Chapter 24

Proteins
Introduction
The three major groups of biological polymers are
polysaccharides, proteins and nucleic acids
Proteins have many diverse functions; they are major
components of the following biomolecules
-Enzymes and hormones which catalyze and regulate biological reactions
-Muscles and tendons which provide the body with means for movement
-Hemoglobin which carries oxygen to all parts of the body
-Antibodies they are integral parts of the immune system
All proteins are polyamides
-Their monomeric units are one of about 20 - amino acids
O

NH2
HO


R
An -amino acid
R is a side chain at the  carbon that determines the identity
of the amino acid (Table 24.1).
O R1 O R4 O

NH NH
H3C NH
NH NH CH3
R1 O R3
O R5

A portion of a protein molecule

Amide (peptide) linkages are shaded.
R1-R5 may be any of the possible side chains.
Chapter 24 2
Proteins have several levels of structure
-Primary structure refers to the exact sequence of amino acids along a protein
chain
-Secondary and tertiary structures refer to the further bending and folding of the
primary structure
-Quaternary structure refers to the aggregation of more than one polyamide chain
All amino acids except glycine are chiral and have the L
configuration (as related to glyceraldhyde) at the  carbon

CO 2H CHO

NH 2 C H HO C H

R CH2OH
An L--amino acid
[usually an (S)--amino acid] L-Glyceraldehyde
[(S)-glyceraldehyde]

CO 2H CHO

NH 2 H HO H
CH2OH
R
Fischer projections for an L--amino acid
and L-glyceraldehyde

Chapter 24 3
Amino acids
Structure and Names
22 amino acids but only 20 amino acids comprise the building
blocks for synthesis of proteins
The remaining 2 amino acids are derived by modification after
biosynthesis of the protein
-Hydroxyproline and cystine are synthesized from proline and cysteine,
respectively, after the protein chain has been synthesized
Cysteine is oxidized under mild conditions to the dissulfide
cystine
-The reaction is reversible
-This linkage is important in maintaining the overall shape of a protein
Disulfide linkage
[O]
2 HO 2C CHCH2 SH HO 2C CHCH2 S S CH2CH CO 2H
[H]
NH2 NH2 NH2
Cysteine Cystine
Essential Amino Acids
Essential amino acids are not made by higher animals and must
be part of the diet
-There are 8 essential amino acids for adult humans (see Table 24.1)
Chapter 24 4
 CO 2H H

NH2 H R C CO 2 H
H 2N
R

pKa1 pKa2 pKa3

Structure of R Name a pl
Abbreviations CO2+ NH3+ R group

Neutral Amino Acids

H Glycine G or Gly 2.3 9.6
Alanine 6.0
CH3 A or Ala 2.3 9.7 6.0
CH(CH3)2 Valineb V or Val 2.3 9.6
L or Leu 6.0
Leucineb 2.4 9.6 6.0
CH2CH(CH3)2 I or Ile 2.4 9.7
CHCH2CH3 Isoleucineb 6.1

CH3

H CH2
Phenylalanineb F or Phe 1.8 9.1
CH2CH2CONH2 Asparagine N or Asn 2.0 8.8
Glutamine Q or Gln 2.2 9.1
H3C CH2

N
Trytophanb W or Trp 2.4 9.4

O
10.6
COH HC CH2 Proline P or Pro 2.0

NH CH2
CH2
(complete structure)

Chapter 24 5
 CO 2H H

NH2 H R C CO 2 H
H 2N
R

pKa1 pKa2 pKa3

Structure of R Name Abbreviationsa
CO2+ NH3+ R group pl
Neutral Amino Acids
CH2OH S or Ser 5.7
Serine 2.2 9.2
CHOH Threonineb T or Thr 10.4 6.5
2.6

