Biokimia1-Prot-Amino Acids, Peptides, and Proteins

Chapter
Chapter 27
27
Amino
Amino Acids,
Acids, Peptides,
Peptides,
and
and Proteins
Proteins
Dr. Wolf's CHM 424 27- 1

27.1
27.1
Classification
Classification of
ofAmino
AminoAcids
Acids

Fundamentals
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
AminoAcids
Acids
+
NH3 an -amino acid that is an
intermediate in the biosynthesis
 of ethylene
CO2–
+ a -amino acid that is one of

– the structural units present in
H3NCH2CH2CO2
 coenzyme A
+ – a -amino acid involved in

H3NCH2CH2CH2CO2 the transmission of nerve
 impulses
The
The 20
20 Key
KeyAmino
Amino Acids
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 27.1.

Table
Table 27.1
27.1
H O
+ –
H3N C C O
R
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
Table 27.1
27.1
H O
+ –
H3N C C O
R
R
The major differences among the side chains

concern:
Size and shape
Electronic characteristics
Table
Table 27.1
27.1
General categories of a-amino acids

nonpolar side chains
polar but nonionized side chains
acidic side chains
basic side chains

Table
Table 27.1
27.1

acidic side chains
basic side chains

Table
Table 27.1
27.1
H O
+ –
Glycine H3N C C O
(Gly or G)
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
Table 27.1
27.1
H O
+ –
H3N C C O
CH3
Alanine
(Ala or A)
Alanine, valine, leucine, and isoleucine have
alkyl groups as side chains, which are nonpolar
and hydrophobic.
Table
Table 27.1
27.1
H O
+ –
H3N C C O
CH(CH3)2
Valine
(Val or V)

Table
Table 27.1
27.1
H O
+ –
H3N C C O
CH2CH(CH3)2
Leucine
(Leu or L)

Table
Table 27.1
27.1
H O
+ –
H3N C C O
CH3CHCH2CH3
Isoleucine
(Ile or I)

Table
Table 27.1
27.1
H O
+ –
H3N C C O
CH3SCH2CH2
Methionine
(Met or M)
The side chain in methionine is nonpolar, but
the presence of sulfur makes it somewhat
polarizable.
Table
Table 27.1
27.1
H O
+ –
H2N C C O
H2C CH2 Proline

C (Pro or P)
H2
Proline is the only amino acid that contains a

secondary amine function. Its side chain is
nonpolar and cyclic.
Table
Table 27.1
27.1
H O
+ –
H3N C C O
CH2
Phenylalanine
(Phe or F)
The side chain in phenylalanine (a nonpolar

amino acid) is a benzyl group.
Table
Table 27.1
27.1
H O
+ –
H3N C C O
Tryptophan
(Trp or W) CH2
The side chain in
tryptophan (a nonpolar
amino acid) is larger
N and more polarizable
than the benzyl group
H of phenylalanine.
Table
Table 27.1
27.1

acidic side chains
basic side chains

Table
Table 27.1
27.1
H O
+ –
H3N C C O
CH2OH
Serine
(Ser or S)
The —CH2OH side chain in serine can be

involved in hydrogen bonding.
Table
Table 27.1
27.1
H O
+ –
H3N C 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
Table 27.1
27.1
H O
+ –
H3N C 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
Table 27.1
27.1
H O
+ –
H3N C C O
Tyrosine
CH2
(Tyr or Y) The side chain of
tyrosine is similar to
that of phenylalanine
but can participate in
hydrogen bonding.
OH
Table
Table 27.1
27.1
H O
+ –
H3N C C O
H2NCCH2 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
Table 27.1
27.1
H O
+ –
H3N C C O
H2NCCH2CH2 Glutamine
(Gln or Q)
O

Table
Table 27.1
27.1

acidic side chains
basic side chains

Table
Table 27.1
27.1
H O
+ –
H3N C C O
– Aspartic Acid
OCCH2
(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
Table 27.1
27.1
H O
+ –
H3N C C O
– Glutamic Acid
OCCH2CH2
(Glu or E)
O

Table
Table 27.1
27.1

acidic side chains
basic side chains

Table
Table 27.1
27.1
H O
+ –
Lysine H3N C C O
(Lys or K) +
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
Table 27.1
27.1
H O
+ –
Arginine H3N C C O
(Arg or R)
CH2CH2CH2NHCNH2
+ NH2

Table
Table 27.1
27.1
H O
+ –
Histidine H3N C C O
(His or H) CH2 Histidine is a basic
amino acid, but less
basic than lysine and
N NH arginine. Histidine can
interact with metal ions
and can help move
protons from one site
Dr. Wolf's CHM 424
to another. 27- 32
27.2
27.2
Stereochemistry
Stereochemistry of
ofAmino
Amino
Acids
Acids

Configuration of -Amino
Configuration of -AminoAcids
Acids
Glycine is achiral. All of the other amino acids

in proteins have the L-configuration at their
carbon.
–
CO2
+
H3N H

27.3
27.3
Acid-Base
Acid-Base Behavior
Behavior of
ofAmino
Amino
Acids
Acids

Recall
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
Properties of
of Glycine
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 •• •• O ••
+ •• •– •• ••
H3NCH2C O• H2NCH2C OH
•• ••

