amino acids

Reginald H. GarrettCharles M. Grisham

Amino Acids

Outline

• What are the structures and properties of amino acids ?

• What are the acid-base properties of amino acids ? • What reactions do amino acids undergo ?• What are the optical and stereochemical properties

of amino acids ? • What are the spectroscopic properties of amino

acids ? • How are amino acid mixtures separated and

analyzed ?

What Are the Structures and Properties of Amino Acids?

• The common amino acids contain a carbon atom alpha to a carboxyl carbon.

• This α-carbon has an amino group attached and an R group (R = H in glycine).

• The are 20 common α-amino acids are used by the ribosomes to make proteins. These 20 have L chirality at the α-carbon.

• Amino acids join together via peptide bonds.• Several modified amino acids occur only rarely

in proteins.• Some amino acids are not found in proteins.


Anatomy of an amino acid. Except for proline and its derivatives, all of the amino acids commonly found in proteins possess this type of structure.


Two amino acids can react with loss of a water molecule to form a covalent bond.

Amino Acids: Atom Naming

• Organic nomenclature: start from one end• Biochemical designation: start from

-carbon and go down the R-group

Amino Acids: Building Blocks of Protein

• Proteins are heteropolymers of -amino acids• Amino acids have properties that are well

suited to carry out a variety of biological functions:• Capacity to polymerize• Useful acid-base properties• Varied physical properties• Varied chemical functionality

The 20 Common Amino Acids

You should know names, structures, approximate α-pKa values, 3-letter and 1-letter codes

Classification based on sidechain structure:• Non-polar amino acids.• Polar, uncharged amino acids.• Acidic amino acids.• Basic amino acids.

Other sidechain structural classifications:• Aromatic, cyclic, hydroxyl, and thiol amino acids.


Some of the nonpolar (hydrophobic) amino acids.


Some of the polar, uncharged amino acids.


The acidic amino acids.


The basic amino acids.

Amino Acids and Their Symbols

Residue Three- letter symbol

One-letter symbol

Mnemonic pKa

Alanine Ala A Alanine Glutamate Glu E GluEtamic

acid 4.3

Glutamine Gln Q Q-tamine Aspartate Asp D AsparDic acid 3.9 Asparagine Asn N AsparagiNe Leucine Leu L Leucine Glycine Gly G Glycine Lysine Lys K Before L 10.5 Serine Serine S Serine Valine Valine V Valine Arginine Arg R aRginine 12.5 Threonine Thr T Threonine Proline Pro P Proline Isoleucine Ile I Isoleucine Methionine Met M Methionine Phenylalanine Phe F Fenylalanine Tyrosine Tyr Y tYrosine 10.1 Cysteine Cys C Cysteine Tryptophan Trp W tWo rings Histidine His H Histidine 6.0

Several Amino Acids Occur Rarely in Proteins

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, epinephrine, histamine, serotonin act as

neurotransmitters and hormones.• Phosphorylated amino acids – a signaling device.

Several Amino Acids Occur Rarely in Proteins

Properties of 20 amino acids

• Hydrophobic• Ala, Val, Leu, Ile, Phe, Pro, Met

• Charged• Arg, Asp, Glu, Lys

• Polar• Ser, Thr, Tyr, Asn, Gln, His, Cys, Trp

Convention of Naming Side Chain Atoms

Properties of Amino Acids

• High melting points, over 200C• More soluble in water than in ether.• Larger dipole moments than simple acids or

simple amines.• Less acidic than most carboxylic acids, less

basic than most amines.

H3N CH

R

C

O

O+ _

pKa = 10 pKb = 12


•Glycine increases main chain flexibility.

•Symmetry at the Ca atom

•Can adopt many different conformations

•Evolutionarily conserved

•Occurs in tight turns

•Alanine is smallish non-polar residue

•Occurs abundantly

•No preference for inside or surface of the protein


•Val, Leu and Ile are branched side chains

•Branching allows limited internal flexibility

•Occur primarily in protein cores

•“Bricks” around which functional parts are assembled


•Phe, Tyr and Trp are the aromatic side chains

•All these contain one methylene group as a spacer

•Side chain flexibility is restricted

•Occur predominantly in the core of proteins

•Tyr can form strong H-bond with its -OH group


•Met and Cys are the sulfur containing side chains

•Met is rather large and flexible

•Met occurs predominantly inside the core

•Cys is special: it can form disulfide crosslinks and is polar


•Asn and Gln have amide in side chain

•Gln has an extra methylene group, rendering the polar group flexible and reducing its interaction with main chain

