Chapter 27: Amino Acids, Peptides, and Proteins.

monomer unit: -amino acids

H NH2
R = side chain
R CO2H

α- Am ino Acid

Biopolymer: the monomeric amino acids are linked through an

amide bond (the carboxylic acids of one AA with the -amino
group of a second)
R1 R1
+ R2 - H2O H
N CO2
H3N CO2 H3N
H3N CO2 C-terminus
O R2
N-terminus

O R2 O R4 O R6 O
H H H H
N N N N
N N N N
H H H H
R1 O R3 O R5 O R7

Peptide or protein (polypeptide)

peptide (< 50 amino acids)

protein (> 50 amino acids) 1
27.1: Classification of Amino Acids. AA’s are classified
according to the location of the amino group.
H H H H H H
H2N C CO2H H2N C C CO2H H2N C C C CO2H
H H H H H H

α-amino acid β-amino acid γ-amino acid

(2-amino carboxylic acid) (3-amino carboxylic acid) (4-amino carboxylic acid)

There are 20 genetically encoded-amino acids found in peptides

and proteins
19 are primary amines, 1 (proline) is a secondary amine
19 are “chiral”, 1 (glycine) is achiral; the natural configuration of
the -carbon is L.
CHO CHO CO2H CO2H
H OH HO H H2N H H2N H
CH2OH CH2OH CH3 R
D-glyceraldehyde L-glyceraldehyde L-alanine

CHO CHO CO2H CO2H

HO H H OH H2N H H2N H
H OH HO H H OH H3C H
CH2OH CH2OH CH3 CH2CH3
D-erythrose L-erythrose L-theronine
2
L-isoleucine
(2S,3R) (2S,3S)
-Amino acids are classified by the properties of their sidechains.
Nonpolar: COO COO
–
COO – –

NH3 NH3 NH3

Glycine (Gly, G) (S)-(+)-Alanine (Ala, A) (S)-(+)-Valine (Val, V)

COO– S COO–
COO–
NH3 NH3
NH3

(S)-(–)-Leucine (Leu, L) (2S,3S)-(+)-Isoleucine (Ile, I) (S)-(–)-Methionine (Met, M)

COO– COO–
N COO–
H NH3 NH3
H N
H
(S)-(–)-Proline (Pro, P) (S)-(–)-Phenylalanine (Phe, F) (S)-(–)-Tryptophan (Trp, W)

Polar but non-ionizable: OH COO–

COO–
HO COO–
NH3
NH3 HO
NH3

(S)-(–)-Serine (Ser, S) (2S,3R)-(–)-Threonine (Thr, T) (S)-(–)-Tyrosine (Tyr, Y)

pKa ~ 13 pKa ~ 13 pKa ~ 10.1

COO– H2N COO– O

HS
COO–
NH3 O NH3 H2N
NH3
(R)-(–)-Cysteine (Cys, C) (S)-(–)-Asparagine (Asn, N) 3
(S)-(+)-Glutamine (Gln, Q)
pKa ~ 8.2
 Acidic: -O COO–
O
COO–
-O
O NH3 NH3

(S)-(+)-Aspartic Acid (Asp, D) (S)-(+)-Glutamic Acid (Glu, E)

pKa ~ 3.6 pKa ~ 4.2

Basic: N H H
H3N COO– H COO– N
COO–
NH3 NH3 H2N N
N H NH3
H
(S)-(+)-Lysine (Lys, K) (S)-(–)-Histidine (His, H) (S)-(+)-Arginine (Arg, R)
pKa ~ 10.5 pKa ~ 6.0 pKa ~ 12.5

27.2: Stereochemistry of Amino Acids: The natural

configuration of the -carbon is L. D-Amino acids are found in
the cell walls of bacteria. The D-amino acids are not genetically
encoded, but derived from the epimerization of L-isomers

4
27.3: Acid-Base Behavior of Amino Acids. Amino acids exist
as a zwitterion: a dipolar ion having both a formal positive and
formal negative charge (overall charge neutral).
R + R _
H2N CO2H H3N CO2
H H
pKa ~ 5 pKa ~ 9

Amino acids are amphoteric: they can react as either an acid or a

base. Ammonium ion acts as an acid, the carboxylate as a base.

