Chapter 25Chapter 25Amino Acids, Peptides, Amino Acids, Peptides,
and Proteinsand Proteins
25.125.1Classification of Amino AcidsClassification 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–
an -amino acid that is anintermediate in the biosynthesisof ethylene
+H3NCH2CH2CO2
–a -amino acid that is one ofthe structural units present incoenzyme A
+H3NCH2CH2CH2CO2
– a -amino acid involved inthe transmission of nerveimpulses
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
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.
C C
O
O–
R
H
H3N+
Table 25.1
C C
O
O–
R
H
H3N+
The major differences among the side chains concern:
Size and shapeElectronic characteristics
Table 25.1
General categories of -amino acids
nonpolar side chainspolar but nonionized side chainsacidic side chainsbasic side chains
Table 25.1
C C
O
O–
H
H
H3N+
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.
Glycine
(Gly or G)
C C
O
O–
CH3
H
H3N+
Table 25.1
Alanine
(Ala or A)
Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar and hydrophobic.
Table 25.1
C C
O
O–
CH(CH3)2
H
H3N+
Valine
(Val or V)
Table 25.1
C C
O
O–
CH2CH(CH3)2
H
H3N+
Leucine
(Leu or L)
Table 25.1
C C
O
O–
CH3CHCH2CH3
H
H3N+
Isoleucine
(Ile or I)
Table 25.1
C C
O
O–
CH3SCH2CH2
H
H3N+
Methionine
(Met or M)
The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat polarizable.
Table 25.1
Proline
C C
O
O–
CH2
H
H2N+
H2CCH2
(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
Phenylalanine
C C
O
O–
CH2
H
H3N+
(Phe or F)
The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group.
Table 25.1
C C
O
O–
CH2
H
H3N+
N
H
Tryptophan
(Trp or W) 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 chainspolar but nonionized side chainsacidic side chainsbasic side chains
Table 25.1
C C
O
O–
CH2OH
H
H3N+
Serine
(Ser or S)
The —CH2OH side chain in serine can be
involved in hydrogen bonding.
Table 25.1
C C
O
O–
CH3CHOH
H
H3N+
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
C C
O
O–
CH2SH
H
H3N+
Cysteine
(Cys or C)
The side chains of two remote cysteines can be joined by forming a covalent S—S bond.
Table 25.1
TyrosineC C
O
O–
CH2
H
H3N+
OH
(Tyr or Y) The side chain of tyrosine is similar to that of phenylalanine but can participate in hydrogen bonding.
Table 25.1
Asparagine
C C
O
O–
H
H3N+
H2NCCH2
O(Asn or N)
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
Glutamine
C C
O
O–
H
H3N+
H2NCCH2CH2
O(Gln or Q)
Table 25.1
General categories of -amino acids
nonpolar side chainspolar but nonionized side chainsacidic side chainsbasic side chains
Table 25.1
Aspartic Acid
C C
O
O–
H
H3N+
OCCH2
O
–
(Asp or D)
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
Glutamic Acid
C C
O
O–
H
H3N+
OCCH2CH2
O
–
(Glu or E)
Table 25.1
General categories of -amino acids
nonpolar side chainspolar but nonionized side chainsacidic side chainsbasic side chains
Table 25.1
C C
O
O–
CH2CH2CH2CH2NH3
H
H3N+
Lysine+(Lys or K)
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
C C
O
O–
CH2CH2CH2NHCNH2
H
H3N+
Arginine
+NH2
(Arg or R)
Table 25.1
Histidine C C
O
O–
H
H3N+
CH2 NHN
(His or H) 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.
25.225.2Stereochemistry of Amino Stereochemistry of Amino
AcidsAcids
Configuration of -Amino Acids
Glycine is achiral. All of the other amino acids in proteins have the L-configuration at their carbon.
H3N+
H
R
CO2–
25.325.3Acid-Base Behavior of Amino Acid-Base Behavior of Amino
AcidsAcids
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
O
OHH2NCH2C••
••
••
•• ••
–••
O
OH3NCH2C ••
••
•• ••+
more consistent with this than 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
–••
O
OH3NCH2C ••
••
•• ••+
more consistent with thiscalled 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
OHH3NCH2C+
••
••
•• ••
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.
O
OHH3NCH2C+
••
••
•• ••
typical ammonium ion: pKa ~9
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.
typical carboxylic acid: pKa ~5
O
OHH3NCH2C+
••
••
•• ••
Acid-Base Properties of Glycine
Therefore, the more stable neutral form of glycine is the zwitterion.