•H- bond donor as well as acceptor


•Asp and Glu are -vely charged at physiological pH

•Althoughly chemically similar, markedly different effect on the conformation and chemical reactivity

•Asp relatively rigid, and found frequently in active sites

•Mostly found on protein surfaces

•Can be effective chelators of metal ions


•Lys and Arg are +vely charged residues

•Long and flexible

•Can form salt bridges or help in catalysis


•Ser and Thr are small and aliphatic

•-OH no more reactive than ethanol

•Frequently form H-bond with main chain


•Pro is imino acid

•Reduces main chain flexibility drastically due to cyclization

•Can often occur in cis- form


•His is a very special residue with pKa of 6.0

•Can be uncharged or charged easily

•Very suitable for catalysis, found in most active centres


What Are Acid-Base Properties of Amino Acids?

Amino Acids are Weak Polyprotic Acids

The degree of dissociation depends on the pH of the medium

• The carboxylic acid group always ionizes first.• Then the side chain (if ionizable) or the α-amino

group. • The order of ionization always proceeds from

most acidic to least acidic.

Amino Acids are Weak Polyprotic Acids

• H2A+ + H2O HA0 + H3O+

• Ka1 = [ HA0 ] [ H3O+ ] __________________________

[H2A+ ]


The second dissociation (the amino group in the case of glycine):

• HA0 + H2O A¯ + H3O+

• Ka2 = [ A¯ ] [ H3O+ ] _______________________

[ HA0 ]



The first dissociation is the carboxylic acid group (using glycine as an example):

+NH3CH2COOH +NH3CH2COO- + H+

(+NH3CH2COO-)(H+)Ka1 = ---------------------------

(+NH3CH2COOH)


The second dissociation is the amino group in the case of glycine:

+NH3CH2COO- NH2CH2COO- + H+

(NH2CH2COO-)(H+)

Ka2 = ---------------------------

(+NH3CH2COO-)


The ionic forms of the amino acids, shown without consideration of any ionizations on the side chain.

pKa Values of the Amino Acids

You should know these numbers and know what they mean

• Alpha carboxyl group: pKa = ~2

• Alpha amino group: pKa = ~9

• These numbers are approximate, but entirely suitable for our purposes.

Zwitterions formation

Although the amino acids are commonly shown as containing an amino group and a carboxyl group, H2NCHRCOOH, certain properties, both physical and chemical, are not consistent with this structure:

1. On contrast to amines and carboxylic acids, the amino acids are non-volatile crystalline solid which melt with decomposition at fairly high temperatures.

2. They are insoluble in non-polar solvents like petroleum ether, benzene, or ether and are appreciably soluble in water.

3. Their aqueous solutions behave like solutions of substances of high dipole moment.


4. Acidity and basicity constants are ridiculously low for –COOH and –NH2 groups. Glycine, e.g., has Ka = 1.6 x 10-10 and Kb = 2.5 x 10-12, whereas most carboxylic acids have Ka’s of about 10-5 and most aliphatic amines have Kb’s of about 10-4.


All these properties are quite consistent with a dipolar ion structure for the amino acids.

CH3N COO-+

Since it exists as internal salt, known as zwitterion,in which both cation and anion are held togetherin the same unit.


Example: glycine exists as

CH3N COO

H

H

-+

Since the zwitterions are held by strong electrostaticattraction, thus m.p. and b.p. are high. Also, itexerts strong attraction to polar water, so it is highly soluble in water, but insoluble in non-polar solvent.


Amino acid with equal number of amino and carboxyl group is neutral when dissolved in water, but in acidic solution, -COO- group is protonated (I.e. exists as a –COOH), and basic solution, -NH3

+ group is free and exists as an –NH2.

Therefore, the acidic group in amino acid is –NH3+

NOT –COOH. The basic group in amino acid is-COO- not –NH2.

