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CHAPER 24
AMINO ACIDS AND PROTEINS
24.1 INTRODUCTION
Of the three groups of biopolymers, protein have the most diverse
function. Most of its molecular weights are much larger. Their shaps
cover a range from the globular protein to the helical coils of a
α–keratin. But all proteins have common features.
Proteins are polyamides and their monomeric units are about
20 different α-amino acids.
Amide links
H
R
R
CH
CH
N
C
H
O
OH
An  -amino acid
R''
R'
N
C
N
H
O
H
R'''
CH
CH
R''''
CH
CH
C
N
C
N
C
N
C
O
H
O
H
O
H
O
A portion of a protien molecular
Primary structure: the exact sequence of the different α-amino acids
along the protein chain.
Second and tertiary structure: the folding of the polyamide chain
which give rise to higher levels of
complexity.
Although hydrolysis of natural occurring proteins may yield as many
as 22 different amino acids, the amino acids have an important
structural feature in common.
24.2 AMINO ACIDS
24.2A STRUCTURES AND NAMES
CO2H
H2N
H
R
R
NAME
ABBREVIATION
-H
Glycine
Gly
6.0
-CH3
Alanine
Ala
6.0
-CH(CH3)2
Valinee
Val
6.0
-CH2CH(CH3)2
Leucinee
Leu
6.0
-CH(CH3)CH2CH3
Isoleucinee
Ile
6.1
pI
R
NAME
ABBREVIATION
pI
Phenylalaninee
Phe
5.5
-CH2 CONH2
Asparagine
Asn
5.4
-CH2CH2CONH2
Glutamine
Gln
5.7
Tryptophane
Trp
5.9
HOOC CH CH2
HN
CH2
C
H2
Proline
Pro
6.3
-CH2OH
Serine
Ser
5.7
H2C
CH2
N
H
(complete structure)
ABBREVIATION
pI
Threoninee
Thr
6.5
Tyrosine
Tyr
5.7
HOOC CH CH2
HN
CH
C
OH
H2
Hyroxyproline
Hyp
6.3
-CH2SH
Cysteine
Cys
5.0
Cystine
Cys-Cys
5.1
-CH2 CH2SCH3
Methioninee
Met
5.8
-CH2COOH
Aspartic acid
Asp
3.0
NAME
R
-CH(CH3 )OH
H2C
OH
(complete structure)
H2
C S
H2
C S
R
NAME
ABBREVIATION
pI
-CH2CH2CO2H
Glutamic acid
Glu
3.2
-CH2(CH2)3NH2
Lysinee
Lys
9.8
-CH2(CH2)2NHCNHNH2
Arginine
Arg
10.8
Histidien
His
7.6
H2
C
N
N
H
The conversion of cysteine to cystine requires addition comment.
it can be reversed by mild reducing agents.
2HO2 CCHCH2SH
NH2
[0]
[H]
HO2CCHCH2S_SCH2CHCO2H
NH2
NH2
Cysteine
Cystine
(°ë ë×°±Ëá)
(ë×°±Ëá)
24.2B ESSENTIAL AMINO ACIDS
For adult humans there are eight essential amino acids. These are
designated with the superscript e in above table.
24.2C AMINO ACIDS AS DIPOLAR IONS
Since amino acids contain both a basic group (-NH2) and an acidic
group (-COOH) , they are amphoteric.
H3N+CHCO2H
R
Cationic form
(ÑôÀë×ÓÐÎʽ£©
- H+
+ H+
H3N+CHCO2R
Dipolar ion
(Á½
¼«Àë×Ó£©
- H+
+ H+
H2NCHCO2R
Anionic form
(ÒõÀë×ÓÐÎʽ£©
In strongly basic solutions all amino present as anions, in acidic
solutions they are present as cations. At some intermediate Ph,
called isoelectric point(pI) the concentration of the dipolar ion is
at its maximum and the concentrations of the anions and cations
are equal.
