Transcript Organic Chemistry Fifth Edition
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 —NH 2 group and a —CO 2 H group, these groups are actually present as —NH 3 + and —CO 2 – respectively.
They are classified as , , ,
etc
. amino acids according the carbon that bears the nitrogen.
Amino Acids
+ N H 3 C O 2 – H 3 + N CH 2 CH 2 C O 2 – H 3 + N 2 CH 2 CH 2 C O 2 – 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 3 N H C R O C O – 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 3 N H C R O C O – 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
Glycine (Gly or G)
Table 25.1
+ H 3 N H 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 3 N H C O C O – CH 3 Alanine (Ala or A) Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar and hydrophobic.
Table 25.1
+ H 3 N H C O C O – CH(CH 3 ) 2 Valine (Val or V)
Table 25.1
+ H 3 N H C O C O – CH 2 CH(CH 3 ) 2 Leucine (Leu or L)
Table 25.1
+ H 3 N H C O C O CH 3 CHCH 2 CH 3 – Isoleucine (Ile or I)
Table 25.1
+ H 3 N H C O C O – CH 3 SCH 2 CH 2 Methionine (Met or M) The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat polarizable.
Table 25.1
H O + H 2 N C C H 2 C C H 2 CH 2 O – Proline (Pro or P) Proline is the only amino acid that contains a secondary amine function. Its side chain is nonpolar and cyclic.
+ H 3 N
Table 25.1
H C O C O – CH 2 Phenylalanine (Phe or F) The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group.
Tryptophan (Trp or W)
Table 25.1
+ H 3 N H C O C O – N CH 2 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 3 N H C O C O – CH 2 OH Serine (Ser or S) The —CH 2 OH side chain in serine can be involved in hydrogen bonding.
Table 25.1
+ H 3 N H C O C CH 3 CHOH O – 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 3 N H C O C O – CH 2 SH Cysteine (Cys or C) The side chains of two remote cysteines can be joined by forming a covalent S —S bond.
Tyrosine (Tyr or Y)
Table 25.1
+ H 3 N H C O C O – CH 2 The side chain of tyrosine is similar to that of phenylalanine but can participate in hydrogen bonding.
OH
Table 25.1
+ H 3 N H C H 2 NCCH 2 O O C O – Asparagine (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
+ H 3 N H C H 2 NCCH 2 CH 2 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 + H 3 N C – OCCH 2 O C O O – Aspartic Acid (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
H + H 3 N C – OCCH 2 CH 2 O C O O – Glutamic Acid (Glu or E)
Table 25.1
General categories of -amino acids nonpolar side chains polar but nonionized side chains acidic side chains basic side chains
Lysine (Lys or K)
Table 25.1
+ H 3 N H O C C O – + CH 2 CH 2 CH 2 CH 2 NH 3 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
Arginine (Arg or R) + H 3 N H C O C O – CH 2 CH 2 CH 2 NHCNH 2 +NH 2
Table 25.1
Histidine (His or H) N + H 3 N H C CH 2 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.
CO 2 – + H 3 N H R
25.3
Acid-Base Behavior of Amino Acids
Recall
While their name implies that amino acids are compounds that contain an —NH 2 group and a —CO 2 H group, these groups are actually present as —NH 3 + and —CO 2 – 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 + H 3 NCH 2 C •• O •• •• – •• H 2 NCH 2 C •• 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 + H 3 N CH 2 C •• O •• •• – called a
zwitterion dipolar ion
or
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).
+ H 3 N CH 2 C •• 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 p
K
a s.
typical ammonium ion: p
K
a ~9 + H 3 N CH 2 C •• OH •• typical carboxylic acid: p
K
a ~5
Acid-Base Properties of Glycine
The more acidic proton belongs to the CO 2 H group. It is the first one removed as the pH is raised.
+ H 3 N CH 2 C •• OH •• typical carboxylic acid: p
K
a ~5
Acid-Base Properties of Glycine
Therefore, the more stable neutral form of glycine is the zwitterion.
+ H 3 N CH 2 C •• O •• •• – + H 3 N CH 2 C •• OH •• typical carboxylic acid: p
K
a ~5
Acid-Base Properties of Glycine
The measured p
K
a 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.