Isoelectric point (pI): The pH at which the amino acid exists

largely in a neutral, zwitterionic form (influenced by the nature
of the sidechain)
_ R
+ R H3O+ HO _
H3N CO2H + R _ H2N CO2
H3N CO2
H H
pKa1 H pKa2
low pH high pH

5
Table 27.2 (p. 1115) & 27.2 (p. 1116)
 pKax + pKay
pI =
2
+ CH3 CH3
+ CH3
H3N CO2H H3N CO2 H2N CO2
H pKa1 H pKa2 H
low pH (2.3) (9.7)
high pH

CO2H CO2H CO2 CO2

CH2 CH2 CH2 CH2
H3N CO2H pKa1 H3N CO2 H3N CO2 H2N CO2
pKa3 pKa2
H (1.9) H (3.6) H H
(9.6)
low pH high pH

NH3 NH3 NH3 NH2

(CH2)4 (CH2)4 (CH2)4 (CH2)4
H3N CO2H H3N CO2 H2N CO2 pKa3 H2N CO2
pKa1 pKa2
H (2.2) H H (10.5) H
(9.0)
low pH high pH

6
Electrophoresis: separation of polar compounds based on their
mobility through a solid support. The separation is based on
charge (pI) or molecular mass.

+ _

+ _
_ _ _ _ + + + +

7
27.5: Synthesis of Amino Acids:
Br NH2
Br2, PBr3 NH3
R-CH2-CO2H R C CO2H R C CO2H
Ch. 19.16 H H

Strecker Synthesis: recall reductive amination

NaB(CN)H3 NH2
O NH3 NH2
R C CO2H
R C CO2H R C CO2H
H
H

NH2 NH2
O NH3 NH2 NaC≡N H3 O +
R C C≡N R C CO 2 H
R C H R C H -or-
H NaOH, H2 O H
N≡C:

Amidomalonate Synthesis: recall the malonic acid synthesis

O O
H3O H2N H
HN CO2Et EtO Na HN CO2Et
C
C C - CO2 RCH2 CO2H
H CO2Et RCH2X RCH2 CO2Et

8
27.5: Reactions of Amino Acids. Amino acids will undergo
reactions characteristic of the amino (amide formation) and
carboxylic acid (ester formation) groups.
H3C
O O O
H2N H H3N H HN H
HOCH2CH3 H3C O CH3

R CO2CH2CH3 H+ R CO2 base R CO2H

27.6: Some Biochemical Reactions of Amino Acids. Many

enzymes involved in amino acid biosynthesis, metabolism and
catabolism are pyridoxal phosphate (vitamin B6) dependent
(please read) R CO -
2
O H racemase,
H CO2-
R CO2- N epimerase R H
OH
+ 2-
O3PO
NH3 OH NH3
PO
N
N D-amino acid
L-amino acid pyridoxal H decarboxylase
phosphate (PLP)
R H
transaminase H
H3N
R CO2-

O
9
27.7: Peptides. Proteins and peptides are polymers made up of
amino acid units (residues) that are linked together through the
formation of amide bonds (peptide bonds) from the amino
group of one residue and the carboxylate of a second residue
HO
+ - H2O H
N CO2H
H2N CO2H H2N
H2N CO2H C-terminus
O
Serine N-terminus OH
Alanine
Ala - Ser
- H2O (A - S)

HO
H
N CO2H
C-terminus By convention, peptide sequences
H2N
N-terminus
O
are written left to right from the
Ser - Ala N-terminus to the C-terminus
(S - A)

O R2 O R4 O R6 O
H H H H
N N N N
N
H
N
H
N
H
N
H
backbone
R1 O R3 O R5 O R7

10
he amide (peptide) bond has C=N double bond character due
resonance resulting in a planar geometry
O R2 _
H H O R2
N N H
N +
H
N
restricts rotations
N N
R1 H O
resistant to hydrolysis
R1 H O
amide bond

