Download Lecture 4: Amino Acids

Lecture 4: Amino Acids
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–
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For the quiz on Wed. (9/7)-NH3+ ~ 9.0, -COO- ~ 2.0,
you must know pKs of side chain groups!
Introduction to amino acid structure (continued)
Amino acid chemistry
Diastereomers
• Special case: 2 asymmetric centers are chemically identical (2
asymmetric centers are mirror images of one another)
• A molecule that is superimposable on its mirror image is
optically inactive (meso form)
Cahn-Ingold-Prelog or (RS) System
• The 4 groups surrounding a chiral center a ranked as follows:
Atoms of higher atomic number bonded to a chiral center are
ranked above those of lower atomic number.
• Priorities of some common functional groups SH > OH > NH2 >
COOH > CHO > CH2OH > C6H5 > CH3 > 2H > 1H
• Prioritized groups are assigned letters W, X, Y, Z, so that W > X
>Y>Z
• Z group has the lowest priority (usually H) and is used to
establish the chiral center.
• If the order of the groups W X Y is clockwise, as viewed
from the direction of Z, the configuration is (R from the latin
rectus, right)
• If the order of the groups W X Y is counterclockwise, as
viewed from the direction of Z, the configuration is (S from the
latin sinister, left)
Cahn-Ingold-Prelog or (RS) System
Cahn-Ingold-Prelog or (RS) System
Cahn-Ingold-Prelog or (RS) System
Prochiral centers have distinguishable
substituents
• Prochiral molecules can be converted from an achiral to chrial
molecule by a single substitution
• Molecules can be assigned a right side and left side for two
chemically identical substituents.
• True for tetrahedral centered molecules
• Example is ethanol
Prochiral centers
Planar objects can also be prochiral
• Stereospecific additions in enzymatic reactions
• If a trigonal carbon is facing the viewer so that the substituents
decrease in a clockwise manner it is the re face
• If a trigonal carbon is facing the viewer so that the substituents
decrease in a counterclockwise manner it is the si face
• Acetaldehyde example
Nomenclature
• Glx can be Glu or Gln
• Asx can be Asp or Asn
• Polypeptide chains are always described from the N-terminus to
the C-terminus
Nomenclature
• Nonhydrogen atoms of the amino acid side chain are named in
sequence with the Greek alphabet
Peptide bonds
•
Proteins are sometimes called polypeptides since they contain many peptide bonds
R1 O
+
H3N
C
OH
C
+
H
H
R2 O
N
C
O-
H
H
+
H3N
C
R1 O
R2 O
C
N
C
H
H
H
C
C
O-
+ H 2O
Structural character of amide groups
• Understanding the chemical character of the amide is
important since the peptide bond is an amide bond.
• These characteristics are true for the amide containing
amino acids as well (Asn, Gln)
• Amides will not ionize but will undergo resonance
-O
O
R
C
NH2
R
C
Resonance forms
NH2
+
Amide has partial charge & double bond
• We can also look at the partial charge and double bond of an amide
as shown below.
• Since the free electrons of the N atom are tied up in forming the
partial double bond, the N atom can not accept a proton (H+).
• This N also has a partial positive charge which will repel protons
and prevent them from binding to the nitrogen (thus no ionization).