Properties
Properties of
of Glycine
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 •• called a zwitterion or
+ dipolar ion
•• •–
H3NCH2C O•
••

Acid-Base
Acid-Base Properties
Properties of
of Glycine
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
of Glycine
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 typical
ammonium •• O •• carboxylic
ion: pKa ~9 + acid: pKa ~5
••
H3NCH2C OH
••
Acid-Base
Properties of
of Glycine
Glycine
The more acidic proton belongs to the CO2H

group. It is the first one removed as the pH is
raised.
typical
•• O •• carboxylic
+ acid: pKa ~5
••
H3NCH2C OH
••
Acid-Base
Properties of
of Glycine
Glycine
Therefore, the more stable neutral form of

glycine is the zwitterion.
•• O ••
+ •• •–
H3NCH2C O•
••
typical
+ acid: pKa ~5
••
H3NCH2C OH
••
Acid-Base
Properties of
of Glycine
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.
typical
+ acid: pKa ~5
••
H3NCH2C OH
••
Acid-Base
Properties of
of Glycine
Glycine
A proton attached to N in the zwitterionic form of

nitrogen can be removed as the pH is increased
further.
•• O •• •• O ••
+ –
•• •– HO •• •• •–
H3NCH2C 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
Isoelectric Point
Point pI
pI
•• O ••
+ ••
H3NCH2C OH The pH at which the
••
concentration of the
pKa = 2.34
zwitterion is a
•• O •• maximum is called the
+ •• •–
isoelectric point. Its
H3NCH2C O• numerical value is the
••
average of the two
pKa = 9.60
pKas.
•• O ••
The pI of glycine is
•• •• •– 5.97.
H2NCH2C O•
Dr. Wolf's CHM 424 •• 27- 45
Acid-Base
Properties of
ofAmino
AminoAcids
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 27.2 gives pKa and pI values for amino
acids with neutral side chains.

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.34
–
Glycine H3N C C O pKa2 = 9.60
pI = 5.97
H

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.34
–
Alanine H3N C C O pKa2 = 9.69
pI = 6.00
CH3

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.32
–
Valine H3N C C O pKa2 = 9.62
pI = 5.96
CH(CH3)2

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.36
–
Leucine H3N C C O pKa2 = 9.60
pI = 5.98
CH2CH(CH3)2

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.36
–
Isoleucine H3N C C O pKa2 = 9.60
pI = 5.98
CH3CHCH2CH3

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.28
–
Methionine H3N C C O pKa2 = 9.21
pI = 5.74
CH3SCH2CH2

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 1.99
–
Proline H2N C C O pKa2 = 10.60
pI = 6.30
H2C CH2
C
H2

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 1.83
–
Phenylalanine H3N C C O pKa2 = 9.13
pI = 5.48
CH2

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.83
–
Tryptophan H3N C C O pKa2 = 9.39
pI = 5.89
CH2
Dr. Wolf's CHM 424

H 27- 55
Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.02
–
Asparagine H3N C C O pKa2 = 8.80
pI = 5.41
H2NCCH2

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.17
–
Glutamine H3N C C O pKa2 = 9.13
pI = 5.65
H2NCCH2CH2

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.21
–
Serine H3N C C O pKa2 = 9.15
pI = 5.68
CH2OH

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 2.09
–
Threonine H3N C C O pKa2 = 9.10
pI = 5.60
CH3CHOH

Table
Table 27.2
27.2
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ –
H3N C C O pKa1 = 2.20
Tyrosine pKa2 = 9.11
CH2 pI = 5.66
Dr. Wolf's CHM 424

OH
27- 60
Table
Table 27.3
27.3
Amino
AminoAcids
Acids with
with Neutral
Neutral Side
Side Chains
Chains
H O
+ pKa1 = 1.96
–
Cysteine H3N C C O pKa2 = 8.18
pI = 5.07
CH2SH

Table
Table 27.3
27.3
Amino
AminoAcids
Acids with
with Ionizable
Ionizable Side
Side Chains
Chains
H O
+ pKa1 = 1.88
–
Aspartic acid H3N C C O pKa2 = 3.65
– pKa3 = 9.60
OCCH2
pI = 2.77
O
For amino acids with acidic side chains, pI is the
average of pKa1 and pKa2.
Table
Table 27.3
27.3
Amino
AminoAcids
Acids with
with Ionizable
Ionizable Side
Side Chains
Chains
H O
+ pKa1 = 2.19
–
Glutamic acid H3N C C O pKa2 = 4.25
– pKa3 = 9.67
OCCH2CH2
pI = 3.22
O