The N-H bond of one amide linkage can form a hydrogen bond
with the C=O of another.
O
H N
R
N-O distance 2.85 - 3.20 Å
N H O N H
O N H O
R R
optimal N-H-O angle is 180 °
H N H N
O O

sulfide bonds: the thiol groups of cysteine can be oxidized to

m disulfides (Cys-S-S-Cys)
1/2 O2 H2O NH2
NH2 S CO2H
2 HO2C S
SH
HO2C NH2
H2
11
 R6 O R8
H H
N N
N N
H H
O O
HS
O R10 R9 O R11 O R13
H H H H
N N N N
N N N N
H H H H
R9 O O O R12 O
S
1/2 O2

SH
R1 O O
H H
N N
N N
H H
O R2 O R4
S
R5 H2 R1 O O R5
H H H H
N N N N
N N N N
H H H H
O O R2 O R4 O

Epidermal Growth Factor (EGF): the miracle of mother’s spit

53 amino acid, 3 disulfide linkages

QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

1986 Nobel Prize in Medicine:

Stanley Cohen 12
Rita Levi-Montalcini
27.8: Introduction to Peptide Structure Determination.
Protein Structure:
primary (1°) structure: the amino acid sequence
econdary (2°): frequently occurring substructures or folds
ertiary (3°): three-dimensional arrangement of all atoms in a
single polypeptide chain
quaternary (4°): overall organization of non-covalently linked
subunits of a functional protein.

1. Determine the amino acids present and their relative ratios

2. Cleave the peptide or protein into smaller peptide fragments
and determine their sequences
3. Cleave the peptide or protein by another method and
determine their sequences. Align the sequences of the
peptide fragments from the two methods

13
 E-A-Y-L-V-C-G-E-R F-V-N-Q-H-L-F
F-V-N-Q-H-L-F-S-H-L-K S-H-L-K-E-A-Y
G-C-F-L-P-K L-V-C-G-E-R-G-C-F
L-G-A L-P-K-L-G-A

F-V-N-Q-H-L-F
F-V-N-Q-H-L-F-S-H-L-K
S-H-L-K-E-A-Y
E-A-Y-L-V-C-G-E-R
L-V-C-G-E-R-G-C-F
G-C-F-L-P-K
L-P-K-L-G-A
L-G-A

F-V-N-Q-H-L-F-S-H-L-K-E-A-Y-L-V-C-G-E-R-G-C-F-L-P-K-L-G-A

14
27.9: Amino Acid Analysis. automated method to determine the
amino acid content of a peptide or protein
Reaction of primary amines with ninhydrin
O O O
NH3 + RCHO + CO2
+ O N
R CO2
O O O
Ninhydrin

Enzymatic
peptide [H] reduce any digestion
-or- disulfide
NH3 individual
-or- amino acids
protein bonds H3O+,  R CO2

liquid derivatize w/ Detected w/

chromatography ninhydrin UV-vis

Different amino
acids have different 1972 Nobel Prize in Chemistry
chromatographic William Stein
mobilities (retention Stanford Moore
times)
15
 ial Hydrolysis of Peptides. Acidic hydrolysis of
eave the amide bonds indiscriminately.
peptidases): Enzymes that catalyzed the hydrolysis
e bonds of peptides and proteins.
cleavage of peptides and proteins at defined sites:
eaves at the C-terminal side of basic residues,
Lys but not His
O R1 O R3 O R1 O R3
H H H H
N N CO2 trypsin N N CO2
H3N N N H3N N O + H3N
H H H
O O O O
H2O

NH3 NH3

rypsin: cleaves at the C-terminal side of aromatic

, Tyr, Trp
O R1 O R3 O R1 O R3
H H H H
N N CO2 chymotrypsin N N CO2
H3N N N H3N N O + H3N
H H H
O O O O
H2O

