3C
CH2
CO2
+
H3NCHCNHCH2CO2CH2CH3
–
CH2C6H5 Br
(86%)
25.16
Carboxyl Group Protection
Protect Carboxyl Groups as Esters
Carboxyl groups are normally protected as
esters.
Deprotection of methyl and ethyl esters is
by hydrolysis in base.
Benzyl esters can be cleaved by
hydrogenolysis.
Hydrogenolysis of Benzyl Esters
O
O
C6H5CH2OC
O
NHCHCNHCH2COCH2C6H5
CH2C6H5
H2, Pd
O
C6H5CH3
CO2
+
–
H3NCHCNHCH2CO
CH2C6H5
(87%)
CH3C6H5
25.17
Peptide Bond Formation
Forming Peptide Bonds
The two major methods are:
1. coupling of suitably protected amino acids
using N,N'-dicyclohexylcarbodiimide (DCCI)
2. via an active ester of the N-terminal amino
acid.
DCCI-Promoted Coupling
O
O
ZNHCHCOH
+ H2NCH2COCH2CH3
CH2C6H5
DCCI, chloroform
O
ZNHCHC
O
NHCH2COCH2CH3
CH2C6H5
(83%)
Mechanism of DCCI-Promoted Coupling
O
+
ZNHCHCOH
C6H11N
C
CH2C6H5
H
C6H11N
O
C
C6H11N
OCCHNHZ
CH2C6H5
NC6H11
Mechanism of DCCI-Promoted Coupling
The species formed by addition of the Zprotected amino acid to DCCI is similar in
structure to an acid anhydride and acts as an
acylating agent.
Attack by the amine function of the carboxylprotected amino acid on the carbonyl group
leads to nucleophilic acyl substitution.
H
C6H11N
O
C
C6H11N
OCCHNHZ
CH2C6H5
Mechanism of DCCI-Promoted Coupling
O
H
C6H11N
C
O +
ZNHCHC
O
NHCH2COCH2CH3
CH2C6H5
C6H11NH
O
H2NCH2COCH2CH3
H
C6H11N
O
C
C6H11N
OCCHNHZ
CH2C6H5
The Active Ester Method
A p-nitrophenyl ester is an example of an "active
ester."
p-Nitrophenyl is a better leaving group than
methyl or ethyl, and p-nitrophenyl esters are
more reactive in nucleophilic acyl substitution.
The Active Ester Method
O
O
ZNHCHCO
NO2 +
H2NCH2COCH2CH3
CH2C6H5
chloroform
O
ZNHCHC
O
NHCH2COCH2CH3 + HO
CH2C6H5
(78%)
NO2
25.18
Solid-Phase Peptide Synthesis:
The Merrifield Method
Solid-Phase Peptide Synthesis
In solid-phase synthesis, the starting material is
bonded to an inert solid support.
Reactants are added in solution.
Reaction occurs at the interface between the
solid and the solution. Because the starting
material is bonded to the solid, any product from
the starting material remains bonded as well.
Purification involves simply washing the
byproducts from the solid support.
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
The solid support is a copolymer of styrene and
divinylbenzene. It is represented above as if it
were polystyrene. Cross-linking with
divinylbenzene simply provides a more rigid
polymer.
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
Treating the polymeric support with
chloromethyl methyl ether (ClCH2OCH3) and
SnCl4 places ClCH2 side chains on some of the
benzene rings.
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
CH2Cl
The side chain chloromethyl group is a benzylic
halide, reactive toward nucleophilic substitution
(SN2).
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
CH2Cl
The chloromethylated resin is treated with the Bocprotected C-terminal amino acid. Nucleophilic
substitution occurs, and the Boc-protected amino
acid is bound to the resin as an ester.
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
–
BocNHCHCO
R
CH
CH2
CH2Cl
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHCO
Next, the Boc
protecting group is
removed with HCl.
R
CH
CH2
CH2
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHCO
DCCI-promoted
coupling adds the
second amino acid.
R
CH
CH2
CH2
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
Remove the Boc
protecting group.
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
Add the next amino
acid and repeat.
The Merrifield Procedure
CH2
CH
CH2
O
CH
CH2
O
+
H3N peptide C NHCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
Remove the peptide
from the resin with
HBr in CF3CO2H.
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
CH
CH2
CH2Br
O
O
+
H3N peptide C NHCHC
R'
O
–
NHCHCO
R
CH
The Merrifield Method
Merrifield automated his solid-phase method.
Synthesized a nonapeptide (bradykinin) in 1962
in 8 days in 68% yield.
Synthesized ribonuclease (124 amino acids) in
1969.
369 reactions; 11,391 steps
Nobel Prize in chemistry: 1984
25.19
Secondary Structures
of Peptides and Proteins
Levels of Protein Structure
Primary structure = the amino acid sequence
plus disulfide links.
