Download Amino Acids and Proteins: →Protein Functions: enzymes, transport

Amino Acids and Proteins:
→Protein Functions: enzymes, transport (hemoglobin-O2 , tranferrin-Fe), protection (MHC
molecules, immunoglobulins), hormones (insulin, glucagons), gene transcription regulation
(histones, DNA binding proteins), structural (collagen, elastin).
Proteins are polymers of amino acids. Protein-over 50 amino acids, peptide-under 50 amino
acids.
→There are 20 common amino acids-coded for by the DNA
Common Amino Acids:
→All have the same general structure:
H
All contain a central a carbon, with a carboxylic acid group
¦
an amino group, and a H atom covalently bonded to the
+
NH3 - C- COO
carbon. The R is a specific chemical group that differs for
¦
each of the 20 common amino acids. The side chain R
R
defines the structural and chemical nature of the amino
acid.
I.
Nonpolar Amino Acids
1) alkyl groups:
Alanine
Ala
A
Valine
Val
V
Leucine
Leu
L
Isoleucine
Ile
I
2) Heterocyclic
Proline
Pro
P
3) Aromatics
Phenylalanine Phe
F
Tryptophan
W
Trp
4) Sulfur containing (thioether)
Methionine Met M
II.
Uncharged Polar Groups-all except G can form hydrogen bonds with water
1) Glycine
Gly
G
2) Hydroxy (alcohol)
Serine
Ser
S
Threonine
III.
Thr
T
3) Sulfhydryl
Cysteine
Cys
C
4) Aromatic
Tyrosine
Tyr
Y
5) Carboxamide
Asparagine Asp
N
Glutamine
Q
Gln
Charged Polar Groups
1) Carboxylic Acids (acids)
Aspartic acid Asp D
Glutamic acid Glu
E
2) Bases
Lysine
Lys
K
Arginine
Arg
R
Histidine
His
H
Derived Amino Acids
The amino acid is post-translationally modified in an enzyme- facilitated reaction. Can
have hydroxylation (hydroxyproline, hydroxylysine), methylation, acetylation, phosphorylation
(only on hydroxyl groups) (see Figure 4.4)
Example:
Cystine- formed by the oxidation of two Cys thiol side chains, joined to form a disulfide covalent
bond (important in protein folding).
R- CH3 - S- S- CH3 - R
Properties of Amino Acids
1) Asymmetry:
The a-carbon of all amino acids (except Gly) is chiral or asymmetric forming
stereoisomers.
COOCOO¦
¦
+
NH3 - C- H
H- C- NH3 +
¦
¦
CH2 OH
CH2 OH
L-serine
D-Serine
In nature, find the L-amino acids.
Usually only one enantiomer is biologically active-because enzymes are so specific.
2) Ultraviolet absorption:
Aromatics absorb light in the near UV (250-300 nm) because of the interaction of
radiation with the electrons in the aromatic ring. Can use UV absorption (spectrophotometer) as
a measure of protein concentration.
All peptides absorb in the far UV (214 nm) because of the amide bond. Can use this fact
to detect all peptides.
3) Amino Acid derivatives:
Many amino acids are the precursors of biologically active molecules (see Figure 4.4).
His → Histamine (decarboxylated histadine)
Tyr → Epinephrine
Trp → Seratonin
4) Reactions of amino acids:
The side chains exhibit specific chemical reactivities, depending on the nature of the
functional group (see figure 4.8). It is the characteristic behavior of the side chain that governs
the reactivity of amino acids incorporated into proteins.
Polymerization of Amino Acids into Peptides and Proteins
Polymerization in cells is catalyzed by enzymes associated with the ribosomes. It is
simply a dehydration reaction (see figure 4.15). The lone pairs on the nitrogen of one amino acid
perform a nucleophilic attack on the carbonyl of the other amino acid resulting in a loss of H2 O.
→Primary structure of protein-amino acid sequence which is predetermined by the DNA
sequence of its gene.. The primary sequence enables the polypeptide chain to fold into a specific
3-D structure (based on intramolecular forces) which gives a protein its chemical and
physiological properties.
→The peptide bond has 2 resonance structures, creating a true structure wit h a partial double
bond (no rotation):
→Can have cis or trans form, but trans is the most stable conformation, so find trans peptide
bonds in proteins.