16
Trypsin and chymotrypsin are endopeptidases
Carboxypeptidase: Cleaves the amide bond of the C-terminal
amino acid (exopeptidase)
27.11: End Group Analysis. The C-terminal AA is identified
by treating with peptide with carboxypeptidase, then analyzing
by liquid chormatography (AA Analysis).
N-labeling: The peptide is first treated with 1-fluoro-2,4-dinitro
benzene (Sanger’s reagent), which selectively reacts with the
N-terminal amino group. The peptide is then hydrolyzed to their
amino acids and the N-terminal amino acid identified as its
N-(2,4-dinitrophenyl) derivative (DNP).
NO2 O2N
R1
H
Δ R1
F H
+ H3N
N CO2
nucle ophilic
N CO 2
N
O arom atic H
O2N NO 2 O
substitution
e nzym atic O2N
dig e stion R1 plus othe r unlabe le d
-or- NH2 + am ino acids
N
H3 O +, Δ NO 2
H
O
17
27.12: Insulin. (please read) Insulin has two peptide chains
(the A chain has 21 amino acids and the B chain has 30 amino
acids) held together by two disulfide linkages
Pepsin: cleaves at the C-terminal side of Phe, Tyr, Leu; but
not at Val or Ala

Pepsin cleavage
Trypsin cleavage
H3O+ cleavage

18
an Degradation and Automated Peptide
hemical method for the sequential cleavage and
on of the amino acids of a peptide, one at a time s
us. Reagent: Ph-N=C=S, phenylisothiocyanate (PITC

S R1 pH 9.0 S R1
H H+
H Ph N CO2
C + N CO2
H2N N N
N H H
Ph O O
H+
H+
Ph N
S H N S O
HN
N CO2 Ph + H2N CO2
HN
OH
R1 -1 peptide with a new
R1
N-terminal amino acid
H+ (repeat degradation cycle)

Ph
N-phenylthiohydantoin: N O
S
separated by liquid chromatography
(based of the R group) and detectedHN
by UV-vis R1

19
Peptide sequencing by Edman degradation:
• Cycle the pH to control the cleavage of the N-terminal amino
acid by PITC.
• Monitor the appearance of the new N-phenylthiohydantoin for
each cycle.
• Good for peptides up to ~ 25 amino acids long.
• Longer peptides and proteins must be cut into smaller fragments
before Edman sequencing.

Tandem mass spectrometry has largely replaced Edman

degradation for peptide sequencing

27.14: The Strategy for Peptide Synthesis:

Chemical synthesis of peptide:
1. Solution phase synthesis
2. Solid-phase synthesis
20
 - H2O H
H
N CO2H
- H2O + H2N
N CO2H
H2N H2N CO2H H2N CO2H
O
O
Ala Val
Val - Ala Ala - Val
(V - A) (A - V)

The need for protecting groups

peptide O
H selectively

Pn OH
+ OPc
coupling Pn
N
N
OPc remove Pn
N H2N H
O
H O - H2O
O

Ala Val Ala - Val

(A - V)

peptide
O coupling O O
H H H
N (-H2O) N N Repeat peptide
H2N OPc Pn N OPc synthesis
H
O Ph O
Ph
Pn OH
Ala - Val N
(A - V) H Phe - Ala - Val
O (F - A - V)
Phe (F)

Orthogonal protecting group strategy: the carboxylate protecting

group must be stable to the reaction conditions for the removal
of the -amino protecting group and ( vice versa) 21
 27.15: Amino Group Protection. The -amino group is
protected as a carbamate.
O
NH3
O Base
O + RO NH
RO Cl OH
O
O
O O O

O NH C6H5 O NH O NH

R R
O O O
tert-butoxycarbonyl benzyloxycarbonyl fluorenylmethylcarbonyl
(t-BOC) (cBz) (FMOC)
removed with removed with mild acid removed with mild base
mild acid or by hydrogenolysis (piperidine)

27.16: Carboxyl Group Protection. Protected as a benzyl

ester; removed by hydrogenolysis
O peptide
O O mild acid
coupling H
C6H5 O N
OH + H2N
O C6H5
C6H5 O N
N
O C6H5
H - H2O H
O O O
Ph
O
O
H OH
N C6H5 O N O O O O
H2N O C6H5 H H H2, Pd/C H
H C6H5 O N N H3N N
O N O
O N O C6H5
peptide H H
- H2O O O O
coupling Ph Ph