Table
Table 27.3
27.3
Amino
AminoAcids
Acids with
with Ionizable
Ionizable Side
Side Chains
Chains
H O
+ pKa1 = 2.18
–
H3N C C O pKa2 = 8.95
+ pKa3 = 10.53
CH2CH2CH2CH2NH3
pI = 9.74
Lysine
For amino acids with basic side chains, pI is the

average of pKa2 and pKa3.
Table
Table 27.3
27.3
Amino
AminoAcids
Acids with
with Ionizable
Ionizable Side
Side Chains
Chains
H O
+ – pKa1 = 2.17
H3N C C O
pKa2 = 9.04
CH2CH2CH2NHCNH2 pKa3 = 12.48
+ NH2 pI = 10.76
Arginine

Table
Table 27.3
27.3
Amino
AminoAcids
Acids with
with Ionizable
Ionizable Side
Side Chains
Chains
H O
pKa1 = 1.82
+ –
Histidine H3N C C O pKa2 = 6.00
pKa3 = 9.17
CH2 pI = 7.59
N NH

27.4
27.4
Synthesis
Synthesis of
ofAmino
AminoAcids
Acids

From -Halo
From -Halo Carboxylic
CarboxylicAcids
Acids
O O
H2O
– +
CH3CHCOH + 2NH3 CH3CHCO NH4Br
Br + NH3
(65-70%)

Strecker
Strecker Synthesis
Synthesis
O
NH4Cl
CH3CH CH3CHC N
NaCN
NH2
1. H2O, HCl, heat
2. HO–
O
– (52-60%)
CH3CHCO
Dr. Wolf's CHM 424

+ NH3
27- 69
Using
Using Diethyl
DiethylAcetamidomalonate
Acetamidomalonate
O O
C C
CH3CH2O C OCH2CH3
CH3CNH H
Can be used in the same manner as diethyl

malonate (Section 21.7).

Example
Example O O
CH3CH2OCCCOCH2CH3
CH3CNH H
O 1. NaOCH2CH3
2. C6H5CH2Cl
O O
CH3CH2OCCCOCH2CH3
CH3CNH CH2C6H5 (90%)
Dr. Wolf's CHM 424 O 27- 71

Example O O
Example
HOCCCOH
H3N CH2C6H5
–CO2 +
O
HBr, H2O, heat
HCCOH
H3N CH2C6H5
+ O O
(65%)
CH3CH2OCCCOCH2CH3
CH3CNH CH2C6H5
Dr. Wolf's CHM 424 O 27- 72

27.5
27.5
Reactions
Reactions of
of Amino
AminoAcids
Acids

Acylation
Acylation of
ofAmino
Amino Group
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
(89-92%)
CH3CNHCH2COH
Esterification
Esterification of
of Carboxyl
Carboxyl Group
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 (90-95%)
Dr. Wolf's CHM 424 CH3 27- 75

Ninhydrin
Ninhydrin Test
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
Dr. Wolf's CHM 424

O O 27- 76
27.6
27.6
Some
Some Biochemical
Biochemical Reactions
Reactions
of
ofAmino
AminoAcids
Acids

Biosynthesis
Biosynthesis of
of LL-Glutamic
-GlutamicAcid
Acid
HO2CCH2CH2CCO2H + NH3
enzymes and
reducing coenzymes
–
HO2CCH2CH2CHCO2
+ NH3
This reaction is the biochemical analog of reductive
amination (Section 22.10).
Transamination
Transamination via
via LL-Glutamic
-GlutamicAcid
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
Transamination via
via LL-Glutamic
-GlutamicAcid
Acid
O
–
+ NH3
enzymes
O
–
HO2CCH2CH2CCO2H + CH3CHCO2
+ NH3
Mechanism
Mechanism
O
–
+ NH3
The first step is imine formation between the

amino group of L-glutamic acid and pyruvic
acid.

Mechanism
Mechanism
O
–
+ NH3
–
HO2CCH2CH2CHCO2
CH3CCO2–
Formation of the imine is followed by proton
removal at one carbon and protonation of
another carbon.
H
–
HO2CCH2CH2CCO2
CH3CCO2–
–
HO2CCH2CH2CCO2
CH3CCO2–
H
–
HO2CCH2CH2CCO2
CH3CCO2–
–
HO2CCH2CH2CCO2
CH3CCO2–
Hydrolysis of the imine function gives

-keto glutarate and L-alanine.

–
HO2CCH2CH2CCO2
CH3CCO2–
H2O
+NH3
– –
HO2CCH2CH2CCO2 + CH3CCO2
O H
Biosynthesis
Biosynthesis of
of LL-Tyrosine
-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
Biosynthesis of
of LL-Tyrosine
-Tyrosine
–
CH2CHCO2
O + NH3
O2, enzyme
–
CH2CHCO2
+ NH3
Biosynthesis
Biosynthesis of
of LL-Tyrosine
-Tyrosine
–
CH2CHCO2
O + NH3
enzyme
–
HO CH2CHCO2
+ NH3
Biosynthesis
Biosynthesis of
of LL-Tyrosine
-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, L-
phenylalanine 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
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
Decarboxylation
N
CH2CH2 NH2
N
H
–CO2, enzymes
N –
CH2CHCO2
N
H + NH3
Neurotransmitters
Neurotransmitters
–
CO2
The chemistry of the +
brain and central H3N H
nervous system is H H
affected by
neurotransmitters.
Several important
neurotransmitters
are biosynthesized
from L-tyrosine. OH
L-Tyrosine
Neurotransmitters
Neurotransmitters
–
CO2
The common name +
of this compound is H3N H
L-DOPA. It occurs H H
naturally in the
brain. It is widely
prescribed to reduce
the symptoms of HO
Parkinsonism.
OH
L-3,4-Dihydroxyphenylalanine
Neurotransmitters
Neurotransmitters
H
Dopamine is formed
by decarboxylation H2N H
of L-DOPA. H H
HO
OH
Dopamine
Neurotransmitters
Neurotransmitters
H
H2N H
H OH
HO
OH
Norepinephrine
Neurotransmitters
Neurotransmitters
H
CH3NH H
H OH
HO
OH
Epinephrine
27.7
27.7
Peptides
Peptides