CH3N COO

H

H

-+ CH3N COOH

H

H

+CNH2 COO

H

H

- HOH- +


Amino acids, as a zwitterions, exhibits both acidic and basic properties in aqueous solutions. In aqueous solution, the ion exists in equilibrium with its cationic form and anionic form simultaneously:

CH3N COO

H

H

-+ CH3N COOH

H

H

+CNH2 COO

H

H

- HOH- +

Isoelectric point and electrophoresis

If an electric field is applied to an aqueous solution of an amino acid, whether there is a migration of the ion or not depends on the pH of the solution.In alkaline solution, the above equilibrium will shift to the left and the concentration of anion will exceed that of cation, and there will be a net migration of the amino acid towards the positive pole.


In acidic solution, the above equilibrium will shift to the right and the concentration of cation will exceed that of anion, and there will be a net migration of the amino acid towards the negative pole.


By adjusting the pH value of the aqueous solution of an amino acid, the concentration of cation can be made equal to that of anion, and there will be no net migration of the amino acid in an electric field. The pH value so adjusted in this case is known as the isoelectric point of the given amino acid. Isoelectric points are characteristic of amino acids. Therefore it is possible to separate different amino acids in a mixture by subjecting the mixture to an electric field and adjusting the pH value, This technique is known as electrophoresis.


Isoelectric Point

• pH at which amino acids exist as the zwitterion (neutral).• Depends on structure of the side chain.• Acidic amino acids, isoelectric pH ~3.• Basic amino acids, isoelectric pH ~9.• Neutral amino acids, isoelectric pH is slightly acidic, 5-6.

Isoelectric Point, pI

The isoelectric point is the pH at which there is zero net charge

Using Gly again:• Charges in the first ionization: +1

0• Charges in the second ionization: 0 -1• So, in the case of glycine, the pH at which

there is most of the zero net charge form occurs half way between the first and second ionizations.

pI = (pKa1 + pKa2)/2 = 5.95

What Are Acid-Base Properties of Amino Acids? Titration of Glycine

pKa Values of the Amino Acids

You should know these numbers and know what they mean

• Arginine, Arg, R: pKa(guanidino group) = 12.5

• Aspartic Acid, Asp, D: pKa = 3.9

• Cysteine, Cys, C: pKa = 8.3

• Glutamic Acid, Glu, E: pKa = 4.3

• Histidine, His, H: pKa = 6.0

• Lysine, Lys, K: pKa = 10.5

• Tyrosine, Tyr, Y: pKa = 10.1

Titrations of polyprotic amino acids

Titration of glutamic acid

Titrations of polyprotic amino acids

Titration of lysine.

A Sample Calculation

What is the pH of a glutamic acid solutionif the alpha carboxyl is 1/4 dissociated?

• pH = 2.2 + (-0.477)• pH = 1.723

• Note that, when the group is ¼ dissociated, ¼ are dissociated and ¾ are not; thus the ratio in the log term is ¼ over ¾ or 1/3.

10

[1]2 log

[3]pH

Another Sample Calculation

What is the pH of a lysine solution if the side chain amino group is 3/4 dissociated?

• 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.

10

[3]10.5 log

[1]pH

Amino Acids as Buffers

From Table 4.1 we can see that Histidine has pKa values of 1.8, 6.0, and 9.2.

It can provide effective buffering at a pH equal to any one of these three pKas.

The titration curve has three regions in which pH is relatively unaffected by addition of acid or base.

What is the zero net charge form of His ?In which ionizations does it appear ?What is the charge on His at pH 4?

• Carboxyl groups form amides & esters• Amino groups form Schiff bases, hydroxy acids (with

Nitrous acid) and amides (by Acetylation)

• Side chains show unique reactivities• Cys residues can form disulfides and can be easily

alkylated• Few reactions are specific to a single kind of side chain

Reactions of Amino Acids


Amino acid composition – an amino acid analysis gives the number of each amino acid in the peptide or protein.

• Peptide bonds are cleaved by acid hydrolysis (6M HCl, 110oC, 18-72 hours). Trp is destroyed.

Amino acid sequence – order of the amino acids in a peptide or protein.• Edman’s reagent (phenylisothiocyanate) is the

currently preferred reagent. It reacts with a free α-amino group of an amino acid or peptide to produce a phenylthiohydantoin (PTH) derivative.

Detecting Amino Acids

Ninhydrin is the classical reagent for detecting amino acids. Reaction requires 2-5 min at 100oC and is sensitive at the nanomole level.

