Download Amino Acids Proteins, and Enzymes

1
Proteins perform many different functions in the body.
2
H
+ │
H3N—C—COO−
│
H
glycine
CH3
+
│
H3N—C—COO−
│
H
alanine
3
Amino acids are classified as
• nonpolar (hydrophobic)
with hydrocarbon side
chains.
• polar (hydrophilic) with
polar or ionic side chains.
• acidic (hydrophilic) with
acidic side chains.
• basic (hydrophilic) with
–NH2 side chains.
Nonpolar
Polar
Acidic
Basic
4
Amino acids
• are chiral except glycine.
• have Fischer projections that are stereoisomers.
• that are L are the only amino acids used in proteins.
COOH
COOH
H2N
H
CH3
L-Alanine
H
NH2
CH3
D-Alanine
COOH
H2N
H
CH2SH
L-Cysteine
COOH
H
NH2
CH2SH
D-Cysteine
5
A zwitterion
• has charged −NH3+ and COO– groups.
• forms when both the –NH2 and the –COOH groups in
an amino acid ionize in water.
• has equal + and – charges at the isoelectric point (pI).
O
║
NH2—CH2—C—OH
glycine
O
║
+
H3N—CH2—C—O–
zwitterion of glycine
6
In solutions more basic than the pI,
• the —NH3+ in the amino acid donates a proton.
+
H3N—CH2—COO–
zwitterion
at pI
Charge: 0
OH–
H2N—CH2—COO–
Negative ion
pH > pI
Charge: 1-
7
In solution more acidic than the pI,
• the COO- in the amino acid accepts a proton.
+
H3N—CH2
zwitterion
at pI
Charge: 0
H+
—COO–
+
H3N—CH2—COOH
Positive ion
pH< pI
Charge: 1+
8
H+
+
H3N–CH2–COOH
positive ion
low pH
OH–
+
H3N–CH2–COO–
zwitterion
pI
H2N–CH2–COO–
negative ion
high pH
9
When an electric current is used to separate a mixture
of amino acids
• the positively charged amino acids move towards the
negative electrode.
• the negatively charged amino acids move toward the
positive electrode.
• an amino acid at its pI does not migrate.
• the amino acids are identified as separate bands on
the filter paper or thin layer plate.
10
With an electric current, a mixture of lysine, aspartate,
and valine are separated.
11
CH3
|
+
H3N—CH—COOH
CH3
|
H2N—CH—COO–
(1)
(2)
Which structure represents:
A. Alanine at a pH above its pI?
B. Alanine at a pH below its pI?
12
CH3
|
+
H3N—CH—COOH
CH3
|
H2N—CH—COO–
(1)
(2)
Which structure represents:
A. Alanine at a pH above its pI?
(2)
B. Alanine at a pH below its pI?
(1)
13
14
 Alanine has a methyl
group for the “R”
 Has 3 carbons
(glycine had just 2)
 NOT essential
 NOT essential
 Essential
 Essential
 Valine is a branched
chain amino acid.
 An "ideal protein"
would contain 70% as
much isoleucine as
lysine.
A rare, inherited metabolic disease in which there is a
failure of oxidative decarboxylation (breakdown) of valine,
leucine and isoleucine results in maple syrup urine
disease, named because of a characteristic odor of the
urine.
 Leucine is abundant in corn protein and many
other grain proteins, and is not therefore a
concern to supplement. Only the L isomer has
biological value. "Ideal protein" would contain
100% as much leucine as lysine.
 Isoleucine is a branched chain amino acid. An "ideal
protein" would contain one-half as much isoleucine as
lysine.
 A rare, inherited metabolic disease in which there is a
failure of oxidative decarboxylation (breakdown) of
valine, leucine and isoleucine results in maple syrup
urine disease, named because of a characteristic odor
of the urine.
 Phenylalanine is not tolerated by people with inborn
errors of PHE metabolism so they must avoid it. Babies
are given a PKU (phenylketonuria) test at birth to detect
phenylalanine metabolites in the urine, so they can be
treated in order to avoid a dangerous condition.