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
Alanine and
and Glycine
Glycine
H O H O
+ – + –
H3N C C O H3N C C O
CH3 H
Dr. Wolf's CHM 424 27- 100

Alanylglycine
Alanylglycine
H O H O
+ –
H3N C C N C C O
CH3 H H
Two -amino acids are joined by a peptide bond

in alanylglycine. It is a dipeptide.
Dr. Wolf's CHM 424 27- 101

Alanylglycine
Alanylglycine
H O H O
+ –
H3N C C N C C O
CH3 H H
N-terminus C-terminus
Ala—Gly
AG
Dr. Wolf's CHM 424 27- 102

Alanylglycine
Alanylglycine and
and glycylalanine
glycylalanine are
are
constitutional
constitutional isomers
isomers
H O H O
+ Alanylglycine
–
H3N C C N C C O Ala—Gly
AG
CH3 H H
H O H O
+ Glycylalanine
– Gly—Ala
H3N C C N C C O
GA
H H CH3
Dr. Wolf's CHM 424 27- 103
Alanylglycine
Alanylglycine
H O H O
+ –
H3N C C N C C O
CH3 H H
The peptide bond is

characterized by a
planar geometry.
Dr. Wolf's CHM 424 27- 104

Higher
Higher Peptides
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.
Dr. Wolf's CHM 424 27- 105

Leucine
Leucine Enkephalin
Enkephalin
Tyr—Gly—Gly—Phe—Leu
YGGFL
Dr. Wolf's CHM 424 27- 106
Oxytocin
Oxytocin
4 5
3
Ile—Gln—Asn C-terminus
2
Tyr Cys—Pro—Leu—GlyNH2
6 7 8 9
1
Cys S S
N-terminus
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.
Dr. Wolf's CHM 424 27- 107
Oxytocin
Oxytocin
S—S bond
An S—S bond between two cysteines is

often referred to as a disulfide bridge.
Dr. Wolf's CHM 424 27- 108
27.8
27.8
Introduction
Introduction to
to Peptide
Peptide
Structure
Structure Determination
Determination
Dr. Wolf's CHM 424 27- 109

Primary
Primary Structure
Structure
The primary structure is the amino acid

sequence plus any disulfide links.
Dr. Wolf's CHM 424 27- 110

Classical
Classical Strategy
Strategy (Sanger)
(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
Dr. Wolf's CHM 424 27- 111
27.9
27.9
Amino
AminoAcid
AcidAnalysis
Analysis
Dr. Wolf's CHM 424 27- 112

Amino
AminoAcid
AcidAnalysis
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.
Dr. Wolf's CHM 424 27- 113
27.10
27.10
Partial
Partial Hydrolysis
Hydrolysis of
of Proteins
Proteins
Dr. Wolf's CHM 424 27- 114

Partial
Partial Hydrolysis
Hydrolysis of
of Peptides
Peptides and
and Proteins
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.
Dr. Wolf's CHM 424 27- 115

Partial
Partial Hydrolysis
Hydrolysis of
of Peptides
Peptides and
and Proteins
Proteins
The enzymes that catalyze the hydrolysis of

peptide bonds are called peptidases, proteases,
or proteolytic enzymes.
Dr. Wolf's CHM 424 27- 116

Trypsin
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
Dr. Wolf's CHM 424 27- 117
Chymotrypsin
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

Dr. Wolf's CHM 424 27- 118
Carboxypeptidase
Carboxypeptidase
Carboxypeptidase is selective for cleaving

the peptide bond to the C-terminal amino acid.
O O O
+ –
H3NCHC protein C NHCHCO
R R
Dr. Wolf's CHM 424 27- 119

27.11
27.11
End
End Group
GroupAnalysis
Analysis
Dr. Wolf's CHM 424 27- 120

End
End Group
GroupAnalysis
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 C-
terminal 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.
Dr. Wolf's CHM 424 27- 121
Sanger's
Sanger's Method
Method
The key reagent in Sanger's method for

identifying the N-terminus is 1-fluoro-2,4-
dinitrobenzene.
1-Fluoro-2,4-dinitrobenzene is very reactive
toward nucleophilic aromatic substitution
(Section 23.5).
NO2
O2N F
Dr. Wolf's CHM 424 27- 122