H3CCHCO2H
CH3
Cationic form
(pKa1 = 2.3£©
OHH+
H3CCHCO2
-
CH3
Dipolar ion
(pKa2 = 9.7£©
OHH+
H3CCHCO2CH3
Anionic form
As the acidity reaches pH 2.3, one half of the cationic form will be
converted to the dipolar ion. As the pH increase to2.3- 9.7 the
predominant form will be the dipolar ion. When pH rise to 9.7,the
dipolar ion will be half-converted to the anionic form. As pH
approached to 14,the anionic form becomes predominant form.
If the side chain of an amino acid contains an extra acidic or basic
group, then the equilibria are more complex.
The isoelectric point (pI) of an amino such as the alanine is the
average of pKa1 and pKa2.
pI = 2.3 +9.7
2
= 6.0
24.3 LABORATORY SYNTHESIS OF α-AMINO ACIDS
A variety of methods have been developed for the laboratory
synthesis of α-amino acids. We shall describe here three general
methods.
24.3A DIRECT AMMONOLYSIS OF AN α-HALO ACID
(1) X , P
R CH2CO2H (2) H2O 4
2
RCHCO2H
X
NH3 (excess)
RCHCO2NH3+
This method is probably used least often because yields tend to be
poor.
24.3B FROM POTASSIUM PHTHALIMIDE
This method is a modification of the Gabriel synthesis of amines.
the yields are usually high and the products are easily purified.
O
N-K+
O
+ ClCH2CO2C2H5
O
N CH2CO2C2H5
O
Ethyl chloroacetate
Potassium phthalimide
(ÂÈ́ú ´× ËáÒÒõ¥£©
(±½ÁÚ¶þ¼×õ£ÑÇ°· ¼Ø£©
(1) KOH / H2O
(2) HCl
CH2CO2 NH3 +
Glycine
(°±»ùÒÒËá)
COOH
+
+ C2H5OH
COOH
Phthalic acid
(ÁÚ±½¶þ¼×Ëá)
24.3C THE STRECKER SYNTHESIS
Treating an aldehyde with ammonia and hydrogen cyanide
produces an α-amino nitrile. Hydrolysis of the nitrile group
of the α-amino nitrile converts the latter to an α-amino acid.
RCHO + NH3 + HCN
RCHCN
H3O+, heat
RCHCO2NH3+
NH2
¦Á-Amino nitrile
¦Á-Amino acid
Mechanism of the first step:
O
RCH + NH3
O-
OH
RCHNH3+
CN
RCH=NH
RCHNH2
-H2O
CN
+
- H
RCHNH
RCHNH2
24.3D RESOLUTION OF DL-AMINO ACIDS
One interesting method for resolving amino acids is based on the use
of enzymes called deacylases.
DL
RCHCO2- (CH3CO)2O
NH3+
DL
RCHCO2H deacylase
CH3COOH
NHCOCH3
CO2+H3N
CO2-
H + H3COCHN
R
L-Amino
H
R
acid
(L-°±»ùËᣩ
D-N-Acylamino
acid
(D-N-õ£°±»ùËᣩ
Easily separated
24.3E STEREOSELECTIVE SYNTHESIS OF AMINO
ACIDS
Producing only the naturally occurring L-amino acid has been
realized through the use of chiral hydrogenation catalysts from
transition metals. One of which is called “(R)-prophos”.
H3 C
H
CH2
(C6H5)2P
P(C6H5)2
(R)-Prophos
[Rh(NBD)2]ClO4 + (R)-prophos
[Rh((R)-prophos)(NBD)]ClO4 + NBD
Chiral rhodium complex
(ÊÖÐÔîî ÅäºÏ Îï £©
Hydrolysis of N-acetyl group under this chiral rhodium complex
yields L-alanine. Because the hydrogenation catalyst is chiral, it
transfers its hydrogen atoms in a stereoselective way. This type
of reaction is called asymmetric synthesis.
