Download amino acids

Reginald H. Garrett
Charles M. Grisham
Chapter 4
Amino Acids
Chapter 4
All objects have mirror
images, and amino
acids exist in mirrorimage forms.
Only the L-isomers of
amino acids occur
commonly in nature.
氨基酸在自然界只有L
異構 型存在
Three Sisters Wilderness, Oregon
Essential Question
• Why are amino acids uniquely suited to their role
as the building blocks of proteins?
• 唯獨 氨基酸 適合擔任構築成 蛋白質的磚頭 (積木)
Ch4 氨基酸之種類結構、性質、分開 分析，組成
蛋白質一級結構
• What are the structures and properties of amino
acids?
• acid-base properties
• reactions amino acids undergo
• optical and stereochemical properties
• spectroscopic properties
• How are amino acid mixtures separated and
analyzed?
• What is the fundamental structural pattern in
proteins?
4.1 Structures and Properties of Amino
Acids
• Amino acids contain a central tetrahedral
carbon atom
• There are 20 common amino acids
• Amino acids can join via peptide bonds
• Several amino acids occur only rarely in
proteins
• Some amino acids are not found in
proteins
4.1 Structures and Properties of Amino Acids
Zwitterions (COO- and NH3+ at neutral pH)
Chiral (asymmetric carbon, tetrahedran)
Head-to-tail ( N’ to C’) react to form peptide
Anatomy of an amino acid
Cα is chiral carbon (asymmetric C, 不對稱碳可接四種基團)
Amino group attached to -carbon (C next to carboxyl group)
- amino acid
Figure 4.1 Except for proline and its derivatives, all of the amino acids
commonly found in proteins possess this type of tetrahedral structure (四
面體).
Peptide Bond Formation
Figure 4.2 Two
amino acids can
react with loss of
a water molecule
to form a covalent
bond, called a
peptide bond
C端和N端接合，
去1分子水
•A di-peptide is formed
from 2 amino acids, A
tripeptide is formed from 3
amino acids…. ;
•“proteins” are
polypeptides”
•A monomeric protein
contains one polypeptide
You should know
names
structures
pKa values
3-letter codes
1-letter codes
The 20 Common Amino Acids
依照獨特側鏈烷基分類
•
•
•
•
Non-polar (非極性)
Polar, uncharged (極性)
Acidic (酸性)
Basic (鹼性)
其他分類方法
•
•
•
•
•
•
Hydrophobic (疏水性)
Hydrophilic (親水性)
Amphipathic (兩性)
Cyclic (環化)
Aromatic (含苯環)
Sulfur-Containing (含硫)
(a) Non-polar (hydrophobic) amino acids-1
白氨酸
丙氨酸
Cyclic
α-Imino acid
脯氨酸
纈草胺酸
Nonpolar a.a. drive protein chains to “fold” (折疊) that is to
form their native functional structure) Aliphatic side chains
(a) Non-polar (hydrophobic) amino acids-2
甲硫氨酸
色氨酸
苯丙氨酸
異白氨酸
Aliphatic side chains
Non-polar (hydrophobic) amino acids (8) -- Ala, Val, Leu, Ile, Met,
Trp (W), Phe (F), Pro
(b) Polar, uncharged amino acids-1
甘氨酸
絲氨酸
天門冬醯氨
麩醯氨
(1) Form H.B. with water (amide, hydroxyl, sulfhydryl group)
(2) Nucleophilic role in enzyme reaction
(3) Glycine has the simplest structure and is not chiral
(b) Polar, uncharged amino acids-2
蘇氨酸
半胱氨酸
Phenolic OH pKa=10.1
general uncharged at ~pH7
酪氨酸
Polar, uncharged amino acids (7) -- Gly, Ser, Thr, Cys, Asn (N), Gln (Q), Tyr (Y)
Figure 4.3
(c) The acidic amino (2) Asp (D), Glu (E)
天門冬氨酸
β- COOH
“-” charge at pH 7
Found in metal binding site
Form aspartate and glutamate
麩氨酸
γ- COOH
The basic amino acids(3) – Lys (K), Arg(R), His
Protonated alkyl amino 離氨酸
guanidino
精氨酸
His pKa 6.0, 10% protonated at pH 7.