Sanger's
Sanger's Method
Method
1-Fluoro-2,4-dinitrobenzene reacts with the

amino nitrogen of the N-terminal amino acid.
NO2
O O O O
–
O2N F + H2NCHC NHCHC NHCH2C NHCHCO
CH(CH3)2 CH2C6H5 CH3
NO2
O O O O
–
O2N NHCHC NHCHC NHCH2C NHCHCO

Dr. Wolf's CHM 424 27- 123
Sanger's
Sanger's Method
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
+ + +
O2N NHCHCOH + H3NCHCO– + H3NCH2CO– + H3NCHCO–
H 3O +
NO2
O O O O
–
O2N NHCHC NHCHC NHCH2C NHCHCO

Dr. Wolf's CHM 424 27- 124
27.12
27.12
Insulin
Insulin
Dr. Wolf's CHM 424 27- 125

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.
Dr. Wolf's CHM 424 27- 126

The
The BB Chain
Chain of
of Bovine
Bovine Insulin
Insulin
Phenylalanine (F) is the N terminus.
Pepsin-catalyzed hydrolysis gave the four peptides:
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Dr. Wolf's CHM 424 27- 127

The
The BB Chain
Chain of
of Bovine
Bovine Insulin
Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Dr. Wolf's CHM 424 27- 128

The
The BB Chain
Chain of
of Bovine
Bovine Insulin
Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
Overlaps between the above peptide sequences were
found in four additional peptides:
SHLV
LVGA
ALT
TLVC
Dr. Wolf's CHM 424 27- 129

The
The BB Chain
Chain of
of Bovine
Bovine Insulin
Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
YTPKA
Dr. Wolf's CHM 424 27- 130

The
The BB Chain
Chain of
of Bovine
Bovine Insulin
Insulin
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.
Dr. Wolf's CHM 424 27- 131
The
The BB Chain
Chain of
of Bovine
Bovine Insulin
Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
Dr. Wolf's CHM 424 27- 132

The
The BB Chain
Chain of
of Bovine
Bovine Insulin
Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALTLVCGERGFFYTPKA
Dr. Wolf's CHM 424 27- 133
Insulin
Insulin
The sequence of the A chain was determined

using the same strategy.
Establishing the disulfide links between cysteine
residues completed the primary structure.
Dr. Wolf's CHM 424 27- 134

Primary
Primary Structure
Structure of
of Bovine
Bovine Insulin
Insulin
N terminus
of A chain S S C terminus
5 15
of A chain
E Q C C S L Y Q L
F I V E N 20
C
S A S V10 YC
S N
S
Q H L
F V N C S
5 G S H L V G A L Y L V
15 C 20
10 G
N terminus
E
of B chain
T Y F F G R
C terminus A K P 25
of B chain 30
Dr. Wolf's CHM 424 27- 135
27.13
27.13
The
The Edman
Edman Degradation
Degradation and
and
Automated
Automated Sequencing
Sequencing of
of
Peptides
Peptides
Dr. Wolf's CHM 424 27- 136

Edman
Edman Degradation
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.
Dr. Wolf's CHM 424 27- 137

Edman
Edman Degradation
Degradation
The key reagent in the Edman degradation is

phenyl isothiocyanate.
N C S
Dr. Wolf's CHM 424 27- 138

Edman
Edman Degradation
Degradation
Phenyl isothiocyanate reacts with the amino

nitrogen of the N-terminal amino acid.
O
+
C6H5N C S + H3NCHC NH peptide
Dr. Wolf's CHM 424 R 27- 139

Edman
Edman Degradation
Degradation
S O
C6H5NHCNHCHC NH peptide
O
+
C6H5N C S + H3NCHC NH peptide
Dr. Wolf's CHM 424 R 27- 140

Edman
Edman Degradation
Degradation
S O
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.
Dr. Wolf's CHM 424 27- 141
Edman
Edman Degradation
Degradation
S O
R
HCl
S
O
C6H5NH C C +
+ H3N peptide
N CH
Dr. Wolf's CHM 424 R 27- 142

Edman
Edman Degradation
Degradation
The product is a thiazolone. Under the

conditions of its formation, the thiazolone
rearranges to a phenylthiohydantoin (PTH)
derivative.
S
O
C6H5NH C C +
+ H3N peptide
N CH
Dr. Wolf's CHM 424 R 27- 143

Edman
Edman Degradation
Degradation
C6H5
The PTH derivative is
N isolated and identified.
S O
C C The remainder of the
peptide is subjected to
HN CH a second Edman
R degradation.
S
O
C6H5NH C C +
+ H3N peptide
N CH
Dr. Wolf's CHM 424 R 27- 144

27.14
27.14
The
The Strategy
Strategy of
of Peptide
Peptide Synthesis
Synthesis
Dr. Wolf's CHM 424 27- 145

General
General Considerations
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
Dr. Wolf's CHM 424 27- 146

General
General Strategy
Strategy
1. Limit the number of possibilities by

"protecting" the nitrogen of one amino acid
and the carboxyl group of the other.
N-Protected C-Protected
phenylalanine glycine
O O
X NHCHCOH H2NCH2C Y
Dr. Wolf's CHM 424

CH2C6H5 27- 147
General
General Strategy
Strategy
2. Couple the two protected amino acids.