CH2=C_CO2H
[Rh((R)-prophos)(H2)(solvent)2]+
H2
NHCOCH3
H3C
COOH
H3COCHN
2-Acetylaminopropenoic acid
N-Acetyl-L-alanine
(2-ÒÒõ£°·»ù±ûÏ©Ëᣩ
(1) OH-, H2O, heat,
(2) H3O+
H
(N-ÒÒõ£»ù-L-±û°· Ëá)
H
H3C
CO2-
+H3N
L-alanine
(L-±û°· Ëá)
24.4 ANALYSIS OF AMINO ACID MIXTURES
Enzymes can cause α-amino acids to polymerize through the
elimination of water:
O
+H N
3
H
C
R
C
O
O-
+
+H N
3
H
C
R'
C
O
O-
H2O
+H N
3
H
C
R
C
O
HN
H
C
C
O-
R
A Dipeptide
(¶þëÄ£©
The –CO-NH- linkage between the amino acids is called a peptide
bond. Amino acid when joined in this way, are called amino acid
residues. The polymers that contains 2,3, a few, or many amino acid
residues are called dipeptides, tripeptides, oligopeptides, and
polypeptides, respectively.
Polypeptides are linear polymers. The free NH3+ group and the
free CO2-group are called the N-terminal and the C-terminal
Residues respectively.
O
+H
3N
H
C
C
R
O
HN
H
C
R¡®
O
*HN
H
C
n
R¡¯¡®
C
C
O-
C-Terminal
N-Terminal
RCHCO2 H
The automatic amino acid
analyzers are based on the
use of insoluble polymers
containing sulfonate groups,
called cation-exchange resins.
CH
SO3-
CH2
CH
SO3-
CH2
CH2
SO3-
R'CHCO2H
NH3 +
R''CHCO2H
NH3+
CH2
HC
NH3 +
SO3-
R'''CHCO2H
NH3+
If the mixture of amino acids pass through a column which is
washed with a buffered solution at a given pH, The individual
amino acids will move down the column at different rates and
ultimately separated.
24.5 AMINO ACID SEQUENCE OF POLYPEPTIDES AND
PROTEINS
The different amino acid sequences of a protein which compose of
20 different amino acids in a single chain of 100 residues are
Amazing large. They are 1.27 X 10130
The methods of determining the amino acid sequence include
Terminal residue analysis, partial hydrolysis and so on.
24.5A TERMINAL RESIDUE ANALYSIS
One very useful method for determining the N-terminal amino acid
residue, called the Sanger method, is based on the use of 2,4dinitrofluorobenzene (DNFB).
O2N
F
+ NH2CHCO-NHCHCO
R
NO2
2,4-Dinitrofluorobenzene
(DNFB)
etc
HCO3(-HF)
R'
Polypeptide
(¶à ëÄ£©
(2,4-¶þÏõ »ù·ú ±½£©
O2N
NHCHCO-NHCHCO
R
NO2
Labeled polypeptide
(ÓбêÖ¾µÄ¶à ëÄ£©
R'
etc
H3O+
O2N
NHCHCO2H + H3N+CHCO2R
NO2
R'
Labeled N-terminal
amino acid
Mixture of
amino acids
(ÓбêÖ¾µÄN-¶Ë°±»ùËᣩ
(»ì ºÏ °±»ùËᣩ
Separated and identify
A second method of N-terminal analysis is the Edman degradation.
This method offers an advantage over the Sanger method in that it
Moves the N-terminal residue and leaves the remainder of the peptide
Chain intact.
N=C=S
+ NH2 CHCO-NHCHCO
R
R'
NHCSNHCHCO_NHCHCO
R
C
N
N
H
H
Unstable intermediate
+
H3 N+CHCOH
R'
Polypeptide with one
less than amino acid
residue
etc
H+
R'
O
CH R
-,
etc OH pH 9
S
rearrangement
heat
C
N
C
NH
CH
R
O
Phenylthiohydantoin
C-terminal residues can identified through the use of digestive
enzymes called carboxypeptidases. These enzymes specifically
catalyze the hydrolysis of the amide bond of the amino acid residue
containing a free –COOH group, liberating it as a free amino acid.