O
OHH3NCH2C+
••
••
•• ••
typical carboxylic acid: pKa ~5
–••
O
OH3NCH2C ••
••
•• ••+
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.
Acid-Base Properties of Glycine
typical carboxylic acid: pKa ~5
O
OHH3NCH2C+
••
••
•• ••
Acid-Base Properties of Glycine
–••
O
OH3NCH2C ••
••
•• ••+
The pKa for removal of this proton is 9.60.This value is about the same as that for NH4
+ (9.3).
HO–
–••
O
OH2NCH2C ••
••
•• ••••
A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased further.
Isoelectric Point (pI)
pKa = 2.34
pKa = 9.60
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.
O
OHH3NCH2C+
••
••
•• ••
–••
O
OH3NCH2C ••
••
•• ••+
–••
O
OH2NCH2C ••
••
•• ••••
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
C C
O
O–
H
H
H3N+
GlycinepKa1 = 2.34pKa2 = 9.60pI = 5.97
Table 25.2 Amino Acids with Neutral Side Chains
AlaninepKa1 = 2.34pKa2 = 9.69pI = 6.00
C C
O
O–
CH3
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
ValinepKa1 = 2.32pKa2 = 9.62pI = 5.96
C C
O
O–
CH(CH3)2
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
LeucinepKa1 = 2.36pKa2 = 9.60pI = 5.98
C C
O
O–
CH2CH(CH3)2
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
IsoleucinepKa1 = 2.36pKa2 = 9.60pI = 6.02
C C
O
O–
CH3CHCH2CH3
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
MethioninepKa1 = 2.28pKa2 = 9.21pI = 5.74
C C
O
O–
CH3SCH2CH2
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
ProlinepKa1 = 1.99pKa2 = 10.60pI = 6.30
C C
O
O–
CH2
H
H2N+
H2CCH2
Table 25.2 Amino Acids with Neutral Side Chains
PhenylalaninepKa1 = 1.83pKa2 = 9.13pI = 5.48
C C
O
O–
CH2
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
TryptophanpKa1 = 2.83pKa2 = 9.39pI = 5.89
C C
O
O–
CH2
H
H3N+
N
H
Table 25.2 Amino Acids with Neutral Side Chains
AsparaginepKa1 = 2.02pKa2 = 8.80pI = 5.41
C C
O
O–
H
H3N+
H2NCCH2
O
Table 25.2 Amino Acids with Neutral Side Chains
GlutaminepKa1 = 2.17pKa2 = 9.13pI = 5.65
C C
O
O–
H
H3N+
H2NCCH2CH2
O
Table 25.2 Amino Acids with Neutral Side Chains
SerinepKa1 = 2.21pKa2 = 9.15pI = 5.68
C C
O
O–
CH2OH
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
ThreoninepKa1 = 2.09pKa2 = 9.10pI = 5.60
C C
O
O–
CH3CHOH
H
H3N+
Table 25.2 Amino Acids with Neutral Side Chains
TyrosinepKa1 = 2.20pKa2 = 9.11pI = 5.66
C C
O
O–
CH2
H
H3N+
OH
Table 25.3 Amino Acids with Ionizable Side Chains
Aspartic acidpKa1 = 1.88pKa2 = 9.60 pKa* = 3.65 pI = 2.77
For amino acids with acidic side chains, pI is the average of pKa1 and pKa*.
C C
O
O–
H
H3N+
OCCH2
O
–
Table 25.3 Amino Acids with Ionizable Side Chains
Glutamic acidpKa1 = 2.19pKa2 = 9.67 pKa* = 4.25pI = 3.22
C C
O
O–
H
H3N+
OCCH2CH2
O
–
Table 25.3 Amino Acids with Ionizable Side Chains
Lysine
pKa1 = 2.18pKa2 = 8.95 pKa* = 10.53pI = 9.74
For amino acids with basic side chains, pI is the average of pKa2 and pKa*.