+ H 3 N CH 2 C •• OH •• typical carboxylic acid: p
K
a ~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. H 3 + N CH 2 C •• O •• •• – HO – •• H 2 N CH 2 C •• O •• •• – The p
K
a for removal of this proton is 9.60.
This value is about the same as that for NH 4 + (9.3).
Isoelectric Point
(
p
I) p
K
a = 2.34
p
K
a = 9.60
+ H 3 N CH 2 C + H 3 N CH 2 C •• H 2 N CH 2 C •• OH •• •• O •• •• – •• 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 p
K
a s.
The p
I
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 p
K
a and p
I
values for amino acids with neutral side chains.
Table 25.2
Amino Acids with Neutral Side Chains Glycine + H 3 N H C O C O – H p
K
a1 p
K
a2 p
I
= 2.34
= 9.60
= 5.97
Table 25.2
Amino Acids with Neutral Side Chains Alanine + H 3 N H C O C O – CH 3 p
K
a1 p
K
a2 p
I
= 2.34
= 9.69
= 6.00
Table 25.2
Amino Acids with Neutral Side Chains Valine + H 3 N H C O C O – CH(CH 3 ) 2 p
K
a1 p
K
a2 p
I
= 2.32
= 9.62
= 5.96
Table 25.2
Amino Acids with Neutral Side Chains Leucine + H 3 N H C O C O – CH 2 CH(CH 3 ) 2 p
K
a1 p
K
a2 p
I
= 2.36
= 9.60
= 5.98
Table 25.2
Amino Acids with Neutral Side Chains Isoleucine + H 3 N H C O C O CH 3 CHCH 2 CH 3 – p
K
a1 p
K
a2 p
I
= 2.36
= 9.60
= 6.02
Table 25.2
Amino Acids with Neutral Side Chains Methionine + H 3 N H C CH 3 SCH 2 CH 2 O C O – p
K
a1 p
K
a2 p
I
= 2.28
= 9.21
= 5.74
Table 25.2
Amino Acids with Neutral Side Chains Proline H O + H 2 N C C H 2 C C H 2 CH 2 O – p
K
a1 p
K
a2 p
I
= 1.99
= 10.60
= 6.30
Table 25.2
Amino Acids with Neutral Side Chains Phenylalanine + H 3 N H C O C O – CH 2 p
K
a1 p
K
a2 p
I
= 1.83
= 9.13
= 5.48
Table 25.2
Amino Acids with Neutral Side Chains Tryptophan + H 3 N H C O C O – CH 2 p
K
a1 p
K
a2 p
I
= 2.83
= 9.39
= 5.89
N H
Table 25.2
Amino Acids with Neutral Side Chains Asparagine + H 3 N H C H 2 NCCH 2 O O C O – p
K
a1 p
K
a2 p
I
= 2.02
= 8.80
= 5.41
Table 25.2
Amino Acids with Neutral Side Chains Glutamine + H 3 N H C H 2 NCCH 2 CH 2 O O C O – p
K
a1 p
K
a2 p
I
= 2.17
= 9.13
= 5.65
Table 25.2
Amino Acids with Neutral Side Chains Serine + H 3 N H C O C O – CH 2 OH p
K
a1 p
K
a2 p
I
= 2.21
= 9.15
= 5.68
Table 25.2
Amino Acids with Neutral Side Chains Threonine + H 3 N H C O C CH 3 CHOH O – p
K
a1 p
K
a2 p
I
= 2.09
= 9.10
= 5.60
Table 25.2
Amino Acids with Neutral Side Chains Tyrosine + H 3 N H C O C O – CH 2 p
K
a1 p
K
a2 p
I
= 2.20
= 9.11
= 5.66
OH
Table 25.3
Amino Acids with Ionizable Side Chains Aspartic acid H + H 3 N C – OCCH 2 O C O O – p
K
a1 p
K
a2 p
K
a* p
I
= 1.88
= 9.60 = 3.65 = 2.77
For amino acids with acidic side chains, p
I
average of p
K
a1 and p
K
a* .