CH2
H CH2 OH Tyrosine Y or Tyr 2.2 9.1 10.1 5.7

COH HC CH2 Hydroxyproline Hyp

1.9 9.7 6.3
NH CH
CH2 OH
(complete structure)
CH2SH Cysreine C or Cys 1.7 10.8 8.3 5.0
CH2S
Cystine Cys-Cys 5.1
1.6 7.9
CH2S 2.3 9.9
CH2CH2SCH3 Methionineb M or Met 2.3 9.2 5.8

Chapter 24 6
 CO 2H H

NH2 H R C CO 2 H
H 2N
R

pKa1 pKa2
pKa3
Structure of R Name Abbreviationsa NH3+ pl
CO2+ R group
R Contains an Acidic (Carboxyl) Group
CH2CO2H Aspartic acid D or Asp 2.1 9.8 3.9 3.0
CH2CH2CO2H Glutamic acid E or Glu 2.2 9.7 4.3 3.2

R Contains a Basic Group

CH2CH2CH2CH2NH2 Lysineb K or Lys 2.2 9.0 10.5o 9.8

NH
R or Arg 2.2 9.0 12.5o
Arginine
CH2CH2CH2NHCNH2 10.8
N
H or His 9.2 6.00 7.6
1.8
Histidine
H CH2
N

a
Single-letter abbreviations are now the most commonly used form in current biochemical literature
b
An essential amino acid
c
pKa is of protonated amine of R group

Chapter 24 7
Amino Acids as Dipolar Ions
In the dry solid state amino acids exist as dipolar ions
(zwitterions)
In aqueous solution an equilibrium exists between the dipolar ion,
the cationic and the anionic forms of the amino acid
-The predominant form depends on the pH of the solution

+ OH- + - OH-
H3NCHCO2H H3O+
H3NCHCO2 H3O+
H2NCHCO2-
R R R
Cationic form Dipolar ion Anionic form
(predominant in (predominant in
strongly acidic strongly basic
solutions, e.g., solutions, e.g.,
at pH 0) at pH 14)

At low pH the amino acid exists primarily in the cationic form

At high pH the amino acid exists primarily in the anionic form
At some intermediate pH called the pI (isoelectric point), the
concentration of the dipolar ion is at a maximum and the
concentrations of anionic and cationic forms are equal
Each individual amino acid has a characteristic pI (see Table 24.1)
-Entire proteins also have a characteristic pI

Chapter 24 8
The amino acid alanine has a neutral side chain and can be used
to illustrate the fundamental behavior of an amino acid at various
pHs
-At low pH alanine exist as the cation
-pKa1 of alanine (for ionization of the carboxylic acid proton) is 2.3, considerably
lower than the pKa of a normal carboxylic acid
CH3 CHC O 2 H CH3CH2 CO 2H

NH3
+
Cationic form of alanine Propanoic acid
pKa1 = 2.3 pKa = 4.89

-pKa2 of alanine (for ionization of a proton from the protonated amino group) is 9.7

- -
OH - OH -
CH3CHCO2 H CH 3 HC CO 2 CH3 HC CO 2
+ +
H 3O H 3O
NH3 NH 3 NH2
+ +

Cationic form Dipolar ion Anionic form

(pKa1 = 2.3) (pKa2 = 9.7)

-The isolectric point, pI, for alanine is the average of the two pKa values i.e. (pKa1 +
pKa2)/2

Chapter 24 9
-When base is slowly added to fully protonated alanine, a pH is reached where half
of the carboxylic acid groups are deprotonated
-This pH of 2.3 is the value of pKa1
-The Henderson-Hasselbach equation predicts this result
HA HA
pKa = pH + log HA = A- and log =0
A- A-
-As more base is added, the pI is reached and the molecule is electrically
neutral;
this point is reached when exactly one equivalent of base is added
-As more base is added and pH 9.7 is reached, half of the the aminium groups
will be deprotonated
-Addition of more base will eventually produce only the anionic amino acid
14
-
CH3 CHCO 2
12
NH2