C C
O
O–
CH2CH2CH2CH2NH3
H
H3N+
+
Table 25.3 Amino Acids with Ionizable Side Chains
Arginine
pKa1 = 2.17pKa2 = 9.04pKa* = 12.48pI = 10.76
C C
O
O–
CH2CH2CH2NHCNH2
H
H3N+
+NH2
Table 25.3 Amino Acids with Ionizable Side Chains
Histidine
pKa1 = 1.82pKa2 = 6.00pKa* = 9.17 pI = 7.59
C C
O
O–
H
H3N+
CH2 NHN
25.425.4Synthesis of Amino AcidsSynthesis of Amino Acids
From -Halo Carboxylic Acids
CH3CHCOH
Br
O
2NH3+H2O
CH3CHCO
NH3
O
+
–
(65-70%)
+ NH4Br
Strecker Synthesis
NH4Cl
NaCNCH3CH
O
CH3CHC
NH2
N
CH3CHCO
NH3
O
+
– (52-60%)
1. H2O, HCl, heat
2. HO–
Using Diethyl Acetamidomalonate
CC
COCH2CH3
H
O O
CH3CH2O
CH3CNH
O
Can be used in the same manner as diethyl malonate (Section 20.11).
Example
1. NaOCH2CH3
2. C6H5CH2Cl
O O
CH3CH2OCCCOCH2CH3
HCH3CNH
O
O O
CH3CH2OCCCOCH2CH3
CH2C6H5CH3CNH
O
(90%)
Example
HBr, H2O, heat
O O
HOCCCOH
CH2C6H5H3N+
O
HCCOH
CH2C6H5H3N+(65%)
–CO2
O O
CH3CH2OCCCOCH2CH3
CH2C6H5CH3CNH
O
25.525.5Reactions of Amino AcidsReactions 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
H3NCH2CO–+
+ CH3COCCH3
O O
CH3CNHCH2COH
O O(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.
+ CH3CH2OH
HCl
O
H3NCHCO–+
CH3
(90-95%)
O
H3NCHCOCH2CH3
+
CH3
–Cl
Ninhydrin Test
Amino acids are detected by the formation of a purple color on treatment with ninhydrin.
OH
O
O
OH+
O
H3NCHCO–+
R
O O
O
N
O
–
O
RCH + CO2 + H2O +
25.625.6Some Biochemical ReactionsSome Biochemical Reactions
of Amino Acidsof Amino Acids
Biosynthesis of L-Glutamic Acid
This reaction is the biochemical analog of reductive amination (Section 21.10).
HO2CCH2CH2CCO2H
O
NH3+
enzymes andreducing coenzymes
HO2CCH2CH2CHCO2
–
NH3+
Transamination via L-Glutamic Acid
L-Glutamic acid acts as a source of the amine group in the biochemical conversion of -ketoacids to other amino acids. In the example to beshown, pyruvic acid is converted to L-alanine.
HO2CCH2CH2CHCO2
–
NH3+
+ CH3CCO2H
O
Transamination via L-Glutamic Acid
HO2CCH2CH2CHCO2
–
NH3+
+ CH3CCO2H
O
enzymes
HO2CCH2CH2CCO2H
O
+ CH3CHCO2
–
NH3+
Mechanism
HO2CCH2CH2CHCO2
–
NH3+
+
The first step is imine formation between theamino group of L-glutamic acid and a coenzyme called pyridoxal phosphate (PLP).
N
-O3POH2C OH
CH3
H O
PLP
Mechanism
HO2CCH2CH2CHCO2
–
NH3+
+
N
-O3POH2C OH
CH3
H O
N
-O3POH2C OH
CH3
H N
HO2CCH2CH2CHCO2––
H
Formation of the imine is followed by proton removal at one carbon and protonation of another carbon.
N
-O3POH2C OH
CH3
H N
HO2CCH2CH2CCO2–
N
-O3POH2C OH
CH3
H NHH
HO2CCH2CH2CCO2–
H
N
-O3POH2C OH
CH3
H N
HO2CCH2CH2CCO2–
Hydrolysis of the imine function gives-ketoglutarate and pyridoxamine phosphate.
N
-O3POH2C OH
CH3
H NHH
HO2CCH2CH2CCO2–
HO2CCH2CH2CCO2
O
–+
H2O
N
-O3POH2C OH
CH3
H NH2H
N
-O3POH2C OH
CH3
H NHH
HO2CCH2CH2CCO2–
+CH3CCO2H
O
The pyridoxamine can do the same sequence of steps in reverse with pyruvate to generate alanine and regenerate PLP.
N
-O3POH2C OH
CH3
H NH2H
+CH3CCO2H
O
CH3CHCO2
–
NH3+
N
-O3POH2C OH
CH3
H NH2H
N
-O3POH2C OH
CH3
H O
L-Tyrosine is biosynthesized from L-phenylalanine.A key step is epoxidation of the aromatic ring to give an arene oxide intermediate.