 PHE is combined with ASP (aspartic acid, another
amino acid) in the common non-nutritive sweetner
aspertame. Equal is another trade name.
 Methionine is a
sulfur-containing
amino acid.
 A portion of the
methionine
requirement can
be met from
cystine, another
sulfur-containing
amino acid.
 An "ideal protein" would contain one-
half as much methionine + cystine as
lysine.
 Unlike most amino acids, both the d
and l isomers of methionine are
biologically active. Consequently, it has
been synthesized and can be
economically purchased for adding to
diets.
 Methionine participates in acting as a
methyl donor, and is involved in the
synthesis of many important
compounds in the body, including
epinephrine and choline.
Cysteine
Cystine
 Tryptophan is the
second most limiting
amino acid in corn
diets for pigs.
 Tryptophan can be used to from the Vitamin niacin but
since feeds are usually low in tryptophan and the
synthesis is insufficient to meet daily needs, it is not relied
upon to meet the animal's need.
 Useful in aiding sleep, tryptophan supplements have been
banned by FDA as dangerous.
 Skatole and indole can be formed from its breakdown in
the large intestine by bacteria, producing foul odors.
 Arginine is a precursor of urea
in the body. Urea is the form
in which nitrogen is removed
from mammals, so normal
breakdown of protein and
deamination of amino acids
will result in urea formation,
requiring arginine.
 Histidine contains an
imidazole group. It is
a precursor of
histamine. An "ideal
protein" would
contain 33% as much
histidine as lysine.
 Lysine is the most limiting amino acid in corn
and most grains for swine and poultry.
 Diets balanced for lysine will contain most of
the other amino acids in excess of
requirement if natural sources of good quality
proteins are used.
 It can be supplemented in the synthetic form
and this is often economical, replacing a
portion of the protein needed.
 Only the L isomer is biologically active.
 Phenylalanine
 Methionine
 Valine
 Histidine
 Threonine
 Arginine
 Tryptophan
 Lysine
 Isoleucine
 Leucine
1. General Features
Nitrogen Balance & Metabolic Pools
2. Degradation
Transamination & Glutamate Dehydrogenases
3. Urea Cycle
4. Sulfur-containing amino acids
5. Creatine & Creatinine
30
31
32
Formation of Peptides
33
A peptide bond
• is an amide bond.
• forms between the carboxyl group of one amino acid
and the amino group of the next amino acid.
34
35
Write the dipeptide Ser-Thr.
36
Write the dipeptide Ser-Thr.
37
A dipeptide
• is named from the free amine (NH3+) using a -yl
ending for the name.
• names the last amino acid with the free carboxyl
group (COO-) by its amino acid name.
38
Write the three-letter abbreviations and names of
the tripeptides that could form from two glycine
and one alanine.
39
Write the names and three-letter abbreviations of the
tripeptides that could form from two glycine and one
alanine.
Glycylglycylalanine
Gly-Gly-Ala
Glycylalanylglycine
Gly-Ala-Gly
Alanylglycylglycine
Ala-Gly-Gly
40
What are the possible tripeptides formed from one
each of leucine, glycine, and alanine?
41
Tripeptides possible from one each of leucine, glycine,
and alanine:
Leu-Gly-Ala
Leu-Ala-Gly
Ala-Leu-Gly
Ala-Gly-Leu
Gly-Ala-Leu
Gly-Leu-Ala
42
Write the three-letter abbreviation and name for the
following tetrapeptide.
CH3
│
CH3
S
│
│
CH–CH3 SH
CH2
│
│
│
CH3 O H CH O H CH2O H CH2 O
+│ ║ │ │ ║ │ │ ║ │ │ ║
H3N–CH–C–N–CH–C–N–CH–C–N–CH–CO–
43
Ala-Leu-Cys-Met
Alanylleucylcysteinylmethionine
CH3
│
CH3
S
│
│
CH–CH3 SH
CH2
│
│
│
CH3 O H CH O H CH2O H CH2 O
+│ ║ │ │ ║ │ │ ║ │ │ ║
H3N–CH–C–N–CH–C–N–CH–C–N–CH–CO–
Ala
Leu
Cys
Met
44
Qualitative Tests for Amino Acids
• There are a number of qualitative tests to
detect the presence of amino acids
• These are largely dependent on the nature
of R-group.