O O
X NHCHC NHCH2C Y
CH2C6H5
O O
X NHCHCOH H2NCH2C Y
Dr. Wolf's CHM 424

CH2C6H5 27- 148
General
General Strategy
Strategy
3. Deprotect the amino group at the N-terminus

and the carboxyl group at the C-terminus.
O O
X NHCHC NHCH2C Y
CH2C6H5
O O
+ –
H3NCHC NHCH2CO
Phe-Gly
CH2C6H5
Dr. Wolf's CHM 424 27- 149
27.15
27.15
Amino
Amino Group
Group Protection
Protection
Dr. Wolf's CHM 424 27- 150

Protect
ProtectAmino
Amino Groups
Groups as
asAmides
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.
Dr. Wolf's CHM 424 27- 151

Protect
ProtectAmino
Amino Groups
Groups as
asAmides
Amides
O O
+ –
CH2OCCl + H3NCHCO
CH2C6H5
1. NaOH, H2O
2. H+
O O
CH2OC NHCHCOH
(82-87%)
CH2C6H5
Dr. Wolf's CHM 424 27- 152
Protect
ProtectAmino
Amino Groups
Groups as
asAmides
Amides
O O
CH2OC NHCHCOH
CH2C6H5
is abbreviated as:
O
ZNHCHCOH or Z-Phe
CH2C6H5
Dr. Wolf's CHM 424 27- 153
Removing
Removing Z-Protection
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
Dr. Wolf's CHM 424 27- 154

Hydrogenolysis
Hydrogenolysis of
of Z-Protecting
Z-Protecting Group
Group
O O
CH2OC NHCHCNHCH2CO2CH2CH3
CH2C6H5
H2, Pd
O
CH3 CO2 H2NCHCNHCH2CO2CH2CH3
CH2C6H5 (100%)
Dr. Wolf's CHM 424 27- 155
HBr
HBr Cleavage
Cleavage of
of Z-Protecting
Z-Protecting Group
Group
O O
CH2OC NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
+
CH2Br CO2 H3NCHCNHCH2CO2CH2CH3
–
CH2C6H5 Br (82%)
Dr. Wolf's CHM 424 27- 156
The
The tert-Butoxycarbonyl
tert-Butoxycarbonyl Protecting
Protecting Group
Group
O O
(CH3)3COC NHCHCOH
CH2C6H5
is abbreviated as:
O
BocNHCHCOH or Boc-Phe
CH2C6H5
Dr. Wolf's CHM 424 27- 157
HBr
HBr Cleavage
Cleavage of
of Boc-Protecting
Boc-Protecting Group
Group
O O
(CH3)3COC NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
H3C O
+
C CH2 CO2 H3NCHCNHCH2CO2CH2CH3
H3C –
CH2C6H5 Br (86%)
Dr. Wolf's CHM 424 27- 158
27.16
27.16
Carboxyl
Carboxyl Group
Group Protection
Protection
Dr. Wolf's CHM 424 27- 159

Protect
Protect Carboxyl
Carboxyl Groups
Groups as
as Esters
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.
Dr. Wolf's CHM 424 27- 160

Hydrogenolysis
Hydrogenolysis of
of Benzyl
Benzyl Esters
Esters
O O O
C6H5CH2OC NHCHCNHCH2COCH2C6H5
CH2C6H5
H2, Pd
O
+ –
C6H5CH3 CO2 H3NCHCNHCH2CO CH3C6H5
CH2C6H5 (87%)
Dr. Wolf's CHM 424 27- 161
27.17
27.17
Peptide
Peptide Bond
Bond Formation
Formation
Dr. Wolf's CHM 424 27- 162

Forming
Forming Peptide
Peptide Bonds
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.
Dr. Wolf's CHM 424 27- 163

DCCI-Promoted
DCCI-Promoted Coupling
Coupling
O O
ZNHCHCOH + H2NCH2COCH2CH3
CH2C6H5
DCCI, chloroform
O O
ZNHCHC NHCH2COCH2CH3
CH2C6H5 (83%)
Dr. Wolf's CHM 424 27- 164
Mechanism
Mechanism of
of DCCI-Promoted
Coupling
O
ZNHCHCOH + C6H11N C NC6H11
CH2C6H5
H O
C6H11N C OCCHNHZ
C6H11N CH2C6H5
Dr. Wolf's CHM 424 27- 165
Mechanism
Mechanism of
of DCCI-Promoted
Coupling
The species formed by addition of the Z-
protected 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 carboxyl-
protected amino acid on the carbonyl group
leads to nucleophilic acyl substitution.
H O
C6H11N C OCCHNHZ
C6H11N CH2C6H5
Dr. Wolf's CHM 424 27- 166
Mechanism
Mechanism of
of DCCI-Promoted
Coupling
H O O
C6H11N C O + ZNHCHC NHCH2COCH2CH3
C6H11NH CH2C6H5
O
H2NCH2COCH2CH3
H O
C6H11N C OCCHNHZ
C6H11N CH2C6H5
Dr. Wolf's CHM 424 27- 167
The
TheActive
Active Ester
Ester Method
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.
Dr. Wolf's CHM 424 27- 168