is the
Table 25.3
Amino Acids with Ionizable Side Chains Glutamic acid H + H 3 N C – OCCH 2 CH 2 O C O O – p
K
a1 p
K
a2 p
K
a* p
I
= 2.19
= 9.67 = 4.25
= 3.22
Table 25.3
Amino Acids with Ionizable Side Chains + H 3 N H O C C O – + CH 2 CH 2 CH 2 CH 2 NH 3 Lysine p
K
a1 p
K
a2 p
K
a* p
I
= 2.18
= 8.95 = 10.53
= 9.74
For amino acids with basic side chains, pI is the average of p
K
a2 and p
K
a* .
Table 25.3
Amino Acids with Ionizable Side Chains + H 3 N H C O C O – CH 2 CH 2 CH 2 NHCNH 2 +NH 2 Arginine p
K
a1 p
K
a2 p
K
a* p
I
= 2.17
= 9.04
= 12.48
= 10.76
Table 25.3
Amino Acids with Ionizable Side Chains Histidine N + H 3 N H C O C CH 2 O – p
K
a1 p
K
a2 p
K
a* p
I
= 1.82
= 6.00
= 9.17 = 7.59
NH
25.4
Synthesis of Amino Acids
From
-Halo Carboxylic Acids
O CH 3 CHCOH + 2 N H 3 H 2 O Br O CH 3 CHCO – + N H 4 Br + N H 3 (65-70%)
Strecker Synthesis
O CH 3 CH N H 4 Cl Na CN CH 3 CH N H 2 C N 1. H 2 O, HCl, heat 2. HO – O CH 3 CH C O – + N H 3 (52-60%)
Using Diethyl Acetamidomalonate
O O C CH 3 CH 2 O CH 3 C N H C H C O OCH 2 CH 3 Can be used in the same manner as diethyl malonate (Section 20.11).
Example
O O CH 3 CH 2 OCCCOCH 2 CH 3 CH 3 C N H H O 1. NaOCH 2 CH 2. C 6 H 5 CH 2 Cl 3 O O CH 3 CH 2 OCCCOCH 2 CH 3 CH 3 C N H CH 2 C 6 H 5 O (90%)
Example
O O –CO 2 H HOCCCOH 3 N + CH 2 C 6 H 5 O HCCOH HBr, H 2 O, heat H 3 N + CH (65%) 2 C 6 H 5 O O CH 3 CH 2 OCCCOCH 2 CH 3 CH 3 C N H CH 2 C 6 H 5 O
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 H 3 + N CH 2 CO – + O O CH 3 COCCH 3 O O CH 3 C N HCH 2 COH (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 H 3 + N CHCO – + CH 3 CH 2 OH CH 3 HCl Cl – O + H 3 N CHCOCH 2 CH 3 CH 3 (90-95%)
Ninhydrin Test
O Amino acids are detected by the formation of a purple color on treatment with
ninhydrin
.
O OH + O H 3 + N CHCO – OH R O O O – RCH + CO 2 + H 2 O + N O O
25.6
Some Biochemical Reactions of Amino Acids
Biosynthesis of L -Glutamic Acid
O HO 2 CCH 2 CH 2 CCO 2 H + NH 3 enzymes and reducing coenzymes HO 2 CCH 2 CH 2 CHCO 2 – + N H 3 This reaction is the biochemical analog of reductive amination (Section 21.10).
Transamination
via
L -Glutamic Acid
O HO 2 CCH 2 CH 2 CHCO 2 – + + N H 3 CH 3 CCO 2 H 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 HO 2 CCH 2 CH 2 CHCO 2 – + + N H 3 CH enzymes 3 CCO 2 H O HO 2 CCH 2 CH 2 CCO 2 H + CH 3 CHCO 2 – + N H 3
Mechanism
HO 2 CCH 2 CH 2 CHCO 2 – + + N H 3 PLP The first step is imine formation between the amino group of L -glutamic acid and a coenzyme called pyridoxal phosphate (PLP).
Mechanism
HO 2 CCH 2 CH 2 CHCO 2 – + + NH 3 HO 2 CCH 2 CH 2 CHCO 2 –
Formation of the imine is followed by proton removal at one carbon and protonation of another carbon.