10
pKa2 = 9.7

8
pH
6 pl = 6.0
-
CH3 CHCO 2
4 +
NH3
pka1 = 2.3
2 CH 3 CHCO 2 H
+
NH 3

0
0 0.5 1.0 1.5 2.0
-
Equivalents of OH
Chapter 24 10
 Lysine, which contains a basic side-chain, has a more complex
equilibrium
-The pI for lysine will be high because of the presence of two basic groups
-The pI for lysine is the average of the monocation (pKa2) and the dipolar ion (pKa3)

+ OH- + - OH- + - OH- -

H3N (CH2)4 CHCO 2H H3N (CH 2) 4 CHCO 2 H3N (CH2)4 CHCO2 H2N (CH2)4 CHCO2
+ H3O+ + H3O+ H3O+
NH3 NH3 NH2 NH2
Dicationic form of Monocationic Dipolar ion Anionic form
lysine form (pKa3 = 10.5)
(pKa1 = 2.2) (pKa2 = 9.0)

Chapter 24 11
Synthesis of a-Amino Acids
The first three methods result in racemic mixtures of amino acids
Direct Ammonolysis of an a-Halo Acid
Yields tend to be poor in this reaction
NH 3 (excess) -
RCHCO 2H R CHCO 2
+
NH 3
X
From Potassium Phthalimide
This is a variation of the Gabriel synthesis and yields are usually
high O

- +
NK + ClCH2 CO2 C2H5

O
Potassium Ethyl
phthalimide chloroacetate
O
CO 2 H
(1) KOH/H2O -
N CH2 CO2 C 2H 5 CH2 CO2 + + C 2H 5 OH
(2) HCl
+
NH3 CO 2 H
O
Glycine Phthalic
(97%)
(85%) acid
Chapter 24 12
 The Strecker Synthesis
Treatment of an aldehyde with ammonia and hydrogen cyanide
yields an -aminonitrile which is hydrolyzed to the -amino acid
-The reaction proceeds via an intermediate imine

O
H2 O+ , heat -
R CH + NH3 + HCN R CHCN R CHCO2
H 2O +
NH2 NH3
-Amino nitrile -Amino acid

-
O O OH
+ -H2O CN- -
H3O+
R CH + NH3 R CHNH3 R CHNH2 RCH NH R CH NH R CH NH2

CN CN
Intermolecular proton
Imine
transfer -Aminonitrile

Chapter 24 13
Resolution of DL-Amino Acids
A racemic amino acid mixture can be resolved by
(1) conversion to a racemic mixture of N-acylamino acids,
followed by
(2) hydrolysis with a deacylase enzyme that selectively deacylates
the L-acylamino acid

- (CH3CO)2O deacylase enzyme

DL- RCHCO 2 DL- RCHCO 2 H CH 3CO 2H

NH 3 CH 3CONH
+
- -
CO2 CO2
+
H3 N H + H NHCOCH3

R R

L-Amino acid D-N-Acylamino acid

Easily separated

Chapter 24 14
Asymmetric Synthesis of Amino Acids
Enantioselective syntheses that produce only the desired
naturally occurring amino acid enantiomers are ideal
One important method involves asymmertic hydrogenation of an
enamide using a chiral transition metal catalyst
-This method was used to synthesize L-dopa, a chiral amino acid used in the
treatment of Parkinson’s disease
Asymmetric Synthesis of L-DOPA

H 3CO COOH H2

NHAc [(Rh(R,R)-DiPAMP)COD]+BF4- (cat)

AcO
H 3CO COOH HO COOH
+
H3O
H NHAc H NH2
AcO HO

100% Yield, 95% ee L-DOPA

OCH3

P P

H3 CO COD = 1,5-Cyclooctadiene
(R,R)-DiPAMP
(chiral ligand for rhodium)
Chapter 24 15
 -A similar method is used to synthesize (S)-phenylalanine, needed for preparation
of Aspartame
Asymmetric Synthesis of Aspartame
COOH (1) (R,R)-PNNP-Rh(I) (cat.), H2, 83% ee COOCH 3
(catalytic asymmetric hydrogenation)
H NH 2
NHAc (2) MeOH, HA