22
27.17: Peptide Bond Formation. Amide formation from the
reaction of an amine with a carboxylic acid is slow. Amide bond
formation (peptide coupling) can be accelerated if the carboxylic
acid is activated. Reagent: dicyclohexylcarbodiimide (DCC)
O O C6H11 C6H11
O O
H NH NH
R O R O R O C R O C
+ +N
N R' H N
C6H11 N C N C6H11 + H C6H11
C6H11 N C N C6H11 C6H11
(DCC) H ••
R'-NH2 "activated acid"
C6H11 O O
O
NH R' + C6H11 C6H11
R O C R N N N
NH H H H
R' HN
+ C6H11 Amide DCU

O O
H H
DCC cBz N CF3CO2H N
cBz OH N OBn H2N OBn
N + H2N
OBn
peptide H
O selectively O
H coupling
O O remove N-
protecting
Ala Val group

DCC O O O O
H H H2, Pd/C H
Ph N N H2N N
cBz N OBn N OH
H H
cBz OH O O
N Ph Ph
H
O Phe - Ala - Val 23
Phe (F) (F - A - V)
• In order to practically synthesize peptides and proteins, time
consuming purifications steps must be avoided until the very
end of the synthesis.
• Large excesses of reagents are used to drive reactions forward
and accelerate the rate of reactions.
• How are the excess reagents and by-products from the reaction,
which will interfere with subsequent coupling steps, removed
without a purification step?

27.18: Solid-Phase Peptide Synthesis: The Merrifield Method.

Peptides and proteins up to ~ 100 residues long are synthesized
on a solid, insoluble, polymer support. Purification is conveniently
accomplished after each step by a simple wash and filtration.

24
The solid support (Merrifield resin): polystyrene polymer
Ph

styrene H3COCH2Cl
initiator Ph Ph Ph Ph Ph ZnCl2
+
CH2Cl
polymerization Ph
Ph Ph Ph Ph
Ph

divinylbenzene Ph
(crosslinker, ~1 %) Ph

O
H
_ N
O BOC
O CF3CO2H
R O
O
O
NH
NH2
R BOC
R

Solid-phase peptide synthesis

FMOC OH purify: purify:

N O O wash & filter
O H H wash & filter N
H2N O N O H H2N O
DCC FMOC N N
O H remove N- H
O protecting O
Val peptide
coupling group

DCC Ph
O
purify: purify by liquid Ph
O
H wash & filter N HF chromatograrphy H
Ph FMOC N O H N OH
N N H2N N
H H remove N- remove N- H
FMOC OH O O protecting protecting or electrophoresis O O
N group group and cleave
H from solid-support
O
Phe (F)
25
lease A- 124 amino acids, catalyzes the hydrolysis
hase synthesis of RNase A:
Synthetic RNase A: 78 % activity
0.4 mg was synthesized
2.9 % overall yield
average yield ~ 97% per coupling step
His-119 A His-12 A

LYS GLU THR ALA ALA ALA LYS PHE GLU ARG
GLN HIS MET ASP SER SER THR SER ALA ALA
SER SER SER ASN TYR CYS ASN GLN MET MET
LYS SER ARG ASN LEU THR LYS ASP ARG CYS
LYS PRO VAL ASN THR PHE VAL HIS GLU SER
LEU ALA ASP VAL GLN ALA VAL CYS SER GLN His-12 B
LYS ASN VAL ALA CYS LYS ASN GLY GLN THR His-119 B
ASN CYS TYR GLN SER TYR SER THR MET SER
ILE THR ASP CYS ARG GLU THR GLY SER SER
LYS TYR PRO ASN CYS ALA TYR LYS THR THR
GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS
GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE pdb code: 1AFL
ASP ALA SER VAL

uce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry:

his development of methodology for chemical synthesis on a solid matrix.” 26
27.19: Secondary Structures of Peptides and Proteins.
-sheet: Two or more extended peptide chain, in which the amide
backbones are associated by hydrogen bonded
N O C
anti-parallel loop R H O R H R H O R