Biosynthesis of L-Tyrosine
CH2CHCO2
–
NH3+
Biosynthesis of L-Tyrosine
CH2CHCO2
–
NH3+
O2, enzyme
CH2CHCO2
–
NH3+
O
Biosynthesis of L-Tyrosine
CH2CHCO2
–
NH3+
O
enzyme
CH2CHCO2
–
NH3+
HO
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, 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 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.
CH2CHCO2
–
NH3+NH
N
Decarboxylation
CH2CHCO2
–
NH3+NH
N
–CO2, enzymes
CH2CH2 NH2
NH
N
Neurotransmitters
The chemistry of the brain and central nervous system is affected by neurotransmitters.
Several important neurotransmitters are biosynthesized from L-tyrosine.
OH
CO2–
HHH
H3N+
L-Tyrosine
H3N
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.
OH
CO2–
HHH
+
L-3,4-Dihydroxyphenylalanine
HO
Neurotransmitters
Dopamine is formed by decarboxylation of L-DOPA.
OH
H
HHH
H2N
HO
Dopamine
Neurotransmitters
OH
H
HHOH
H2N
HO
Norepinephrine
Neurotransmitters
OH
H
HHOH
CH3NH
HO
Epinephrine
25.725.7PeptidesPeptides
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
CH3
O
C+
H
C O–
H3N
O
C
H
H
CH3N+
O–
Alanylglycine
CH3
O
CH3N+
H
C
O
CN
H
H
C O–
H
Two -amino acids are joined by a peptide bond in alanylglycine. It is a dipeptide.
Alanylglycine
Ala—Gly
AG
N-terminus C-terminusCH3
O
CH3N+
H
C
O
CN
H
H
C O–
H
Alanylglycine and glycylalanine are constitutional isomers
H
O
CH3N+
H
C
O
CN
H
CH3
C O–
H
AlanylglycineAla—Gly
AG
GlycylalanineGly—Ala
GA
CH3
O
CH3N+
H
C
O
CN
H
H
C O–
H
Alanylglycine
The peptide bond is characterized by a planar geometry.
CH3
O
CH3N+
H
C
O
CN
H
H
C O–
H
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—LeuYGGFL
Oxytocin
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.
N-terminus
C-terminusIle—Gln—Asn
Tyr
Cys S S
Cys—Pro—Leu—GlyNH2
1
2
34 5
6 7 8 9
Oxytocin
S—S bond
An S—S bond between two cysteines isoften referred to as a disulfide bridge.
25.825.8Introduction to Peptide Introduction to Peptide Structure DeterminationStructure 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.
25.925.9Amino Acid AnalysisAmino 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.
25.1025.10Partial Hydrolysis of PeptidesPartial 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.
NHCHC
O
R'
NHCHC
O
R"
NHCHC
O
R
lysine or arginine
Chymotrypsin
Chymotrypsin is selective for cleaving the peptidebond to the carboxyl group of amino acids withan aromatic side chain.
NHCHC
O
R'
NHCHC
O
R"
NHCHC
O
R
phenylalanine, tyrosine, tryptophan
Carboxypeptidase
proteinH3NCHC
O
R
+NHCHCO
O
R
–C
O
Carboxypeptidase is selective for cleavingthe peptide bond to the C-terminal amino acid.
25.1125.11End Group AnalysisEnd 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 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.
Sanger's 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 (Chapter 12).
FO2N
NO2
Sanger's Method
1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid.
FO2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
H2NCHC
O OOO
CH(CH3)2
–+
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–
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.
O2N
NO2
NHCH2C NHCHCO
CH3
NHCHC
CH2C6H5
NHCHC
O OOO
CH(CH3)2
–
H3O+
O
O2N
NO2
NHCHCOH
CH(CH3)2
H3NCHCO–
CH3
+H3NCH2CO–
O O
+
O
H3NCHCO–
CH2C6H5
++ +
+
25.1225.12InsulinInsulin
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:
SHLVLVGAALTTLVC
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLVLVGA
VGAL
ALY
YLVCVCGERGF
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:
SHLVLVGAALTTLVC
Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence.