(S)-Phenylalanine methyl ester

(97% ee after recrystallization)
Ph Ph
N N
HOOC
H 3C H 2P 2P P PH 2 2 CH 3 COOH

H2N H
HH
(R,R)-PNNP
(chiral ligand for rhodium)
L-Aspartic acid

H NH 2
NH
COOH

HOOC H O

Aspartame

Chapter 24 16
Polypeptides and Proteins
Enzymes polymerize amino acids by forming amide linkages
The polymer is called a peptide and the amide linkages are called
peptide bonds or peptide linkages
Each amino acid in the peptide is called an amino acid residue
Proteins can contain one or more polypeptide chains and other
associated molecules or metal ions
O O
+ - + -
H3 N CH C O + H 3N HC C O

R R'
[-H2O]

O O
+
H3 N -
CH C NH CH C O

R R'
A dipeptide

Chapter 24 17
 Polypeptides are customarily written with the N-terminal residue
to the left
-Three letter or one letter abbreviations are usually used as a short hand to
indicate the sequence of a polypeptide

O O O
+
H3 N CH -
C NH CH C NH CH C O

R R' R''
n

N-Terminal residue C- Terminal residue

O O
O O
+ -
+ - H3N CH C NH CH2 C O
H3N CH2 C NH HC C O
HC
CH
H 3C CH3 H3 C CH3
Glycylvaline Valylglycine
(Gly . Val) (Val . Gly)

Chapter 24 18
Hydrolysis
A polypeptide can be hydrolyzed by refluxing with 6M
hydrochloric acid for 24h
The individual amino acids can be separated from each other
using a cation-exchange resin
-An acidic solution of the amino acids is passed through the cation-exchange
column; the strength of adsorption varies with the basicity of each amino acid (the
most basic are held most strongly)
-Washing the column with a sequence of buffered solutions causes the amino
acids to move through it at different rates
+
- H 3N CH CO 2H
HC SO 3

R
CH2
+
H3 N CH CO 2H
-
HC SO 3
R'
CH2
+
- H3 N CH CO 2H
HC SO 3

R''
CH2
+
- H3 N CH CO 2H
HC SO 3

R'''
Chapter 24 19
 In the original method, the column eluant is treated with
ninhydrin, a dye used for detecting and quantifying each amino
acid as it comes off the column
O O

C OH
+H2O
O C
-H2O
C OH

O O
Indane-1,2,3-trione Ninhydrin

O -
O O
O
-
2 O + RCHCO2 N + RCH + CO2
(-H3O+)
+
H3N
O O O
Purple anion

In modern practice, analysis of amino acid mixtures is routinely

accomplished using high performance liquid chromatography
(HPLC)

Chapter 24 20
Primary Structure of Polypeptides and Proteins
The sequence of amino acids in a polypeptide is called its primary
structure
-Several methods exist to elucidate the primary structure of peptides
Edman Degradation
Edman degradation involve sequential cleavage and identification
of N-terminal amino acids
Edman degradation works well for polypeptide sequence analyses
up to approximately 60 amino acid residues
-The N-terminal residue of the polypeptide reacts with phenyl isothiocyanate
-The resulting phenylthiocarbamyl derivative is cleaved from the peptide chain
-The unstable product rearranges to a stable phenylthiohydantoin (PTH) which is
purified by HPLC and identified by comparison with PTH standards

OH-,pH 9
N C S + H2N CH CO HN CH CO etc
S R R'
HA
NH C NHCHCO HN CH CO etc
R R'
Labeled polypeptide

Chapter 24 21
Sanger N-Terminal Analysis
The N-terminal end of the polypeptide is labeled with 2,4-
dinitrofluorobenzene and the polypeptide is hydrolyzed
-The labeled N-terminal amino acid is separated from the mixture and identified