His side chains role as proton donor
and acceptor in enzyme reaction.
His-containing peptides are
biological buffers.
Arg, Lys side chains involved in
electrostatic interactions.
imidazole
組氨酸
Others ways to classify amino acids
• Hydrophobic (疏水性)
• Gly, Ala, Val, Leu, Ile, Phe, Pro
• Hydrophilic (親水性)
• Ser, Thr, Cys, Asp, Asn, Glu, Gln, Arg,
His
• Amphipathic (兩性)
• Met, Lys, Tyr, Trp
• Trp is sometimes considered as a borderline member
of the group because it also can interact with H2O via
the N-H moiety of the indole ring
Others ways to classify amino acids
• Aromatic amino acids (含苯環)
• Tyr, Trp, Phe
• Cyclic Amino Acid (1 環形 as an imino acid)
• His
• Sulfur-containing (含硫)
• Met, Cys
• Thioester S in Met can be an effective metal ligand
• Thiolated anion in Cys is the most potent nucleophiles
Several Amino Acids Occur Rarely in Proteins
蛋白質內的稀有氨基酸
We'll see some of these in later chapters
•
•
•
•
•
•
Selenocysteine in many organisms
Pyrrolysine in several archaeal species
Hydroxylysine, hydroxyproline - collagen
Carboxyglutamate - blood-clotting proteins
Pyroglutamate – in bacteriorhodopsin
GABA (γ-aminobutyric acid, decarboyxlation of glutamic acid),
epinephrine, histamine, serotonin act as
neurotransmitters and hormones
• Phosphorylated amino acids – a signaling
device
稀有氨基酸在結締組織, 血栓蛋白, 古細菌細胞膜
Figure 4.4
Selenocysteine (Sec) in lower eukaryotes
Hydroxylysine and hydroxyproline found in
connective-tissue proteins
carboxy-glutamate found in blood-clotting
proteins
pyroglutamate found in bacteriorhodopsin
稀有氨基酸 是神經傳導物與賀爾蒙
Tryptophan
Glutamic acid
Histidine
Tyrosine
Figure 4.4 (c) Several amino acids that act as
neurotransmitters and hormones.
Reginald H. Garrett
Charles M. Grisham
4.2 What Are Acid-Base
Properties of Amino Acids?
Acid-Base Properties of Amino Acids-1
Figure 4.5 The ionic forms of the amino acids, shown
without consideration of any ionizations on the side chain.
Acid-Base Properties of Amino Acids-2
• Amino Acids are Weak Polyprotic Acids
• The degree of dissociation depends on the pH
of the medium
• H2A+ + H2O  HA0 + H3O+
[ HA0 ][ H 3 0 ]
K a1 

[H2 A ]
Acid-Base Properties of Amino Acids-3
The second dissociation (the amino group in the case
of glycine):
• HA0 + H2O  A¯ + H3O+

Ka 2

[ A ][ H 3O ]

0
[ HA ]
pKa Values of the Amino Acids
• These numbers are approximate, but entirely suitable for our
purposes
K1 for common amino acids are 0.4 – 1.0 x 10 -2
α-COOH range pKa 2.0-2.4,
α-NH3 pKa 9.0-9.8
.
Acid-Base Properties of Amino Acids-4
pI
(isoelectric point)
In a protein,
if acidic groups
predominate, the pI will be
low. –acidic protein.