The
TheActive
Active Ester
Ester Method
Method
O O
ZNHCHCO NO2 + H2NCH2COCH2CH3
CH2C6H5
chloroform
O O
ZNHCHC NHCH2COCH2CH3 + HO NO2
CH2C6H5 (78%)
Dr. Wolf's CHM 424 27- 169
27.18
27.18
Solid-Phase
Solid-Phase Peptide
Peptide Synthesis:
Synthesis:
The
The Merrifield
Merrifield Method
Method
Dr. Wolf's CHM 424 27- 170

Solid-Phase
Solid-Phase Peptide
Peptide Synthesis
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.
Dr. Wolf's CHM 424 27- 171

The
The Solid
Solid Support
Support
CH2 CH2 CH2 CH2

CH CH CH 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.
Dr. Wolf's CHM 424 27- 172
The
The Solid
Solid Support
Support
CH2 CH2 CH2 CH2

CH CH CH CH
Treating the polymeric support with

chloromethyl methyl ether (ClCH2OCH3) and
SnCl4 places ClCH2 side chains on some of the
benzene rings.
Dr. Wolf's CHM 424 27- 173
The
The Solid
Solid Support
Support
CH2 CH2 CH2 CH2

CH CH CH CH
CH2Cl
The side chain chloromethyl group is a benzylic

halide, reactive toward nucleophilic substitution
(SN2).
Dr. Wolf's CHM 424 27- 174
The
The Solid
Solid Support
Support
CH2 CH2 CH2 CH2

CH CH CH CH
CH2Cl
The chloromethylated resin is treated with the Boc-
protected C-terminal amino acid. Nucleophilic
substitution occurs, and the Boc-protected amino
acid is bound to the resin as an ester.
Dr. Wolf's CHM 424 27- 175
The
The Merrifield
Merrifield Procedure
Procedure
CH2 CH2 CH2 CH2

CH CH CH CH
O CH2Cl
–
BocNHCHCO
R
Dr. Wolf's CHM 424 27- 176

The
The Merrifield
Procedure
CH2 CH2 CH2 CH2

CH CH CH CH
O
CH2
BocNHCHCO
Next, the Boc R

protecting group is
removed with HCl.
Dr. Wolf's CHM 424 27- 177
The
The Merrifield
Procedure
CH2 CH2 CH2 CH2

CH CH CH CH
O
CH2
H2NCHCO
DCCI-promoted R
coupling adds the
second amino acid
Dr. Wolf's CHM 424 27- 178
The
The Merrifield
Procedure
CH2 CH2 CH2 CH2

CH CH CH CH
O O
CH2
BocNHCHC NHCHCO
R' R
Remove the Boc
protecting group.
Dr. Wolf's CHM 424 27- 179
The
The Merrifield
Procedure
CH2 CH2 CH2 CH2

CH CH CH CH
O O
CH2
H2NCHC NHCHCO
R' R
Add the next amino
acid and repeat.
Dr. Wolf's CHM 424 27- 180
The
The Merrifield
Procedure
CH2 CH2 CH2 CH2

CH CH CH CH
O O O
CH2
+
H3N peptide C NHCHC NHCHCO
R' R Remove the peptide
from the resin with
HBr in CF3CO2H
Dr. Wolf's CHM 424 27- 181
The
The Merrifield
Procedure
CH2 CH2 CH2 CH2

CH CH CH CH
CH2Br
O O O
+ –
H3N peptide C NHCHC NHCHCO
R' R
Dr. Wolf's CHM 424 27- 182

The
The Merrifield
Merrifield Method
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
Dr. Wolf's CHM 424 27- 183

27.19
27.19
Secondary
Secondary Structures
Structures
of
of Peptides
Peptides and
and Proteins
Proteins
Dr. Wolf's CHM 424 27- 184

Levels
Levels of
of Protein
Protein Structure
Structure
Primary structure = the amino acid sequence

plus disulfide links
Secondary structure = conformational
relationship between nearest neighbor amino
acids
 helix
pleated  sheet
Dr. Wolf's CHM 424 27- 185

Levels
Levels of
of Protein
Protein Structure
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
Dr. Wolf's CHM 424 27- 186

Pleated  Sheet
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.
Dr. Wolf's CHM 424 27- 187
Pleated  Sheet
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.
Dr. Wolf's CHM 424 27- 188
 Helix
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.
Dr. Wolf's CHM 424 27- 189

27.20
27.20
Tertiary
Tertiary Structure
Structure
of
of Peptides
Peptides and
and Proteins
Proteins
Dr. Wolf's CHM 424 27- 190

Tertiary
Tertiary Structure
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
Dr. Wolf's CHM 424 27- 191

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.
Dr. Wolf's CHM 424 27- 192

Carboxypeptidase
Carboxypeptidase
Disulfide bond
Zn2+
Arg-145
N-terminus
C-terminus
tube model ribbon model