Ruhemann’s Purple570 nm

OH

O

O

OHNH2-CH-COOH

CH3

+

O

O

O

O

NCO2CH3

CHO+ +2

Note: The product from Pro isYellow and absorbs at 440 nm.

N-Terminal Reagents

DNFB - Sanger’s reagent (dinitrofluorobenzene)

DANSYL choride (dimethylaminonaphthalenesulfonyl chloride)

NO2

NO2

F

DNFB

SO2Cl

N(CH3)2

DANSYL-Cl

Other Reagents

C-terminal analysis:Hydrazine

Disulfide reduction:Dithiothreitol - Cleland’s Reagent

Thiols reactions:Iodoacetate

5,5’-dithiobis-(2-nitrobenzoic acid) - Ellman’s reagent

NH2-NH2

HO C H

H C OH

Cleland's

CH2-SH

CH2-SH

NO2 S S

COOH

NO2

COOHEllman's

I-CH2-COOH

Edman’s Reaction

Edman’s reagent reacts with the N-terminal residue of a peptide or protein and cleaves the peptide bond forming a cyclic thiazoline derivative that reacts in weak aqueous acid to form a PTH-amino acid. This reaction can proceed down the chain cleaving successive residues. Samples are used to identify each residue.

Oxidation of Cysteine to Cystine

Cysteine residues react with each other to form disulfides. This will occur with O2 in air.

Stereochemistry of Amino Acids

• All common AA except glycine are chiral at the α-carbon atom.

• L-amino acids predominate in nature and are the only ones used in ribosomal protein synthesis.

• D,L-nomenclature is based on D- and L-glyceraldehyde.

• R,S-nomenclature system is more convenient, since amino acids like isoleucine and threonine (with two chiral centers) can be named unambiguously.

Stereochemistry of Amino Acids

Discovery of Optically Active Molecules and Determination of Absolute Configuration

Emil Fischer deduced the structure of glucose in 1891. Fischer’s proposed structure was confirmed by J. M. Bijvoet in 1951 (by X-ray diffraction).

Rules for Description of Chiral Centers in the (R,S) System

Naming a chiral center in the (R,S) system is accomplished by viewing the molecule from the chiral center to the atom with the lowest priority. The priorities of the functional groups are:SH > OH > NH2 > COOH > CHO > CH2OH > CH3

Rules for Description of Chiral Centers in the (R,S) System

Spectroscopic Properties

• All amino acids absorb in the infrared (bond vibrations).

• Only Phe, Tyr, and Trp absorb in the UV (electronic transitions between energy levels).

• Absorbance at 280 nm is a good method for determining protein concentration.

• NMR spectra are characteristic of each residue in a protein, and high resolution NMR measurements can be used to elucidate three-dimensional structures of proteins.


The UV spectra of the aromatic amino acids at pH 6.

Beer’s Law:

A = εcl

Spectroscopic Detection of Aromatic Amino Acids• The aromatic amino acids

absorb light in the UV region

• Proteins typically have UV absorbance maxima around 275-280 nm

• Tryptophan and tyrosine are the strongest chromophores

• Concentration can be determined by UV-visible spectrophotometry using

Beers law: A = ·c·l


Proton NMR spectra of several amino acids.


A plot of C13 chemical shifts versus pH for the carbons of lysine.

Separation of Amino Acids

• Mikhail Tswett, a Russian botanist, first separated colorful plant pigments by ‘chromatography’.

• Many chromatographic methods exist for separation of amino acid mixtures:• Ion exchange chromatography.• High-performance liquid chromatography.


Gradient separation of common PTH-amino acids

Amino Acids: Classification

Common amino acids can be placed in five basic groups depending on their R substituents:

• Nonpolar, aliphatic (7)

• Aromatic (3)

• Polar, uncharged (5)

• Positively charged (3)

• Negatively charged (2)

Aliphatic Amino Acids

• http://en.wikipedia.org/wiki/File:Aa.svg

Aromatic Amino Acids


Charged Amino Acids


Polar Amino Acids


Special Amino Acids


End Chapter 4Amino Acids

amino acids

Documents

polyprotic amino acids

h3n

basic amino acids

aromatic amino acids

uncharged amino acids

acidic amino acids

polar amino acids

carboxylic acid group