H HO 2 CCH 2 CH 2 CCO 2 –
HO 2 CCH 2 CH 2 CCO 2 – H HO 2 CCH 2 CH 2 CCO 2 –
HO 2 CCH 2 CH 2 CCO 2 – Hydrolysis of the imine function gives -ketoglutarate and pyridoxamine phosphate.
HO 2 CCH 2 CH 2 CCO 2 – HO 2 CCH 2 CH 2 CCO 2 – + O H 2 O
The pyridoxamine can do the same sequence of steps in reverse with pyruvate to generate alanine and regenerate PLP.
O CH 3 CCO 2 H +
CH 3 CHCO 2 – + N H 3 O CH 3 CCO 2 H +
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.
CH 2 CHCO 2 – + N H 3
Biosynthesis of L -Tyrosine
O CH 2 CHCO 2 – + N H 3 O 2 , enzyme CH 2 CHCO 2 – + N H 3
H O
Biosynthesis of L -Tyrosine
O CH 2 CHCO 2 – + N H 3 enzyme CH 2 CHCO 2 – + N H 3
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.
N N H CH 2 CHCO 2 – + N H 3
Decarboxylation
N CH 2 CH 2 N H 2 N H N N H –CO 2 , enzymes CH 2 CHCO 2 – + N H 3
Neurotransmitters
The chemistry of the brain and central nervous system is affected by neurotransmitters.
Several important neurotransmitters are biosynthesized from L -tyrosine.
+ H 3 N H CO 2 – 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.
H HO 3 + N H CO 2 – H H OH L -3,4-Dihydroxyphenylalanine
Neurotransmitters
Dopamine is formed by decarboxylation of L -DOPA.
H 2 N H
H
H H HO OH Dopamine
Neurotransmitters
H 2 N H
H
H OH HO OH Norepinephrine
Neurotransmitters
CH 3 N H H
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 O + H 3 N C CH 3 C O – H + H 3 N C H O C O –
Alanylglycine
H O + H 3 N C CH 3 C N H H C H O C O – Two -amino acids are joined by a peptide bond in alanylglycine. It is a
dipeptide
.
Alanylglycine
H O H + H 3 N C C N C N-terminus CH 3 H Ala —Gly H AG O C O – C-terminus
Alanylglycine and glycylalanine are constitutional isomers
H O + H 3 N C CH 3 C N H H C H O C O – Alanylglycine Ala —Gly AG H + H 3 N C H O C N H H C CH 3 O C O – Glycylalanine Gly —Ala GA
Alanylglycine
H O + H 3 N C CH 3 C N H H C 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 4 5 Ile —Gln—Asn 2 Tyr 1 Cys N-terminus S S C-terminus Cys —Pro—Leu—GlyNH 2 6 7 8 9 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 p acids.
I
among the various amino 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 N HCHC R O N HCHC R' O N HCHC 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 N HCHC R O N HCHC R' O N HCHC R" phenylalanine, tyrosine, tryptophan
Carboxypeptidase
Carboxypeptidase is selective for cleaving the peptide bond to the C-terminal amino acid.
+ R O H 3 N CHC protein O C O N HCHCO – 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 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).
NO 2 O 2 N F
Sanger's Method
O 2 N 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid.
NO 2 O O O F + H 2 N CHC N HCHC N CH(CH 3 ) 2 CH 2 C 6 H 5 HCH 2 C O N HCHCO – CH 3 O 2 N NO 2 O O O N HCHC N HCHC N HCH 2 C CH(CH 3 ) 2 CH 2 C 6 H 5 O N HCHCO – CH 3
Sanger's Method
O 2 N 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.
NO 2 O N HCHCOH + O + H 3 N CHCO – + O + H 3 N CH 2 CO – O + + H 3 N CHCO – CH(CH 3 ) 2 CH 2 C 6 H 5 CH 3 O 2 N NO 2 O H 3 O + O O N HCHC N HCHC N HCH 2 C CH(CH 3 ) 2 CH 2 C 6 H 5 O N HCHCO – CH 3
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
F F V N Q H L
5 N terminus of B chain
I V E
S 5
Q C C
S
C G
S
A
of B chain
S
S
S H L
10 C terminus
C V
10
S A
30
V L Y G A
C terminus 15 of A chain
Q L E L Y
15
L N
S 20
Y C N
S
V G
20
K P T Y F F
25
G R E
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.