Dr. Wolf's CHM 424 27- 193
What
What happens
happens at
at the
the active
active site?
site?
•• •
O• O H2N
+
H3N peptide C NHCHC – + C Arg-145
R O H2N
Dr. Wolf's CHM 424 27- 194

What
What happens
happens at
at the
the active
active site?
site?
•• •
O• O H2N
+
R O H2N
The peptide or protein is bound at the active site

by electrostatic attraction between its negatively
charged carboxylate ion and arginine-145.
Dr. Wolf's CHM 424 27- 195

What
What happens
happens at
at the
the active
active site?
site?
Zn2+
•• •
O• O H2N
+
R O H2N
Zn2+ acts as a Lewis acid toward the carbonyl

oxygen, increasing the positive character of the
carbonyl carbon.
Dr. Wolf's CHM 424 27- 196

What
What happens
happens at
at the
the active
active site?
site?
Zn2+
•• •
O• O H2N
+
R O H2N
H
• O•
• •
H
Water attacks the carbonyl carbon. Nucleophilic
acyl substitution occurs.
Dr. Wolf's CHM 424 27- 197
What
What happens
happens at
at the
the active
active site?
site?
Zn2+
H2N
+ C Arg-145
•• •
O• H2N
+ •• –
H3N peptide C O ••
•• O
+
H3NCHC –
R O
Dr. Wolf's CHM 424 27- 198
27.21
27.21
Coenzymes
Coenzymes
Dr. Wolf's CHM 424 27- 199

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.
Dr. Wolf's CHM 424 27- 200

Coenzymes
Coenzymes
NADH, coenzyme A and coenzyme B12 are

examples of coenzymes.
Heme is another example.
Dr. Wolf's CHM 424 27- 201

Heme
Heme
H 2C CH CH3
H 3C CH CH2
N N
Fe
N N
H 3C CH3
HO2CCH2CH2 CH2CH2CO2H
Molecule surrounding iron is a

type of porphyrin.
Dr. Wolf's CHM 424 27- 202
Myoglobin
Myoglobin
C-terminus Heme
N-terminus
Heme is the coenzyme that binds oxygen in myoglobin

(oxygen storage in muscles) and hemoglobin (oxygen
transport).
Dr. Wolf's CHM 424 27- 203
27.22
27.22
Protein
Protein Quaternary
Quaternary Structure:
Structure:
Hemoglobin
Hemoglobin
Dr. Wolf's CHM 424 27- 204

Protein
Protein Quaternary
Quaternary Structure
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.
Dr. Wolf's CHM 424 27- 205

End
End of
of Chapter
Chapter 27
27
Dr. Wolf's CHM 424 27- 206

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Chapter

Dr. Wolf's CHM 424 27- 1

Dr. Wolf's CHM 424 27- 2

While their name implies that amino acids are

Dr. Wolf's CHM 424 27- 3

+ a -amino acid that is one of

+ – a -amino acid involved in

More than 700 amino acids occur naturally, but

Dr. Wolf's CHM 424 27- 5

The amino acids obtained by hydrolysis of

The major differences among the side chains

General categories of a-amino acids

Dr. Wolf's CHM 424 27- 8

General categories of a-amino acids

Dr. Wolf's CHM 424 27- 9

Glycine is the simplest amino acid. It is the only

Dr. Wolf's CHM 424 27- 12

Dr. Wolf's CHM 424 27- 13

Dr. Wolf's CHM 424 27- 14

H2C CH2 Proline

Proline is the only amino acid that contains a

The side chain in phenylalanine (a nonpolar

General categories of a-amino acids

Dr. Wolf's CHM 424 27- 19

The —CH2OH side chain in serine can be

The side chains of two remote cysteines can be

The side chains of asparagine and glutamine

Dr. Wolf's CHM 424 27- 25

General categories of a-amino acids

Dr. Wolf's CHM 424 27- 26

Aspartic acid and glutamic acid (next slide) exist

Dr. Wolf's CHM 424 27- 28

General categories of a-amino acids

Dr. Wolf's CHM 424 27- 29

Lysine and arginine (next slide) exist as their

Dr. Wolf's CHM 424 27- 31

Dr. Wolf's CHM 424 27- 33

Glycine is achiral. All of the other amino acids

Dr. Wolf's CHM 424 27- 34

Dr. Wolf's CHM 424 27- 35

While their name implies that amino acids are

How do we know this?

Dr. Wolf's CHM 424 27- 36

more consistent with this than this

Dr. Wolf's CHM 424 27- 37

more consistent with this

Dr. Wolf's CHM 424 27- 38

The zwitterionic structure of glycine also follows

Now ask yourself "As the pH is raised, which is

The more acidic proton belongs to the CO2H

Therefore, the more stable neutral form of

The measured pKa of glycine is 2.34.

A proton attached to N in the zwitterionic form of

One way in which amino acids differ is in

Dr. Wolf's CHM 424 27- 46

Dr. Wolf's CHM 424 27- 47

Dr. Wolf's CHM 424 27- 48