HCO3-
O2N F + H2N CH CO HNCH CO etc
(-HF )
NO2 R R'
2,4-Dinitrofluorobemzene Polypeptide
(DNFB)

H3O+
O2N NHCHCO NHCHCO etc

NO2 R R'
Labeled polypeptide

+ -
O2N NHCHCO2H + H3N CH CO2
R R'
NO2
Labeled N- terminal amino Mixture of
acid amino acids

Separate and identify

The Sanger method is not as widely used as the Edman method

Chapter 24 22
Examples of Polypeptide and Protein Primary
Structure
Oxytocin and Vasopressin
Oxytocin stimulates uterine contractions during childbirth
Vasopressin causes contraction of peripheral blood vessels and a
resultant increase in blood pressure
-The two polypeptides are nonapeptides and differ in only 2 amino acid residues
Oxytocin
H 3C CH3 O NH2
CH C
O O CH2 O O CH2

H2N CCH2NH C CH NH C N C CH NH C CH NH O
O
C
O CH2
CH CH2CH2C NH2
Leu S NH
S
C O
CH2
CH CH CH2CH3
H 2N CH C NH CH C NH CH3

O CH2 O
Ile

OH

Chapter 24 23
Vasopressin
H2N NH
C
NH
O NH2
CH2

CH2 C
O O O O CH2
CH2
H2N CCH2NH C CH NH C N C CH NH C CH NH O
O
Arg C
O CH2
CH CH2CH2C NH2
S NH
S
C O
CH2
CH CH2
H2 N CH C NH CH C NH
Phe

O CH2 O

OH

Chapter 24 24
Insulin
Insulin is a hormone which regulates glucose metabolism
-Insulin deficiency in humans is the major cause of diabetes mellitus
-The structure of bovine insulin (shown below) was determined in 1953 by Sanger
-Human insulin differs from bovine insulin at only three amino acids in its
sequence
Ser
S Cys
S Val
A chain
Gly IIe Val Glu Gln Cys Ser Leu
Cys Ala
Tyr
S
Gln
B chain S
Leu
Phe Val Asn Gln His Leu Cys
Gly Glu

Glu Val Leu His Ser Asn

Ala Tyr
Leu Tyr Leu Val Cys S S Cys
Gly Asn
Ala Lys Pro Thr Tyr Phe Phe Gly Arg Glu
Chapter 24 25
Polypeptide and Protein Synthesis
Laboratory synthesis of polypeptides requires orchestration of
blocking and activating groups to achieve selective amide bond
formation
-Amino groups must be blocked until their reactivity as a nucleophile is desired
-Carboxylic acid groups must be activated for acyl substitution at the appropriate
time
Amino groups are usually blocked using one of the following:
-A benzyloxycarbonyl group (a “Z” group)
-A di-tert-butyloxycarbonyl group (a “Boc” group)
-An 9-fluorenylmethoxycarbonyl group (an “Fmoc” group)
Methods for installing and removing Z, Boc, and Fmoc groups are
shown below:
Tert - Butyloxycarbonyl Group

O Protection O
base
(CH 3)3C OCOC(CH 3)3 + H 2N R
o
(CH 3)3C OC NHR + (CH 3)3C OH
25 C
Di-tert-butyl carbonate tert - Butyloxycarbonyl
or Boc group
HCl or CF3CO2H
Deprotection in
acetic acid, 25 oC

(CH 3)3C CH2 + CO 2 + H 2N R

Chapter 24 26
 Methods for installing and removing Z, Boc, and Fmoc groups are
shown below:
9-Fluorenylmetholxycarbonyl Group
O

Protection (introduction of Fmoc group)

O Cl
O
(1) aq Na2CO3, O
CH2 +
+ H 3N
– dioxane, 0o
Fmoc NH
O (2) H3O+ OH
R
R
9-Fluoromethyl Amino acid Fmoc-protected amino acid
carbonochloridate (side chain protected in (stable in acid)
advance if neccessary)
O