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLVLVGA
VGAL
ALY
YLVCVCGERGF
GFFYTPK
YTPKA
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLVLVGA
VGAL
ALY
YLVCVCGERGF
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
N terminus of B chain
C terminus of B chain
C terminus of A chain
C
S
S5
5
15
10
15
20
20
2530
S
10
S SSF
F F
F
V N Q H L
C C
C
C
VV
VVG
G
G
S
S
S
H L L
L
G A
A
AC
L Y
Y
E
E L E
R
Y
YI Q
K P T
QN
N
C
25.1325.13The Edman Degradation and The Edman Degradation and
Automated Sequencing of Automated Sequencing of PeptidesPeptides
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.
peptideH3NCHC
O
R
+NHC6H5N C S +
Edman Degradation
peptideC6H5NHCNHCHC
O
R
NH
S
peptideH3NCHC
O
R
+NHC6H5N C S +
Edman Degradation
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.
peptideC6H5NHCNHCHC
O
R
NH
S
Edman Degradation
HCl
peptideH3N+
+C6H5NH C
SC
N CH
R
O
peptideC6H5NHCNHCHC
O
R
NH
S
Edman Degradation
The product is a thiazolone. Under theconditions of its formation, the thiazolonerearranges to a phenylthiohydantoin (PTH)derivative.
peptideH3N+
+C6H5NH C
SC
N CH
R
O
Edman Degradation
CCN
HN CH
R
OS
C6H5The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation.
peptideH3N+
+C6H5NH C
SC
N CH
R
O
25.1425.14The Strategy of Peptide SynthesisThe 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-Protectedphenylalanine
C-Protectedglycine
NHCHCOH
CH2C6H5
O
X H2NCH2C
O
Y
General Strategy
2. Couple the two protected amino acids.
NHCH2C
O
YNHCHC
CH2C6H5
O
X
NHCHCOH
CH2C6H5
O
X H2NCH2C
O
Y
General Strategy
3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus.
NHCH2CO
O
H3NCHC
CH2C6H5
O+ –
Phe-Gly
NHCH2C
O
YNHCHC
CH2C6H5
O
X
25.1525.15Amino Group ProtectionAmino Group Protection
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
Protect Amino Groups as Amides CH2OCCl
O
+ H3NCHCO
CH2C6H5
O–+
1. NaOH, H2O
2. H+
NHCHCOH
CH2C6H5
O CH2OC
O
(82-87%)
Protect Amino Groups as Amides
NHCHCOH
CH2C6H5
O CH2OC
O
is abbreviated as:
ZNHCHCOH
CH2C6H5
O
or Z-Phe
An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by:
a) hydrogenolysis
b) cleavage with HBr in acetic acid
Removing Z-Protection
Hydrogenolysis of Z-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5
O CH2OC
O
H2, Pd
H2NCHCNHCH2CO2CH2CH3
CH2C6H5
O CH3 CO2
(100%)
HBr Cleavage of Z-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5
O CH2OC
O
HBr
H3NCHCNHCH2CO2CH2CH3
CH2C6H5
O CH2Br CO2
(82%)
+
Br–
The tert-Butoxycarbonyl Protecting Group
NHCHCOH
CH2C6H5
O
(CH3)3COC
O
is abbreviated as:
BocNHCHCOH
CH2C6H5
O
or Boc-Phe
HBr Cleavage of Boc-Protecting Group
NHCHCNHCH2CO2CH2CH3
CH2C6H5
O
(CH3)3COC
O
HBr
H3NCHCNHCH2CO2CH2CH3
CH2C6H5
O
CO2
(86%)
+
Br–CH2C
H3C
H3C
25.1625.16Carboxyl Group ProtectionCarboxyl Group Protection
Carboxyl groups are normally protected as esters.
Deprotection of methyl and ethyl esters isby hydrolysis in base.
Benzyl esters can be cleaved byhydrogenolysis.
Protect Carboxyl Groups as Esters
Hydrogenolysis of Benzyl Esters
NHCHCNHCH2COCH2C6H5
CH2C6H5
O
C6H5CH2OC
O O
H2, Pd
H3NCHCNHCH2CO
CH2C6H5
O
C6H5CH3 CO2
(87%)
+ –CH3C6H5
25.1725.17Peptide Bond FormationPeptide Bond Formation
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.
Forming Peptide Bonds
DCCI-Promoted Coupling
ZNHCHCOH
CH2C6H5
O
+ H2NCH2COCH2CH3
O
DCCI, chloroform
ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
O
(83%)
Mechanism of DCCI-Promoted Coupling
ZNHCHCOH
CH2C6H5
O
+ C6H11N C NC6H11
CH2C6H5
O
C6H11N C
C6H11N
H
OCCHNHZ
Mechanism of DCCI-Promoted Coupling
CH2C6H5
O
C6H11N C
C6H11N
H
OCCHNHZ
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.