or N N N O
N N N N
turn
NC H O R H O R H O R H O

O H R O H O H R O H

N N N N
O N N N
CN R O H R O H R O H R

CN

parallel NC
O R H O R H O R H O

NC H
N N N N
N N N O

R H O R H O R H O R
crossover
O R H O R H O R H O
H
N N N N
N N N O

O R
NC
R H O R H O R H

27
NC
-helix: 3.6 amino acids per coil, 5.4 Å
C 3.6 AA
QuickTime™ and a
5.4 Å
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
QuickTime™ and a TIFF (Uncompressed) decompressor
QuickTime™ and a
are needed to see this picture.
TIFF (Uncompressed) decompressor
TIFF (Uncompressed) decompressor are needed to see this picture.
are needed to see this picture.
28
 myoglobin
pdb code: 1WLA

Bacteriorhodopsin
pdb code: 1AP9

Anti-parallel
-sheets
of lectin
Parallel -sheets pdb code: 2LAL
carbonic anhydrase
pdb code: 1QRM 29
 27.20: Tertiary Structure of polypeptides and Proteins.
Fibrous. Polypeptides strands that “bundle” to form elongated
fibrous assemblies; insoluble;
Globular. Proteins that fold into a “spherical” conformation .
Hydrophobic effect. Proteins will fold so that hydrophobic amino
acids are on the inside (shielded from water) and hydrophilic
amino acids are on the outside (exposed to water).

s • Tyr • Leu • Glu • Phe • Ile • Ser • Asp • Ala • Ile • Ile • His •Val • His
30• Ser •
Enzymes: proteins that catalyze biochemical reactions.
• by bringing the reactive atoms together in the optimal geometry
for the reaction.
• lowering the activation energy (G‡) by stabilizing the
transition state and/or high energy intermediate.
• many enzymes use the functional groups of the amino acid
sidechain to carry out the reactions
Proteases (peptidases): catalyzes the hydrolysis of peptide bonds
O R O R O protease O R O R O
H H H H
H3N N N H3N N + HN N
N N N CO2 H2O N O 3 N CO2
H H H
H O R O R H O R O R

Four classes of proteases:

Serine (trypsin): aspartate-histidine-serine
Aspartyl (HIV protease, renin): two aspartates
Cysteine (papain, caspase): histidine-cysteine
Metallo (Zn2+) (carboxypeptidase, ACE): glutamate
31
Mechanism of carboxylpeptidase, metalloprotease (p. 1151)
Mechanism of a serine protease (trypsin, chymotrypsin):
oxy-anion
hole NH HN
NH O
HN NH HN
O O Ser192O R1
R2 R1 NHR2
R1 N H
Ser195 H O O
O Ser195 acyl-enzyme
H intermediate
H - R2-NH2
H
His57 N His57 His57 N
N

N N N
H H H
Asp102 CO2- Asp102 CO2- Asp102 CO2-

NH HN
O Ser195
Ser192O R1 O
O H
+ RCO2H
H
H
His57 His57 N
N

N N
H H
Asp102 CO2- Asp102 CO2-

32
27.21: Coenzymes. Some reactions require additional organic
molecules or metal ions. These are referred to as cofactors or
coenzymes. O
OH H O O NH2
S O O P O O NH2
HO
P OH P OH
N N O O O
+ HO HO
H2N
N N NH2
N
O N C N
NH2 Pyridoxal Phophates
Thiamin Diphosphate Co
(vitamin B1) (vitamin B6) O H N N
H2N NH2
H2N N N O
O
N
N HN
N NH
N
Fe N O HN H
N HO H
H O
N N CO2- -O P O
O
O O CO2- O H H
HO
O Folic Acid OH
OH (vitamin B9)
Vitamin B12
Heme NH2 (cyanocobalamin)
N N
O
OH O
O N
HO O P O P O O N
HN NH O- O-
OH
HO OH
N N O
S CO2H
NH
N
Biotin
(vitamin B7) O
Flavin Ade nine Dipho s phate (FAD)
(Vitamin B 2 )

27.22: Protein Quaternary Structure. (please read) 33