Secondary structure = conformational
relationship between nearest neighbor amino
acids.
 helix
pleated  sheet
Levels of Protein Structure
The -helix and pleated  sheet are both
characterized by:
Planar geometry of peptide bond
Anti conformation of main chain
Hydrogen bonds between N—H and O=C
Pleated  Sheet
Shown is a  sheet of protein chains composed of
alternating glycine and alanine residues.
Adjacent chains are antiparallel.
Hydrogen bonds between chains.
van der Waals forces produce pleated effect.
Pleated  Sheet
 Sheet is most commonly seen with amino acids
having small side chains (glycine, alanine, serine).
80% of fibroin (main protein in silk) is repeating
sequence of —Gly—Ser—Gly—Ala—Gly—Ala—.
 Sheet is flexible, but resists stretching.
 Helix
Shown is an  helix of a protein
in which all of the amino acids
are L-alanine.
Helix is right-handed with 3.6
amino acids per turn.
Hydrogen bonds are within a
single chain.
Protein of muscle (myosin) and
wool (-keratin) contain large
regions of -helix. Chain can
be stretched.
25.20
Tertiary Structure
of Polypeptides and Proteins
Tertiary Structure
Refers to overall shape (how the chain is folded).
Fibrous proteins (hair, tendons, wool) have
elongated shapes.
Globular proteins are approximately spherical.
Most enzymes are globular proteins.
An example is carboxypeptidase.
Carboxypeptidase
Carboxypeptidase is an enzyme that catalyzes
the hydrolysis of proteins at their C-terminus.
It is a metalloenzyme containing Zn2+ at its
active site.
An amino acid with a positively charged side
chain (Arg-145) is near the active site.
Carboxypeptidase
Disulfide bond
Zn2+
Arg-145
N-terminus
C-terminus
Tube model
Ribbon model
What Happens at the Active Site?
•• •
O•
+
H3N
peptide
C
O
NHCHC –
R
O
H2N
+ C
H2N
Arg-145
What Happens at the Active Site?
•• •
O•
+
H3N
peptide
C
O
NHCHC –
R
O
H2N
+ C
Arg-145
H2N
The peptide or protein is bound at the active site
by electrostatic attraction between its negatively
charged carboxylate ion and arginine-145.
What Happens at the Active Site?
•• •
O•
+
H3N
peptide
C
Zn2+
O
NHCHC –
R
O
H2N
+ C
Arg-145
H2N
Zn2+ acts as a Lewis acid toward the carbonyl
oxygen, increasing the positive character of the
carbonyl carbon.
What Happens at the Active Site?
Zn2+
•• •
O•
+
H3N
peptide
C
O
NHCHC –
R
O
H2N
+ C
Arg-145
H2N
H
• O•
• •
H
Water attacks the carbonyl carbon. Nucleophilic
acyl substitution occurs.
What Happens at the Active Site?
Zn2+
H2N
+ C
•• •
O•
+
H3N
peptide
C
•• –
O ••
••
H2N
O
+
H3NCHC –
R
O
Arg-145
25.21
Coenzymes
Coenzymes
The range of chemical reactions that amino acid
side chains can participate in is relatively
limited.
Acid-base (transfer and accept protons)
Nucleophilic acyl substitution
Many other biological processes, such as
oxidation-reduction, require coenzymes,
cofactors, or prosthetic groups in order to occur.
Coenzymes
NADH, coenzyme A and coenzyme B12 are
examples of coenzymes.
Heme is another example.
Heme
H 2C
CH
H 3C
CH3
N
N
CH
CH2
Fe
N
H 3C
HO2CCH2CH2
N
CH3
CH2CH2CO2H
Molecule surrounding iron is a
type of porphyrin.
Myoglobin
C-terminus
Heme
N-terminus
Heme is the coenzyme that binds oxygen in myoglobin
(oxygen storage in muscles) and hemoglobin (oxygen
transport).
25.22
Protein Quaternary Structure:
Hemoglobin
Protein Quaternary Structure
Some proteins are assemblies of two or more
chains. The way in which these chains are
organized is called the quaternary structure.
Hemoglobin, for example, consists of 4
subunits.
There are 2  chains (identical) and 2  chains
(also identical).
Each subunit contains one heme and each
protein is about the size of myoglobin.
25.23
G-Coupled Protein Receptors
G-Coupled Protein Receptors
GCPRs (the “G” stands for guanine in “guanine
nucleotide-binding proteins”) occur throughout
the body and function as “molecular switches”
that regulate many physiological processes.
GCPRs span the cell membrane and when
they bind their specific ligand (a small organic
molecule, lipid, peptide, ion, etc.), they undergo
a conformational change, which results in the
transduction of a signal across the membrane.
The Core of Modern Medicine
GCPRs are the target for many therapeutic
agents in the treatment of cancer, cardiac
malfunction, inflammation, pain, obesity, diabetes
and disorders of the central nervous system.