Download essential amino acids

Amino Acids and Proteins
© E.V. Blackburn, 2008
Structure of amino acids
The hydrolysis of most proteins produces about
twenty different amino acids.
The acids have an amino group bonded to the  carbon:H O
R C C
NH 2OH
an -amino acid
© E.V. Blackburn, 2008
Amino acid classification
The 20 amino acids are further classified:
• neutral - one amino and one carboxyl group
• acidic - one amino and two carboxyl groups
• basic - two amino and one carboxyl group
© E.V. Blackburn, 2008
Neutral amino acids
Name
Alanine
Asparagine
Cysteine
Glutamine
Glycine
Isoleucine
Leucine
Methionine
Phenylalanine
Proline
Symbol
Structure
Ala or A CH3CH(NH2)CO2H
Asn or N H2NCOCH2CH(NH2)CO2H
Cys or C HSCH2CH(NH2)CO2H
Gln or Q H2NCOCH2CH2CH(NH2)CO2H
Gly or G CH2(NH2)CO2H
Ile or I CH3CH2CH(CH3)CH(NH2)CO2H
Leu or L (CH3)2CHCH2CH(NH2)CO2H
Met or M CH3SCH2CH2CH(NH2)CO2H
Phe or F C6H5CH2CH(NH2)CO2H
Pro or P
N CO2H
H
© E.V. Blackburn, 2008
Neutral amino acids
Name
Symbol
Serine
Threonine
Ser or S
Thr or T
Tryptophane
Try or W
Structure
HOCH2CH(NH2)CO2H
HOCH(CH3)CH(NH2)CO2H
CH 2CH(NH 2)CO 2H
N
H
Tyrosine
Tyr or Y HO
Valine
Val or V
CH 2CH(NH 2)CO 2H
(CH3)2CHCH(NH2)CO2H
© E.V. Blackburn, 2008
Acidic amino acid
Name
Symbol
Structure
Aspartic acid
Asp or D HO2CCH2CH(NH2)CO2H
Glutamic acid
Glu or E
HO2CCH2CH2CH(NH2)CO2H
© E.V. Blackburn, 2008
Basic amino acids
Name
Symbol
Arginine
Arg or R
Histidine
His or H
Structure
HN=C-H(CH2)3CH(NH2)CO2H
NH2
CH 2CH(NH 2)CO 2H
N
Lysine
Lys or K
NH
H2N(CH2)4CH(NH2)CO2H
© E.V. Blackburn, 2008
Essential amino acids
Humans can synthesize only 10 of these amino acids. The
remaining amino acids must be obtained from dietary
sources and are so called essential amino acids.
These are: Isoleucine, Leucine, Methionine,
Phenylalanine, Threonine, Tryptophan, Valine, Arginine,
Histidine, and Lysine.
© E.V. Blackburn, 2008
Stereochemistry of -Amino
Acids
Most naturally occurring amino acids have the same
configuration about the  carbon:
CHO
HO
H
CH 2OH
CO 2H
H 2N
H
CH 2OH
L-(-)-glyceraldehyde
L-(-)-serine
CO 2H
H 2N
H
R
natural amino acids
© E.V. Blackburn, 2008
Acid-base Properties of Amino
Acids
RCHCO2H
RCHCO2-
H+
NH2
-amino acid
NH3
+
zwitterion
OH-
H
+
RCHCO2H
NH3
+
OH-
RCHCO2NH2
© E.V. Blackburn, 2008
Physical Properties
• high melting points with decomposition
• very water soluble
• insoluble in non-polar organic solvents such as
ether
© E.V. Blackburn, 2008
Isoelectric Points
- the pH of a dilute aqueous solution of the amino acid at
which the amino acid is exactly neutral.
The isoelectric point of neutral amino acids lies between
pH = 4.8 and 6.3. For the basic amino acids, it is in the
range 7.8 to 10.8. The range is 2.7 to 3.2 for acidic
amino acids.
© E.V. Blackburn, 2008
Isoelectric Points
RCHCO2H
NH2
-amino acid
-
RCHCO2
NH3
+
zwitterion
H+
H+
OH-
RCHCO 2H
NH 3
+
OH-
RCHCO2NH2
At a pH above the isoelectric point, amino acids form anions;
at a lower pH, protonation occurs and cations form. An amino
acid is the least soluble at its isoelectric point.
© E.V. Blackburn, 2008
Electrophoresis
Electrophoresis uses isoelectric point differences to separate
amino acids and proteins.
• The amino acid is spotted on a strip of paper (or gel) on a
center line.
• The strip is moistened with a buffer solution of a given pH.
• Electrodes are attached to the ends of the slip and a
potential applied.
• Depending on the amino acid’s charge, it will migrate either
to the positive or negative electrode. The rate and direction
of migration depends on the buffer pH and the isoelectric
point of the amino acid.
© E.V. Blackburn, 2008
Synthesis of amino acids - the
Hell-Volhard-Zelinsky reaction
1. Br2/PBr3
CH3CH2CO2H
2. H2O
CH3CHBrCO2H
excess
NH3
CH3CHCO2H
NH2
alanine
© E.V. Blackburn, 2008
Synthesis of amino acids - the
Strecker synthesis
O NH4Cl/KCN
R CH
H2O
NH3 adds first to give an
imine then CN- attacks
RHC C N
NH2
H3O+
RCHCO2H
NH2
© E.V. Blackburn, 2008
The Peptide Bond
R O
R O
R
C C N C C N C
H
H H
H H
The principal chain contains the amide bonds; the
substituents, R, make up the side chains. The
individual amino acids which make up the peptide
are often referred to as amino acid residues.