If basic groups
predominate, the pI will be
high. –basic protein
Acid-Base Properties of Amino Acids-5
R-grop
* Glutamic acid (acidic, pI~3)
γ- COOH pK3 9.7
* Lysine (basic, pI ~10)
ε-NH3+ pK3 10.5
pKa Values of the Amino Acids
You should know these numbers and know what
they mean
• Arginine, Arg, R: pKa(guanidino group) = 12.5
• Cysteine, Cys, C: pKa = 8.3
• Aspartic Acid, Asp, D: pKa = 3.9
• Glutamic Acid, Glu, E: pKa = 4.3
• Histidine, His, H: pKa = 6.0
Cysteine, Cys, C: pKa = 8.3
Form intra-chain (分子內)
and inter-chain (分子間)
disulfide bonds. Play an
important structural role
R- group pKa Values of the Amino Acids
You should know these numbers and know
what they mean
• Lysine, Lys, K: pKa = 10.5
• Serine, Ser, S: pKa = 13
• Threonine, Thr, T: pKa = 13
• Tyrosine, Tyr, Y: pKa = 10.1
Titrations of polyprotic amino acids
* Glutamic acid (acidic, pI~3)
* Lysine (basic, pI ~10)
Titration of glutamic acid and lysine
Ch2 Henderson-Hasselbalch
equation:
• Describe the dissociation of some weak acid, HA,
occurring with an acid dissociation constant, Ka.
HA --> H+ + AKa = [H+][ A-]/[ HA]
[H+] = Ka [ HA]/ [ A-]
log [H+] = log Ka + log [ HA]/ [ A-]
pH = -log [H+]
• define pKa=-log Ka
pH = pKa + log[A-]/[ HA]
A Sample Calculation
What is the pH of a glutamic acid solution
if the alpha carboxyl is 1/4 dissociated?
[1]
pH  2  log10
[3]
•pH = 2 + (-0.477)
•pH = 1.523
•Note that, when the group is ¼ dissociated, 1/4 is
dissociated and ¾ are not; thus the ratio in the log
term is ¼ over ¾ or 1/3.
Another Sample Calculation
What is the pH of a lysine solution if the
side chain amino group is 3/4
dissociated?
[3]
pH  10.5  log10
[1]
• pH = 10.5 + (0.477)
• pH = 10.977 = 11.0
• Note that, when the group is ¾ dissociated, ¾ is
dissociated and ¼ is not; thus the ratio in the log
term is ¾ over ¼ or 3/1.
Reactions of Amino Acids
Reactions of Amino Acids
• (1) Carboxyl groups form amides & esters
• (2) Amino groups form Schiff bases and amides
• (3) Edman reagent (phenylisothiocyanate)
reacts with the α-amino group (N-terminal) of an
amino acid or peptide to produce a
phenylthiohydantoin (PTH) derivative.
• (4) Side chains show unique reactivities
• Cys residues can form disulfides (-SS-) and
can be easily alkylated (-S-R’)
• Few reactions are specific to a single kind of
side chain
Reactive α-amino group and Edman reagent form
PTH-amino acid
phenylisothiocyanate
phenylthiohydantoin
Figure 4.8 (a) Edman’s reagent reacts with the N-terminal
amino acid of a peptide or protein to form a cyclic thiazoline
derivative that reacts in weak aqueous acid to form a
PTH-amino acid.
Cysteine –SH residues in proteins react with
each other to form disulfides (-S-S-)
Figure 4.8 (b).
e.g. Two cysteines form a cystine
Reactive amino groups and chromophore in
Green Fluorescent Protein (GFT 綠螢光蛋白)
A jellyfish (Aequorea victoria) native to
the northwest Pacific Ocean contains a
green fluorescent protein. GFP is a
naturally fluorescent protein.
Genetic engineering techniques can be
used to “tag” virtually any protein,
structure, or organelle in a cell. The
GFP chromophore lies in the center of
a β-barrel protein structure.
The prosthetic group (helper molecule,
輔基 )of GFP is an oxidative product of
the sequence –FSYGVQ-.
serine-tyrosine-glycine form pigment
complex
–FSYGVQ- chromophore form complex
autocatalytically produce Green Fluorescent
(自催化)
The prosthetic group (helper molecule, 輔基 )of GFP is an
oxidative product of the sequence –FSYGVQ-.
Genetically engineered GFP into yellow fluorescent
protein (YFP, 黃螢光蛋白)
Amino acid substitutions
in GFP can tune the color
of emitted light. Shown
here is an image of
African green monkey
kidney cells expressing
yellow fluorescent protein
(YFP) fused to α-tubulin,
a cytoskeletal protein.