Mechanism of DCCI-Promoted Coupling
H2NCH2COCH2CH3
O
C6H11N C
C6H11NH
H
O + ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
O
CH2C6H5
O
C6H11N C
C6H11N
H
OCCHNHZ
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
The Active Ester Method
ZNHCHCO
CH2C6H5
O
+ H2NCH2COCH2CH3
O NO2
chloroform
ZNHCHC
CH2C6H5
O
NHCH2COCH2CH3
O
(78%)
+ HO
NO2
25.1825.18Solid-Phase Peptide Synthesis:Solid-Phase Peptide Synthesis:
The Merrifield MethodThe 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
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.
CH2 CH2 CH2 CH2CH CH CH CH
The Solid Support
Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings.
CH2 CH2 CH2 CH2CH CH CH CH
The Solid Support
The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2).
CH2 CH2 CH2 CH2CH CH CH CH
CH2Cl
The Solid Support
CH2 CH2 CH2 CH2CH 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.
The Merrifield Procedure
CH2 CH2 CH2 CH2CH CH CH CH
CH2Cl
BocNHCHCO
R
O–
The Merrifield Procedure
BocNHCHCO
R
O
CH2 CH2 CH2 CH2CH CH CH CH
CH2
Next, the Boc protecting group is removed with HCl.
The Merrifield Procedure
H2NCHCO
R
CH2 CH2 CH2 CH2CH CH CH CH
CH2
O
DCCI-promoted coupling adds the second amino acid.
The Merrifield Procedure
NHCHCO
R
OCH2
CH2 CH2 CH2 CH2CH CH CH CH BocNHCHC
R'
O
Remove the Boc protecting group.
The Merrifield Procedure
CH2
CH2 CH2 CH2 CH2CH CH CH CH
NHCHCO
R
O
H2NCHC
R'
O
Add the next amino acid and repeat.
The Merrifield Procedure
Remove the peptide from the resin with HBr in CF3CO2H.
CH2 CH2 CH2 CH2CH CH CH CH
CH2
NHCHCO
R
O
NHCHC
R'
O
C
O+
H3N peptide
The Merrifield Procedure CH2 CH2 CH2 CH2CH CH CH CH
CH2Br
NHCHCO
R
O
NHCHC
R'
O
C
O+
H3N peptide–
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
25.1925.19Secondary StructuresSecondary Structures
of Peptides and Proteinsof 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.
helixpleated sheet
Levels of Protein Structure
Planar geometry of peptide bondAnti conformation of main chainHydrogen bonds between N—H and O=C
The -helix and pleated sheet are both characterized by:
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.
25.2025.20Tertiary StructureTertiary Structure
of Polypeptides and Proteinsof 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
N-terminus
C-terminus
Zn2+
Arg-145
Tube model Ribbon model
What Happens at the Active Site?
H3N peptide
O
NHCHC+
C
•• ••
R
O
O
–
H2N
H2N
C Arg-145+
What Happens at the Active Site?
H3N peptide
O
NHCHC+
C
•• ••
R
O
O
–
H2N
H2N
C Arg-145+
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?
H3N peptide
O
NHCHC+
C
•• ••
R
O
O
–
H2N
H2N
C Arg-145+
ZnZn2+2+
Zn2+ acts as a Lewis acid toward the carbonyl oxygen, increasing the positive character of the carbonyl carbon.
What Happens at the Active Site?
H3N peptide
O
NHCHC+
C
•• ••
R
O
O
–
H2N
H2N
C Arg-145+
ZnZn2+2+
Water attacks the carbonyl carbon. Nucleophilic acyl substitution occurs.
O•• ••
H
H
What Happens at the Active Site?
ZnZn2+2+
H2N
C Arg-145+
H3N peptide
O+
C
•• ••
O ••••
••
–
H3NCHC
R
O
O
–
H2N
+
25.2125.21CoenzymesCoenzymes
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 N
N N
N
Fe
H3C
H3C CH3
CH3
CH2CH2CO2H
CH CH2
H2C CH
HO2CCH2CH2
Molecule surrounding iron is a type of porphyrin.
Myoglobin
N-terminusN-terminus
C-terminusC-terminus Heme
Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen transport).
25.2225.22Protein Quaternary Structure:Protein Quaternary Structure:
HemoglobinHemoglobin
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.
25.2325.23G-Coupled Protein ReceptorsG-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.