C 6 H 5 N C S + O + H 3 N CHC R N H peptide
Edman Degradation
S O C 6 H 5 NHC N HCHC R N H peptide C 6 H 5 N C S + O + H 3 N CHC R N H peptide
Edman Degradation
S O C 6 H 5 NHC N HCHC N H 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 C 6 H 5 NHC N HCHC R N H HCl C 6 H 5 NH C N S C CH R O + + H 3 N peptide peptide
Edman Degradation
The product is a thiazolone. Under the conditions of its formation, the thiazolone rearranges to a phenylthiohydantoin (PTH) derivative.
C 6 H 5 NH C N S C CH R O + + H 3 N peptide
S
Edman Degradation
C HN C 6 H 5 N C CH R O The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation.
C 6 H 5 NH C N S C CH R O + + H 3 N 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 X O N HCHCOH CH 2 C 6 H 5 C-Protected glycine O H 2 N CH 2 C Y
General Strategy
2. Couple the two protected amino acids.
O O X N HCHC N HCH 2 C CH 2 C 6 H 5 Y X O N HCHCOH CH 2 C 6 H 5 O H 2 N CH 2 C Y
General Strategy
3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus.
O O X N HCHC N HCH 2 C CH 2 C 6 H 5 Y + O H 3 N CHC O N HCH 2 CO – CH 2 C 6 H 5 Phe-Gly
25.15
Amino Group Protection
Protect Amino Groups as Amides
Amino groups are normally protected by converting them to amides.
Benzyloxycarbonyl (C 6 H 5 CH 2 O —) 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 CH 2 OCCl O CH 2 OC + O + H 3 N CHCO – CH 2 C 6 H 5 1. NaOH, H 2 O 2. H + O N HCHCOH CH 2 C 6 H 5 (82-87%)
Protect Amino Groups as Amides
CH 2 O OC O N HCHCOH CH 2 C 6 H 5 is abbreviated as: O Z N HCHCOH CH 2 C 6 H 5 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 CH 2 OC O N HCHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 H 2 , Pd CH 3 CO 2 O H 2 N CHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 (100%)
HBr Cleavage of Z-Protecting Group
O CH 2 OC O N HCHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 HBr CH 2 Br CO 2 CH O + H 3 N CHC N HCH 2 CO 2 CH 2 CH 3 2 C 6 H 5 Br – (82%)
The
tert-
Butoxycarbonyl Protecting Group
(CH 3 ) 3 O COC O N HCHCOH CH 2 C 6 H 5 is abbreviated as: O Boc N HCHCOH CH 2 C 6 H 5 or Boc-Phe
HBr Cleavage of Boc-Protecting Group
O (CH 3 ) 3 C OC O N HCHC N HCH 2 CO 2 CH 2 CH 3 CH 2 C 6 H 5 HBr H 3 C H 3 C C CH 2 CO 2 CH O + H 3 N CHC N HCH 2 CO 2 CH 2 CH 3 2 C 6 H 5 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 C 6 H 5 CH 2 OC O O N HCHC N HCH 2 CO CH 2 C 6 H 5 CH 2 C 6 H 5 H 2 , Pd C 6 H 5 CH 3 CO 2 O + H 3 N CHC N HCH 2 CO – CH 2 C 6 H 5 (87%) CH 3 C 6 H 5
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 Z N HCHCOH CH 2 C 6 H 5 + O H 2 N CH 2 COCH 2 CH 3 DCCI, chloroform O O Z N HCHC N HCH 2 COCH 2 CH 3 CH 2 C 6 H 5 (83%)
Mechanism of DCCI-Promoted Coupling
O Z N HCHCOH CH 2 C 6 H 5 + C 6 H 11 N C NC 6 H 11 H C 6 H 11 N C C 6 H 11 N O OCCH N HZ CH 2 C 6 H 5
Mechanism 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 C 6 H 11 N C C 6 H 11 N OCCH N HZ CH 2 C 6 H 5
Mechanism of DCCI-Promoted Coupling
H C 6 H 11 N C C 6 H 11 NH O + O O Z N HCHC N HCH 2 COCH 2 CH 3 CH 2 C 6 H 5 O H 2 N CH 2 COCH 2 CH 3 H C 6 H 11 N C C 6 H 11 N O OCCH N HZ CH 2 C 6 H 5
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.