Fmoc = H 2C O C

(9-Fluorenylmethoxycarbonyl group)
Deprotection (removed of Fmoc group)

O NC5H10
O
+
Fmoc NH + DMF
H 3N
– + CO2 +
OH N O
R H R
Piperidine Unprotected (Byproduct)
(C5H11N) amino acid

Chapter 24 27
Carboxylic acid groups are usually activated by
conversion to a mixed anhydride:
-Ethyl chloroformate can be used

O O O
(1) (C2H5)3N
Z NHCH C OH Z NHCH C O C O C2H 5
(2) ClCO 2C2H 5
R R
"Mixed anhydride"

+
-
O H3N CHCO2
O
R'
Z NHCH C O C O C 2H5

R
O

Z NHCH C NHCH CO2H + CO2 + H 5C2 OH

R R'

Chapter 24 28
An Example of Laboratory Peptide Synthesis:
Synthesis of Ala-Leu
O
- OH- (1 ) (C 2H 5) 3N
CH 3 CH CO 2 + C 6 H 5 CH 2 O C Cl
o CH 3 CH CO 2 H ( 2) ClCO2C2 H5
25 C
NH 3
NH
+
C O
Ala Benzyl chloroformate C 6 H 5 CH 2 O

+ Z-Ala
NH
3 -
O O (CH3 )2CH CH CH CO
2 2
Leu
CH 3 CH C O C O C6H 5

NH

C O CO 2 + C 2 H 5 OH

C 6 H 5 CH 2 O
O
Mixed anhydride H2/Pd
of Z-Ala CH 3 CH C NH CH CO 2 H

CH 2
NH

C O CH
H 3C CH 3
C 6 H 5 CH 2 O
Z-Ala Leu
O
-
CH 3 CH C NH CH CO 2 + CH 3 + CO
2
CH 2
N H3
+
CH
H 3C CH 3
Ala Leu

Chapter 24 29
Polymer O
O CH2OH + HO C CH NH Fmoc
R

Diisopropylcarbodiimide
Step 1 Attaches C-terminal
(Fmoc protected) amino
Polymer O acid residue to resin
O CH2 O C CH NH Fmoc
Step 2 Purifies resin with
R
attched residue by
Piperidine in DMF washing
Step 3 Removes Fmoc protecting
Polymer O
group
O CH2 O C CH NH
2
R Step 4 Purifies by washing
O
HOC CH NH Fmoc Step 5 Adds next (protected)
R' amino acid residue
and
Diisopropylcarbodiimide

Polymer O O

O CH2 O C CH NH CHC NH Fmoc Step 6 Putifies by washing

R R'

Piperidine in DMF Step 7 Removes protecting

Repetitions of steps 47
group
CF3 CO2 H
Final Step Detaches completed
Polymer O O O polypeptide
O CH2 Br + HO C CH NH C CHNH C CHNH etc.
R R' R''

Chapter 24 30
The extended polypeptide chains in b-pleated sheets form
hydrogen bonds to adjacent polypeptide chains
-Slight bond rotations are necessary between amide groups to avoid unfavorable
steric interactions between peptide side chains, leading to the pleated structure
-The -pleated sheet is the predominant structure in silk fibroin

H O H R H O

N C C C
N
C N N
C C

H O H
R H R H
crowding crowding
R H H
O H R
O

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

Hypothetical flat-sheet structure

(not formed because of steric hindrance)

Chapter 24 31
Each of the four protein subunits carries a heme group
-The four heme groups are shown in purple
-Each heme group can bind one oxygen molecule in a reversible complex

CH3 CH CH2

H H
C C
CH3 CH3
N

N Fe N
O
HO C CH2CH2 N CH CH2
C C
H H
CH2 CH3

CH2
C OH
O

Chapter 24 32