24.5B PARTIAL HYDROLYSIS
Break the polypeptide chain into small fragments, then examine the
structure of these smaller fragments to determine the original
polypeptide. For example:
We are given a pentapeptide known to contain valine(two residues),
lucine, Histidine, and phenylalanine. Then the molecular formular:
Val2, Leu, His, Phe
By using DNFB and carboxypeptidase we discover that valine and
leucine are the N-terminal and C-terminal, respectively.
Val ( Val, His, Phe) Leu
We then subject the pentapeptide to partial acid hydrolysis and obtain
the following dipeptides.
Val·His + His·Val + Val·Phe + Phe·Leu
The points of overlap of the dipeptides tell us that the original
Pentapeptide must have been the following:
Val· His· Val· Phe· Leu
24.6 PRIMARY ATRUCTURES OF POLYPEPTIDES
AND PROTEINS
The covalent structure of a protein or polypeptide is called primary
structure. Chemists have had remarkable success in determining
the primary structure.
24.6A OXYTOCIN AND VASOPRESSIN
Oxytocin and vasopressin are two rather polypeptides with
strikingly similar structures. But these two polypeptides have
quite different physiological effects.
Oxytocin occurs only in the female of a species and stimulates
uterine contraction during childbirth. Vasopressin occurs in male
and female. Its major function is as an antidiuretic.
24.6B INSULIN
Insulin, a hormone secreted by the pancreas, regulates glucose
metabolism.
Bocine insulin has a total of 51 amino acid residues in two polypeptide chains, called A and B chains. These chains are joined by
two disulfide linkage.
Human insulin differs from bovine insulin at only three amino
acids residues. Insulin from most mammals has a similar structure.
24.6C OTHER POLYPEPTIDES AND PROTEINS
Successful sequential analyses have now been achieved with
hundreds of other polypeptides and proteins including the
following:
(1) Bovine ribonuclease.
(2) human hemoglobin.
(3) bovine trypsinogen and chymotrypsinogen.
(4) gamma globulin.
24.7 POLYPEPTIDE AND PROTEIN SYNTHESIS
We must first “activate” the carboxyl group of an acid by converting
it to an anhydride or acid chloride and then allow it react with an
amine. But when both the acid group and the amino group are present
in the same molecular, the problem becomes more complicate.
24.7A PROTECTING GROUPS
We must “protect” the amino group by converting it to some other
group of low nucleophilicity-one that will not react with a reactive
acyl derivative. Then remove the protecting group.
The reagents are benzyl chloroformate and di-tert-butyl carbonate:
C6H5CH2OCOCL
(CH3)3COCOOC(CH3)3
Benzyl chloroformate
Di-tert-butyl carbonate
(ÜлùÂȼ×Ëáõ¥£©
(¶þÊ嶡 »ù̼ËáÑΣ©
Both reagents react with the following amino group to form
derivatives that are unreactive toward further acylation.
OH25¡æ
CH2OCOCl + H2NR
Benzyl chloroformate
CH2OCONHR + Cl-
Benzyloxycarbonyl
or Z group
HBr
acetic acid
(cold)
H2 / Pd
CH3 + CO2 +H2NR
CH2Br + CO2 +H2NR
O
(CH3)3COCOC(CH3)3 + H2NR
O
base
25¡æ
(CH3 )3COCNHR + (CH3)3 CHO
tert-Butyloxycarbonyl
or boc group
HCl or CF3CO2H
acetic acid, 25¡æ
(CH3 )2C=CH2 + CO2 +H2NR
Remove of the benzyl group with hydrogen and a catalyst depends
on the fact that benzyl-oxygen bonds are weaker and are subject to
hydrogenolysis at low temperatures.
O
C6H5CH2OCR
O
H2 / Pd
25¡æ
C6H5CH3 + HOCR
24.7B ACTIVATION OF THE CARBOXYL GROUP
A much better method is to convert the carboxyl group of the
“protected” amino acid to a mixed anhydride using ethyl chloroFormate.