In some proteins, two or more polypeptide chains
are joined together by disulfide bridges.
© E.V. Blackburn, 2008
Nomenclature
Starting from the free amine end of the peptide, the
names of the amino acids are joined together with each
amino acid being considered as a substituent of the
following amino acid. The name ends with the name of
the final amino acid:
(CH 3)2CH
CH 3
C6H5CH 2 O
CH 2 O HCOH
H3NCH C NHCH C NHCHCO 2+
phenylalanylleucylthreonine
Phe-Leu-Thr
© E.V. Blackburn, 2008
Aspartame
• an artificial sweetener sold under the trade name
Nutrasweet
O
O
+
H3N-CH-C-NH-CH-C-OCH 3
CH2-C6H5
CH2
-
CO2
aspartylphenylalanine methyl ester
Asp-Phe-OCH3
• the ester ending is indicated by -OCH3 in the abridged
notation.
© E.V. Blackburn, 2008
Angiotensin II
Angiotensin II is a blood pressure regulating hormone. It
contains 8 amino acid residues. It is possible to arrange
these in 40,320 different ways only one of which
corresponds to the hormone!
Its structure is actually: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe.
© E.V. Blackburn, 2008
Sequential Degradation of
Peptides
Step 1: breaking disulfide linkages and purification of products
Many polypeptides consist of two or more chains which are
joined together by disulfide bridges. These bridges must be
broken and the fragment products separated. This is done by
an oxidation:
O
S S
chain A
HCOOH
chain B
SO3H
chain A
HO3S
chain B
© E.V. Blackburn, 2008
Purification
The various methods used to separate polypeptides
depend on size, solubility in a given solvent, charge or
ability to become bonded to a support material
(chromatography).
• dialysis - filtration through a semi-permeable
membrane
• ion exchange chromatography
• electrophoresis
• chromatography
© E.V. Blackburn, 2008
Step 2. What amino acids are
present?
The polypeptide is completely hydrolyzed (HCl 6M, 110C,
24hr) to give a mixture of the free amino acids:
O
O
H 2O
+
H3N-CH-C-NH-CH-C-OH
HCl 6M
CH3
CH(CH3)2
110o, 22hr
O
O
Ala-Val
+
+
H3N-CH-C-OH + H3N-CH-C-OH
CH 3
Ala
CH(CH 3)2
Val
© E.V. Blackburn, 2008
Amino Acid Analysis
The mixture is separated using an automated system.
The apparatus includes an ion exchange column with a
negatively charged support (usually carboxylate or
sulfonate). The amino acid mixture, in weakly acidic
solution, is added to the column.
According to structure, the amino acids are more or less
protonated and so will be retained more or less strongly on
the column. The pH of the eluant is gradually increased
which results in deprotonation and the subsequent elution of
the amino acids.
Elution begins with the strongest acid and ends with the
weakest.
© E.V. Blackburn, 2008
Amino Acid Analysis
The eluant then passes into an analyzer which holds a
special indicator, ninhydrin. The amino acid eluant
forms a violet-purple color by reaction with the ninhydrin
and the color’s intensity is proportional to the quantity of
acid present.
Asp
Ser Glu
Thr
Gly Ala
Pro
pH
© E.V. Blackburn, 2008
Step 3. The sequential analysis of
amino acids starting at the free
amino end
There are a number of methods used to determine the
N-terminal amino acid of a peptide. They are all based
on the fact that it is the only amino acid with a free amino
group.
© E.V. Blackburn, 2008
Sanger Degradation
F
NO2
H2NCH2CONH
NO2
NHCH2CONH
NO2
NO2
© E.V. Blackburn, 2008
Edman Degradation
Phenyl isothiocyanate, C6H5N=C=S, is a reagent which
permits the progressive removal and identification of the
N-terminal amino acid. The rest of the chain is not
affected by the reaction therefore progressive removal is
possible.
© E.V. Blackburn, 2008
Edman Degradation
O
Ph-N=C=S + H2N-CH-C-NH-Pep
R
S
O
Ph-NH-C- HN-CH-C-NH-Pep
R
O
Ph-NH
H
N R
a thiazolinone
S
pyridine
H2O/Me 2NPh
CF 3CO 2H
O
H2O/H+
+
Ph N
+ H3N-Pep
CH-R
NH
S
phenylthiohydantoin
derivative
© E.V. Blackburn, 2008
Specific Cleavage of Peptides
A number of enzymes catalyze the hydrolysis of
peptide bonds at specific points in an amino acid
sequence. These enzymes are called proteases,
peptidases or proteolytic enzymes.
Trypsin is an example. It catalyzes the hydrolysis of
polypeptides at the acyl group of arginine or lysine
residues.
Chymotrypsin cleaves at the acyl side of the aryl
substituted acids phenylalanine, tyrosine and tryptophan.
© E.V. Blackburn, 2008
C-terminal residue
The enzyme carboxypeptidase cleaves the C-terminal
amide bond in a peptide. The peptide is incubated with
the enzyme until the first amino acid is detected.
© E.V. Blackburn, 2008
A peptide!
Treatment of a peptide with 2,4-dinitrofluorobenzene followed
by hydrolysis gives N-dinitrophenylvaline. Leucine is the free
acid.
The peptide contains one of each of the following amino acid
residues Leu, Ser, Phe, Pro, Tyr, Lys, Gly and Val.
Partial hydrolysis gives four peptides which contain the
indicated amino acids:
peptide A:- Leu, Ser, Phe peptide B:- Ser, Pro, Tyr, Lys
peptide C:- Tyr, Lys, Gly
peptide D:- Lys, Val, Gly
© E.V. Blackburn, 2008