取代 FSYGVQ
Green  yellow
4.4 Stereochemistry of Amino Acids
• All but glycine are chiral
• L-amino acids predominate in nature
• D,L-nomenclature is based on D- and Lglyceraldehyde
• R,S-nomenclature system is superior,
since amino acids like isoleucine and
threonine (with two chiral centers) can be
named unambiguously
Stereochemistry of Amino Acids
4.5 Spectroscopic Properties
• All amino acids absorb at infrared wavelengths
• Only Phe, Tyr, and Trp absorb UV. Absorbance
at 280 nm is a good diagnostic device for amino
acids
• 非破壞性測量蛋白質濃度方法之一
• NMR spectra are characteristic of each residue in
a protein, and high resolution NMR
measurements can be used to elucidate threedimensional structures of proteins
Spectroscopic Properties-aromatic a. a.
Figure 4.10
Under pH 6
Trp absorbs hundreds
of times more strongly
than Phe. (取log值)
Amino acids absorb
only weakly in the UV.
Commonly measure at
UV210 ,UV230, UV280
Proton NMR spectra of several amino acids
Figure 4.11 Spectroscopic Properties (from Aldrich library of NMR spectra)
Zero on the chemical shift scale is defined by the resonance of tetramethylsilane (TMS ).
Because highly sensitive to teir environment, the chemical shift of individual NMR signals can
detect the pH-dependent ionization of amino acids.
A plot of chemical shifts versus pH for
the carbons of lysine (13C NMR)
Figure 4.12.
Spectroscopic
Properties
Changes in chemical
shift are most
pronounced for atoms
near the titration groups.
Lysine (basic, pI ~10)
α-COOH pK1 2.2
α- NH3+ pK2 9.0
ε-NH3+ pK3 10.5
Such measurements have been very useful for
studies of the ionization behavior of amino acids
in proteins.
4.6 Separation of Amino Acids
• ‘chromatography’
• Method based on partition and separation properties of
proteins in solid phase and mobile phase
• Many chromatographic methods exist for separation of amino
acid mixtures
• Ion exchange chromatography (IEC)
• Electrical charge (Anionic, Cationic)
• Affinity chromatography
• Hydrophobic Interaction chromatography
• High-performance liquid chromatography (HPLC)
Separation of Amino Acids
Figure 4.13 Gradient
separation of
common PTH-amino
acids
4.7 What is the Fundamental Structural
Pattern in Proteins?
• Proteins are unbranched polymers of amino acids
• Amino acids join head-to-tail through formation of
covalent peptide bonds
• Peptide bond formation results in release of water
• The peptide backbone of a protein consists of the
repeated sequence –N-Cα-Co• “N” is the amide nitrogen of the amino acid
• “Cα” is the alpha-C of the amino acid
(接側鏈side chain, R-group) (R1, R2, R3即reside 1, 2, 3)
• “Co” is the carbonyl carbon of the amino acid
4.7 What is the Fundamental Structural
Pattern in Proteins?
(N端)––N-Cα-Co-N-Cα-Co- (C端)
Figure 4.14 Peptide formation is the creation of an amide
bond between the carboxyl group of one amino acid and
the amino group of another amino acid.
The Peptide Bond
• Is usually found in the trans conformation
• Has partial (40%) double bond character
• Is about 0.133 nm long - shorter than a typical
single bond (N-Cα 0.145 nm)but longer than a double bond (
C=O 0.123 nm)
• Due to the double bond character, the six atoms of the
peptide bond group are always planar
• N partially positive; O partially negative
The trans conformation of the peptide bond
Figure 4.15
Cα-C0
Co-N
N-Cα
Peptide backbone is a repeating –N-Cα-Co- sequence,
such as (N端)––N-Cα-Co-N-Cα-Co- (C端)
Length of a peptide bond -Co-N- 0.133 nm
The peptide bond has partial of 40% double
bond character C=O
Figure 4.16 (a) One of the postulated resonance forms is
shown here.