Exception: Pro-because the NH group is attached in a ring structure, cis and trans have
about equal energy. Therefore, whether it is cis or trans at the Pro position depends on the
overall protein 3-D structure.
→Write primary structures sequentially from the N-terminus to the C-terminus consistent with
the order of amino acids.
The sequence of this peptide is written as: GFKN or Gly-Phe-Lys-Asn
Peptide Synthesis in Lab
Peptides are chemically synthesized in the lab by linking one amino acid at a time.
Why?
1) To investigate properties of peptides by varying side chains.
Example: Substance P (neurotransmitter) RPKPQFFGLM Can determine which amino
acids are critical to function by making peptides with altered amino acids.
2) Obtain peptide with unique properties like nonstandard side chains, isotopic labels, fluorescent
labels, etc.
3) Manufacture pharmacologically active peptides that are biologically scarce or nonexistent.
4) Production of synthetic vaccines.
Vaccines- virus that has been killed or attenuated to stimulate immune response system to
produce antibodies. These can cause disease and it is difficult to culture viruses.
Preparing vaccines with synthetic peptides of viral antigenic determinants would avoid
these problems.
→Aspartame is a dipeptide: Asp-Phe-OMe.
Would get 6 products for this reaction. The goal is to achieve regioselectivity without
racemization (only L amino acids). So must use protecting groups to only allow the desired
reaction.
Protect reactive side chains:
-OH
Y, S, T
t-butyl group
-COOH
D, E
t-butyl group
-SH
C
trityl group
R, K
BOC
Charge and Chemical Properties:
→At physiological pH:
basic (R, K)
+ charged
acidic (D, E)
- charged
pH= pKa +log B/A The pKa depends on environment, so usually given a pKa range.
In a protein: ΣLys+ΣArg
>1 basic
ΣAsp+ΣGlu
<1 acidic
Can calculate the ionic form of a molecule at a given pH by:
pH=pKa+logB/A
A molecule will have different physiological activities at different pH values.
Example His An enzyme requires the His to be in the base form to be active
(activity= B/(B+A) What is the activity of this enzyme at pH=6.0? (the pKa of the His side chain
is 6.0)
6.0=6.0+logB/A
so B/A=1 so B/(B+A) is ½ or 50%
What is the activity at pH=8.0?
8.0=6.0+logB/A so B/A is 100/1 so B/(B+A) is 100/101 or 99%
At what pH is the activity of the enzyme 75%? So B is 75 and A is 25
X=6.0+log75/25 X is 6.5
Titrations of Amino Acids:
→ All of the amino acids contain at least two dissociable hydrogens. At low pH, both the
carboxyl and the amino groups are protonated and the molecule has a net positive charge. As the
pH increases, the carboxyl group dissociates (pKa 2.3), yielding a neutral zwitterionic species.
As the pH further increases, the amino group dissociates )pKa 9.6) to yield a negatively charged
molecule (see figures 4.5 and 4.6). The isoelectric point is the pH at which the amino acid is
neutral.
pI= average of the 2 pKs values that control the boundaries of the Zwitterion
pI= (2.3+9.6)/2= 5.95
→ Some amino acids also contain ionizable side chains (R, K, H, D, E, C, and Y).
So, Glu has 3 ionizable groups:
See Figure 4.7 for the titration curve of Glu.
pI= (4.3+2.2)/2= 3.3
→Note: At low pH values, amino acids, peptides, and proteins will have a net + charge. At high
pH values, amino acids, peptides, and proteins will have a net – charge. The magnitude of the
charge depends on the difference between pH and pI.
→ Calculating the pI for proteins based on pKa would be difficult, so experimentally measure
the pI by determining the pH at which the protein is neutral and will no linger move in an electric
field.
Calculating the Charge of a Peptide:
A peptide contains a chargeable C-terminal carboxy group, a chargeable N-terminal
amino group, and the chargeable side chains of certain amino acids (R,K,H,D,E,C,Y). To
determine the charge of a peptide at a given pH, one must determine the charge of each of these
groups based on their pKa values.
R,K,H,N-terminus is either + charged at pH<pKa or neutral at pH>pKa
D,E,C,Y,C-terminus is either neutral at pH<pKa or – charged at pH>pKa
Example: What is the charge of the peptide RSTDY at pH=3?
At pH=8.0?