Exp. 1
Ninhydrin Reaction
• A color reaction given by amino acids and peptides
on heating with the chemical ninhydrin.
• The technique is widely used for the detection and
quantitation (measurement) of amino acids and
peptides.
• Ninhydrin is a powerful oxidizing agent which reacts
with all amino acids between pH 4-8 to produce a
purple colored-compound.
• The reaction is also given by primary amines and
ammonia but without the liberation of Co2
• The amino acids proline and hydroxyproline also
reacts but produce a yellow color.
Exp. 1
Ninhydrin Reaction
■ Method:
• 1 ml AA + 1 ml NH
• heat in boiling WB for 5min.
• Purple color.
Exp. 1
Ninhydrin Reaction
■ Method:
• α-amino acid + 2 ninhydrin --->
CO2 + aldehyde + final complex (purple) + 3H2O
• In summary, ninhydrin, which is originally
yellow, reacts with amino acid and turns deep
purple. It is this purple color that is detected in
this method.
Exp. 2 Xanthoproteic Reaction
• This reaction involves the nitration of benzene
nucleus in alkaline medium. As a result AAs that
contain aromatic nucleus undergo this reaction.
• Aromatic AAs form yellow nitro derivative on
heating with conc. nitric acid, the salts of these
derivatives are orange.
Phenylalanine
Tryptophan
Tyrosine
Exp. 2 Xanthoproteic Reaction
■ Method:
•
•
•
•
1 ml AA + 1 ml conc. HNO3
heat the mixture in WB for 30s
cool
add drop-wise 40% NaOH to
render the solution alkaline
• Yellow to orange color.
Exp. 2 Xanthoproteic Reaction
Tryptophan
Tyrosine
(a)Nitrated tyrosine and tryptophan (b)
Exp. 3 Millon Reaction
• This reaction is used to detect the
presence
of
phenol
(hydroxybenzene) which reacts with
Millon's reagent to form red
complexes.
• The only phenolic AA is tyrosine.
Tyrosine
Exp. 3 Millon Reaction
■ Method:
•
•
•
•
•
1 ml AA + 5 drops of Millon reagent
heat the mixture in BWB for 10min
cool too room temp
add 5 drops of NaNO2
Brick red color.
Exp. 4 Hopkin-cole Reaction
• This reaction is used to detect the
presence of indol group
• The indol group of tryptophan reacts
with glyoxalic acid in the presence
of conc. H2SO4 to give purple color.
Tryptophan
Exp. 4 Hopkin-cole Reaction
■ Method:
• 1 ml AA + 1 ml Hopkin-cole
reagent
• mix well
• Carefully pour conc. H2SO4
down the side of the tube so as
to form two layers
• Purple ring at the interface.
Exp. 5
Sulfur Reaction
• This reaction is specific to detect the
presence of sulfur.
• The sulfur of cystein and cystine is
converted to inorganic sulfide with
conc. NaOH. Lead acetate is added
and a ppt of black lead sulfide
indicates a +ve reaction.
Cystein
Exp. 5
Sulfur Reaction
• 2 ml AA + 1 ml 40% NaOH + 1-3 drops of
lead acetate solution
• heat the mixture in WB for 3min
• Cool
• observe any change
• Black ppt.
Exp. 6
Sakaguchi Reaction
• This reaction is used to detect
the presence of guanidine
group.
• The only AA that contains
guanidine group is arginine
which reacts with α-naphthol
and an oxidizing agent such as
bromide water to give a red
color.
Arginine
Exp. 6
Sakaguchi Reaction
• 2 ml AA + 1 ml 2M NaOH + 1 ml ethanolic
0.02% α-naphthol
• mix well
• cool in ice
• add 1 ml of alkaline hypochlorite solution
• Red color