O
R
C

NH2
Amide character in the peptide bond
• Since the peptide bond is also an amide it also undergoes
resonance.
+
H3N
R1 O
R2 O
C
N
C
H
H
H
C
C
O-
• Therefore, peptides are rigid due to resonance around the amide
bond, having ≈ 40% double-bond character.
• This restricts the rotation due to delocalization of electrons and
overlap of the O-C-N  orbitals.
Amide character in the peptide bond
• The double bond character results in a planar form around the
peptide bond.
Structural hierarchy in proteins
• Primary structure (1º structure)-for a protein is the
amino acid sequence of its polypeptide chain(s).
• Secondary structure (2º structure)-the local spatial
arrangement of a polypeptide’s backbone atoms without
regard to the conformations of their side chains.
• Tertiary structure (3º structure)-refers to the 3dimensional structure of an entire polypeptide (close to
secondary structure).
• Quaternary structure (4º structure)-The spatial
arrangement of a protein’s subunits
– Most protein is made up of two or more polypeptide chains
(subunits) associated through noncovalent interactions.
Structural hierarchy in proteins
Primary structure (1º structure) of
proteins
• Primary structure (1º structure)-for a protein is the amino acid
sequence of its polypeptide chain(s).
• Amino acid sequence of a protein determines
– three-dimensional conformation.
– Resulting functional specificity (molecular mechanism of action)
• Sequence comparisons among analogous proteins are important in
comparing how proteins function and have indicated evolutionary
relationships among proteins
• Amino acid sequence analyses have important clinical applications
because many diseases are caused by mutations that lead to an
amino acid change in a protein.
• Therefore, amino acid sequence analysis is an important tool for
research.
General approach for the analysis of the
amino acid sequence of a protein
•
•
•
•
•
•
Purify protein to homogeneity
Break disulfide bonds
Determine the aa composition
Identify the N-terminal sequence
Identify the C-terminal sequence
Break the polypeptide into fragments by internal
cleavage (Trypsin, chymotrypsin, pepsin, CNBr).
• Determine the amino acid sequence of each fragment.
• Repeat using different enzymes or CNBr.
• Overlap and align fragments.
Breaking disulfide bonds
•
•
Recall that cysteine (Cys-SH HS-Cys) can convert to cystine (Cys-S-S-Cys)
in the presence of air (oxidation) and will convert back if reduced.
We can also prevent the formation of the disulfide bond by modifying the
SH group of Cys.
+
H3N
H
H
ox.
-OOC
C
+
H3N
Cysteine
CH2 SH
red. OOC
C
+
H3N
CH2 C
CH2 S-S
Cystine
H
COO-
+
H3N
Cysteine reactions
H
2
HS
CH2 CH2 OH
+
-OOC
-mercaptoethanol
C
+
H3N
CH2 C
CH2 S-S
Cystine
H
2 -OOC C
+
H3N
CH2 SH
Cysteine
+
S-CH2-CH2-OH
S-CH2-CH2-OH
H
COO-
Cysteine reactions
HS
CH2-CH-CH-CH2 SH
OH OH
-OOC
+
H3N
CH2 SH
Cysteine
C
+
H3N
H
2 -OOC C
H
+
Dithiothreitol
Dithioerythritol
Cleland’s reagent
+
+
H3N
CH2 C
CH2 S-S
Cystine
HO
S
HO
S
H
COO-
Cysteine reactions
H
ICH2COOIodoacetate
+ -OOC
C
+
H3N
R-group
CH2 SH
Cysteine
H
-OOC
C
+
H3N
CH2 S CH2COO-
+
Carboxymethylcysteine
HI
General approach for the analysis of the
amino acid sequence of a protein
•
•
•
•
•
•
Purify protein to homogeneity
Break disulfide bonds
Determine the aa composition
Identify the N-terminal sequence
Identify the C-terminal sequence
Break the polypeptide into fragments by internal
cleavage (Trypsin, chymotrypsin, pepsin, CNBr).
• Determine the amino acid sequence of each fragment.
• Repeat using different enzymes or CNBr.
• Overlap and align fragments.
N-terminus identification
• Sanger’s reagent - (fluorodintrobenzene) FDNB
• Dansylation - (1-dimethyl-amino-naphthalene-5-sulfonyl
chloride) Dansyl Chloride
• Edman degradation
– Invented by Pehr Edman
– Phenylisothiocyanate (PITC, Edman’s Reagent)
Sanger’s reagent (fluorodintrobenzene) FDNB
O 2N
F
FDNB
The reaction with FDNB is an aromatic
nucleophillic substitution reaction.
O 2N
..
R1 O
R2 O
O
N
C
C
C
C
H N
+
NO2
H H
HF
base
H H
polypeptide
O
O
R
R
1
2
H
O
N
C
C
C
C
N
NO2
H
H H
Sanger’s reagent will also react with other amino groups (epsilon amino group in-lysine). But only one alpha amino group will be
labeled by this reagent. Aromatic amino groups are more stable than the peptide bond.
Reaction with Dansyl Chloride
H3C
N
O
H3C
S
Cl
+
O
Dansyl Chloride
H3C
R2 O
O
N
C
C
C
C
N
H
H H
H H
HCl
base
H3C
N
..
R1 O
polypeptide
O
H R1 O
S
N C C N C C O-
O
H
R2 O
H H
From this we know the N-terminal amino acid and the amino acid composition but not the sequence.