The peptide bond has partial of 40%
double bond character C=N
Figure 4.16 (b) The peptide bond has partial double
bond character. One of the postulated resonance
forms is shown here.
The peptide bond is a resonance hybrid of
C=O and C=N
Figure 4.16 (c) The peptide bond is best described as a
resonance hybrid of the forms shown on the two previous
slides.
The coplanar relationship of the 6 atoms
in the amide group – Amide plane
The coplanar relationship of the atoms in the amide group
is highlighted here by an imaginary shaded plane lying
between adjacent α-carbons.
“Peptides”根據含多少氨基酸殘基 分類
Terminology (如何稱呼?)
• Short polymers of amino acids
• Each unit is called a residue
• 為何稱為”殘基 “
• What is left after the release of H2O when an amino acid
forms a peptide link upon joining the peptide chain
• 2 residues - dipeptide
• 3 residues – tripeptide
• 12-20 residues - oligopeptide
• many - polypeptide
“Protein” contains one or more polypeptide
chains
Terminology (如何稱呼?)
•
•
•
•
One polypeptide chain - a monomeric protein
More than one - multimeric protein
Homomultimer - one kind of chain
Heteromultimer - two or more different chains
•
•
Hemoglobin, for example, is a heterotetramer
It has two alpha chains and two beta chains
α2β2
Proteins - Large and Small
一般含100-2000 amino acid residues
• Insulin (αβ) - A chain of 21 residues, B chain of 30
residues -total mol. wt. of 5,733
• Glutamine synthetase (α12) –
• 12 subunits of 468 residues each
• 12 x 468 = 7488 a.a.
• - total mol. wt. of 600,000 ( 600 kDa)
• Connectin proteins - alpha - MW 2.8 million (
• beta connectin - MW of 2.1 million, with a length of 1000
nm -it can stretch to 3000 nm
Size of Protein Molecules 100-2000 a.a.
Average Mr in eukaryotes ~ 31.7 kDa, containing 270 a.a.
Size of Protein Molecules
From Table 4.2 Size of protein molecules.
Molecular weights: Insulin, 5,733;
Cytochrome c, 12,500;
Ribonuclease, 12,640;
Lysozyme, 13,930;
Myoglobin, 16,980.
Size of Protein Molecules
From: Table 4.2 Size of Protein Molecules
Molecular weights: Hemoglobin, 64,500;
Immunoglobulin, 149,900;
Glutamine synthetase, 600,000. (12-mer)
The Sequence of Amino Acids in a Protein
• Is a unique characteristic of every protein
• Is encoded by the nucleotide sequence of DNA
• Is thus a form of genetic information
• Is read from the amino terminus to the
carboxyl terminus
Aspartame is a dipeptide
• Aspartame is a methyl ester of the dipeptide of L-aspartic acid and Lphenylalanine.
• An artificial sweeteners “Equal” ,“Nutrasweet” and ‘Canderol”, is
approximately 200 times sweeter than sucrose.
http://en.wikipedia.org/wiki/Aspartame
• using an enzyme from Bacillus thermoproteolyticus to catalyze the
condensation of the chemically altered amino acids will produce high
yields without the bitter β-form byproduct.
• may hydrolyze into its constituent amino acids under conditions of
elevated temperature or high pH. At room temperature, it is most stable at
pH 4.3, where its half-life is nearly 300 days. At pH 7, however, its half-life
is only a few days
阿斯巴甜是一種含L-天冬
氨酸和L-苯丙氨酸的二肽
甲酯
Glutathione (GSH) – a tripeptide
• A tripeptide containing the amino acids glutamate, cysteine, and
glycine, linked together in that order.
• free thiol group provides protection for cells against oxidative stress.
• For example, it can help to keep cysteine thiol groups in proteins in
the reduced state. If two thiol groups become oxidized, they can be
reduced nonenzymatically by glutathione. (還原劑)
• 2 GSH + H2O2  GSSG + 2 H2O (by GSH peroxidase)