O
Z_NHCHC_OH
R
O
(1) (C2H5)3N
(2)ClCO2C2H5
O
Z_NHCH_C_O_C_OC2H5
R
Mixed anhydride
(»ì ºÏ ôû£©
The mixed anhydride can be used to acylate another amino acid and
form a peptide linkage.
O
O
+
Z_NHCH_C_O_C_ OC2H5 H3N CHR'CO2
R
O
Z_NHCH_C_NHCHCO2H
R
R'
+ CO2 +C2H5 OH
Dicyclohexylcarbodiimide can also be used to activate the carboxyl
group of an amino acid.
24.7C PEPTIDE SYNTHESIS
The principle involve here can,of course, be extended to the
synthesis of much longer polypeptide chains.
O
CH3CHCO2 -
+
C6H5CH2OC
OH25¡æ
Cl
CH3CH
NH
NH3+
C
benzyl chloroformate
Ala
(Âȱ½¼×Ëáõ¥)
Z-Ala
O
OCH2C6H5
NH3+
CH3CHCOOCOOC2H5H
NH
O
(CH3)2CHCH2CHCO2CO2 + C2H5OH
C O
(1) (C2H5)3N
(2) ClCO2C2H5
CO2H
CH3CH C
CH2
NH
C
OCH2C6H5
NHCHCH2O2 H
O
CH
H3C
OCH2C6H5
O
H2 / Pd
CH3CH C
NHCHCO2-
NH3+
CH2
H3C
CH
CH3
+
CH3
+ CO2
CH3
24.7D AUTOMATED PEPTIDE SYNTHESIS
The Merrifield method for automated synthesis:
O
O
CH2Cl + HOCCHNHCOC(CH3)3
base
O
Step 1
Attaches C-terminal
(protect) amino acid
residue to resin
Step 2
Purifies resin with
attached residue by
washing
Step 3
Removes protecting
group
Step 4
Purifies by washing
Step 5
Adds nest (protect)
amino acid residue
R
O
CH2OCCHNHCOC(CH3)3
R
CF3CO2H / CH2Cl2
O
CH2OCCHNH2
R
HOCOCHR'NHCOOC(CH3)3
dicyclohexylcarbodiimide
O
O
O
CH2 OCCHNHC CHNHCOC(CH3 )3
R'
R
Step 6
Purifies by washing
Step 7
Removes protecting
group
Final step
Detaches completed
polypeptide
CF3CO2H / CH2Cl2
Repetitions of step 4-7
HBr / CF3CO2H
O
O
O
CH2 Br + HOCCHNHC CHNHCCHNH
R
R'
R''
etc
24.8 SECONDARY AND TERTIARY STUCTURES
OF PROTEINS
24.8A SECONDARY STRUCTURE
The secondary structure of a protein is defined by the local Conformation of its polypeptide backbone. These local conformation have
come to be specified terms of regular folding patterns.
Polypeptide chain of a natural protein can interact with itself two
major ways: through formation of a β-pleated sheet and an α helix.
H
O
N
C
crowding
H
R
C
C
N
C
R H
H
O
R H
O
H
C
C
N
C
N
O
H
C
H
R
H
O
N
C
C
N
R H
H
crowding
O
R H
C
C
C
N
O
H
Fully extended polypeptide chains could conceivably form a flatsheet structure (above).
Slight rotation of bonds can transform a flat-sheet structure into
the β-pleated sheet or β configuration.
The α helix structure is a right-handed helix with 3.6amino acid
residues per turn in naturally occurring. It is the predominant
structure of the polypeptide.α
Helices and pleated sheets account for only about one half of the
average globular protein. The remaining polypeptide segments
have what is called a coil or loop conformation.
24.8B TERTIARY STRUCTURE
The tertiary structure of a protein is its three-dimensional shape that
arises from further foldings of its polypeptide chains, foldings
superimposed on the coils of the α helixes.
24.9 INTRODUCTION TO ENZYMES
Enzymes have the ability to bring about vast increases in the rates
of reaction. Enzymes also show remarkable specificity for their
reactants and for their products.