O Z N HCHC O CH 2 C 6 H 5
The Active Ester Method
NO 2 + O H 2 N CH 2 COCH 2 CH 3 chloroform O O Z N HCHC N HCH 2 COCH 2 CH 3 + H O CH 2 C 6 H 5 (78%) NO 2
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
CH 2 CH CH 2 CH CH 2 CH CH 2 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
CH 2 CH CH 2 CH CH 2 CH CH 2 CH Treating the polymeric support with chloromethyl methyl ether (ClCH 2 OCH 3 ) and SnCl 4 places ClCH 2 side chains on some of the benzene rings.
The Solid Support
CH 2 CH CH 2 CH CH 2 CH CH 2 CH CH 2 Cl The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (S N 2).
The Solid Support
CH 2 CH CH 2 CH CH 2 CH CH 2 CH CH 2 Cl 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
CH 2 CH CH 2 CH CH 2 CH CH 2 CH O Boc N HCHCO – CH 2 Cl R
The Merrifield Procedure
CH 2 CH CH 2 CH CH 2 CH CH 2 CH Next, the Boc protecting group is removed with HCl.
O Boc N HCHCO R CH 2
The Merrifield Procedure
CH 2 CH CH 2 CH CH 2 CH CH 2 CH DCCI-promoted coupling adds the second amino acid.
O H 2 N CHCO R CH 2
The Merrifield Procedure
CH 2 CH CH 2 CH CH 2 CH CH 2 CH O Boc N HCHC R' O CH 2 N HCHCO R Remove the Boc protecting group.
The Merrifield Procedure
CH 2 CH CH 2 CH CH 2 CH CH 2 CH O H 2 N CHC R' O CH 2 N HCHCO R Add the next amino acid and repeat.
The Merrifield Procedure
CH 2 CH CH 2 CH CH 2 CH CH 2 CH O + H 3 N peptide C O N HCHC R' O CH 2 N HCHCO R Remove the peptide from the resin with HBr in CF 3 CO 2 H.
The Merrifield Procedure
CH 2 CH CH 2 CH CH 2 CH CH 2 CH O + H 3 N peptide C O N HCHC R' O N HCHCO – CH 2 Br R
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 characterized by: sheet are both 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 Zn 2+ at its active site.
An amino acid with a positively charged side chain (Arg-145) is near the active site.
Carboxypeptidase
Arg-145 Disulfide bond Zn 2+ N-terminus Tube model C-terminus Ribbon model
What Happens at the Active Site?
+ H 3 N peptide O • C N HCHC O – R O H 2 N + C H 2 N Arg-145
What Happens at the Active Site?
+ H 3 N peptide O • C N HCHC O – R O H 2 N + C H 2 N 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?
+ H 3 N peptide O • C Zn 2+ N HCHC O – R O H 2 N + C H 2 N Arg-145 Zn 2+ acts as a Lewis acid toward the carbonyl oxygen, increasing the positive character of the carbonyl carbon.
What Happens at the Active Site?
+ H 3 N peptide O • C Zn 2+ N HCHC O – R O H H 2 N + C H 2 N Arg-145 H Water attacks the carbonyl carbon. Nucleophilic acyl substitution occurs.
What Happens at the Active Site?
+ H 3 N peptide Zn 2+ H 2 N + C O • C •• O •• – H 2 + H 3 N CHC O – N 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 B 12 examples of coenzymes.
are Heme is another example.
Heme
H 2 C CH H 3 C N N N Fe N H 3 C CH 3 CH CH 2 CH 3 HO 2 CCH 2 CH 2 CH 2 CH 2 CO 2 H 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 (also identical).
chains 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.