The enzyme and the substrate combine to form an enzyme-substrate
complex.
Enzyme + Substrate
enzyme-substrate
complex
Enzyme + Product
Almost all enzymes are proteins. Reactions catalyzed by enzymes
are completely stereospecific, and this specificity comes from the
way enzymes bind their substrates.
Some enzymes will accept only one compound as its substrate,
others will accept a range of compounds with similar groups.
Inhibitor: a compound that can alter the activity of an enzyme.
competitive inhibitor: a compound that competes directly with
the substrate for the active site.
Some enzymes require the presence of a cofactor. Others may
require the presence of an organic molecule called a coenzyme.
Many of the water-soluble vitamins are the precursors of
coenzymes.
O
C
N
Niacin
(ÄáÑÇÐÂ)
OH
OH
H
N
HO
O
CH3
OH
O
CH3
Pantothenic Acid
(·º Ëá)
24.10 LYSOZYME: MODE OF ACTION OF AN ENZYME
Lysozyme is made up of 129 amino acid residues. Three short
segments of the chain between residues 5-15, 24-34, 88-96 have
the structure of an α helix; the residues between 41-45, and 50-54
form pleated sheets; and a hairpin turn occurs at residues 46-49.
The remaining polypeptide segments of lysozyme have a coil or
loop conformation.
Lysozyme’s substrate is a polysaccharide of amino sugar that makes
up part of the bacterial cell wall.
24.11 SERINE PROTEASES
Serine proteases: the digestive enzymes secreted by the pancreas into
the small intestines to catalyze the hydrolysis of
peptide bonds.
The digestive enzymes includes chymotrypsin, trypsin, and elastin.
The catalytic triad of chymotrypsin cause cleavage of a peptide
bond by acylation of the serine residue 195 of chymotrysin. Near
the active site is a hydrophobic binding site that accommodates
nonpolar side chains of the protein.
His
Asp
57
102
O
H2C
CH2
Ser
C
195
O-
H
N
N
CH2
H
O
H
Hydrophobic pocket
binding site
O
N
C
R'
R
His
Asp
57
102
O
H2C
CH2
Ser
C
O-
195
H
N
N+
CH2
H
O
H
ON
R'
C
R
Tetrahedral
intermediate
His
Asp
57
102
O
H2C
CH2
Ser
C
195
-
O
H
N
N
H
CH2
H
O
N
C
R'
Acetylated
serine
resifue
O
R
His
Asp
57
102
O
H2C
CH2
Ser
C
-
O
195
H
N
N+
CH2
H
O
H
O
C
R
O-
Tetrahedral
intermediate
His
Asp
57
102
O
H2C
CH2
Ser
C
-
O
195
H
N
N
CH2
O
H
H
+
O
O
C
R
Regeneration of the active site of chymotrypsin. Water causes
hydrolysis of the acyl-serine bond.
Compounds such as diisopropylphosphofluoridate (DIPF) that
irreversibly inhibit serine proteases. It has been shown that they
do this by reacting only with Ser 195.
Ser
195
CH2OH + F
CH(CH3)2
CH(CH3)2
O
O
P
Ser
O
195
CH2
O
P
O
O
O
CH(CH3)2
CH(CH3)2
(DIPF)
DIP-Enzyme
24.12 HEMOGLOBIN: A CONJUGATED PROTEIN
CH=CH2
H3C
H
H
C
C
H3C
CH3
N
Hemoglobin: a protein
can carry oxygen.
N Fe N
N
HOOCH2CH2C
CH
H
HOOCH2CH2C
CH=CH2
C
H
CH3
The iron of the heme group is in the 2+ oxidation state and it forms
a coordinate bond to a nitrogen of the imidazole group of histidine
of the polypeptide chain. This leaves one valence of the ferrous ion
combine with oxygen as follows:
N
O2
N
Fe
N
N
N
A portion of oxygenated
hemoglobin
When the heme combing with oxygen the ferrous ion does not
become readily oxidized to the ferric state.