Reginald H. Garrett Charles 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 -carbon has an amino group attached and an R group (R = H in glycine). The are 20 common -amino acids are used by the ribosomes to make proteins. These 20 have L chirality at the -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.

What Are the Structures and Properties of Amino Acids?

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.

What Are the Structures and Properties of Amino Acids?

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 -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.

The 20 Common Amino Acids

Some of the nonpolar (hydrophobic) amino acids.

The 20 Common Amino Acids

Some of the nonpolar (hydrophobic) amino acids.

The 20 Common Amino Acids

Some of the polar, uncharged amino acids.

The 20 Common Amino Acids

Some of the polar, uncharged amino acids.

The 20 Common Amino Acids

The acidic amino acids.

The 20 Common Amino Acids

The basic amino acids.

Amino Acids and Their Symbols

Residue Alanine Glutamate Glutamine Aspartate Asparagine Leucine Glycine Lysine Serine Valine Arginine Threonine Proline Isoleucine Methionine Phenylalanine Tyrosine Cysteine Tryptophan Histidine Three- letter symbol Ala Glu Gln Asp Asn Leu Gly Lys Serine Valine Arg Thr Pro Ile Met Phe Tyr Cys Trp His One-letter symbol A E Q D N L G K S V R T P I M F Y C W H Mnemonic Alanine GluEtamic acid Q-tamine AsparDic acid AsparagiNe Leucine Glycine Before L Serine Valine aRginine Threonine Proline Isoleucine Methionine Fenylalanine tYrosine Cysteine tWo rings Histidine pKa 4.3

3.9

10.5

12.5

10.1

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

Several Amino Acids Occur Rarely in Proteins

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.

pKa = 10

_ + H3N CH C O R

pKb = 12

Properties of Amino Acids

Glycine increases main chain flexibility.

Symmetry at the Ca atom Can adopt many different conformations

Evolutionarily conserved
Occurs in tight turns

Properties of Amino Acids

Alanine is smallish non-polar residue Occurs abundantly No preference for inside or surface of the protein

Properties of Amino Acids

Val, Leu and Ile are branched side chains Branching allows limited internal flexibility Occur primarily in protein cores Bricks around assembled which functional parts are

Properties of Amino Acids

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

Properties of Amino Acids

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

Properties of Amino Acids

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

Properties of Amino Acids

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

Properties of Amino Acids

Lys and Arg are +vely charged residues Long and flexible

Can form salt bridges or help in catalysis

Properties of Amino Acids

Ser and Thr are small and aliphatic -OH no more reactive than ethanol Frequently form H-bond with main chain

Properties of Amino Acids

Pro is imino acid Reduces main cyclization chain flexibility drastically due to

Can often occur in cis- form

Properties of Amino Acids

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 -amino group. The order of ionization always proceeds from most acidic to least acidic.

What Are Acid-Base Properties of Amino Acids?

Amino Acids are Weak Polyprotic Acids H2A+ + H2O HA0 + H3O+ Ka1 = [ HA0 ] [ H3O+ ]

__________________________

[H2A+ ]

What Are Acid-Base Properties of Amino Acids?

The second dissociation (the amino group in the case of glycine): HA0 + H2O A + H3O+ Ka2 = [ A ] [ H3O+ ]

_______________________

[ HA0 ]

What Are Acid-Base Properties of Amino Acids?

The first dissociation is the carboxylic acid group (using glycine as an example):
+NH CH COOH 3 2

+NH3CH2COO- + H+

(+NH3CH2COO-)(H+) Ka1 = --------------------------(+NH3CH2COOH)

What Are Acid-Base Properties of Amino Acids?

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

+NH CH COO3 2

NH2CH2COO- + H+

(NH2CH2COO-)(H+) Ka2 = --------------------------(+NH3CH2COO-)

What Are Acid-Base Properties of Amino Acids?

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: Alpha amino group: pKa = ~2 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: On contrast to amines and carboxylic acids, the amino acids are non-volatile crystalline solid which melt with decomposition at fairly high temperatures.

1.

Zwitterions formation
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.

Zwitterions formation
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 Kas of about 10-5 and most aliphatic amines have Kbs of about 10-4.

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

+ H3N C

COO

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

Zwitterions formation
Example: glycine exists as
H + H3N C H
Since the zwitterions are held by strong electrostatic attraction, thus m.p. and b.p. are high. Also, it exerts strong attraction to polar water, so it is highly soluble in water, but insoluble in non-polar solvent.

COO

Zwitterions formation
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.
H H2N C H COO OH H + H3N C H COO H H+ + H3N C H COOH

Therefore, the acidic group in amino acid is NH3+ NOT COOH. The basic group in amino acid is -COO- not NH2.

Isoelectric point and electrophoresis

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:
H H2N C H COO H + H3N C H COO H H+ + H3N C H COOH

OH -

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.

Isoelectric point and electrophoresis

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.

Isoelectric point and electrophoresis

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

What Are Acid-Base Properties of Amino Acids?

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 solution if the alpha carboxyl is 1/4 dissociated?

[1] pH 2 log10 [3]

pH = 2.2 + (-0.477) pH = 1.723 Note that, when the group is dissociated, are dissociated and are not; thus the ratio in the log term is over 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 dissociated, is dissociated and is not; thus the ratio in the log term is over or 3/1.

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?

Reactions of Amino Acids

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

Reactions of Amino Acids

Reactions of Amino Acids

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. Edmans reagent (phenylisothiocyanate) is the currently preferred reagent. It reacts with a free -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.
O 2 O O O N O O OH

+ NH2-CH-COOH OH CH3

CHO + CO2 + CH3

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

Ruhemanns Purple 570 nm

N-Terminal Reagents
F NO2

DNFB - Sangers reagent (dinitrofluorobenzene)

NO2 DNFB

DANSYL choride (dimethylaminonaphthalenesulfonyl chloride)

N(CH 3)2 DANSYL- Cl SO2Cl

Other Reagents
C-terminal analysis: Hydrazine
NH 2-NH 2
CH2-SH HO C H H C OH

Disulfide reduction: Dithiothreitol - Clelands Reagent

Thiols reactions: Iodoacetate

I-CH2-COOH

CH2-SH Cleland's

5,5-dithiobis-(2-nitrobenzoic acid) Ellmans reagent

NO2 COOH S S Ellman's NO2 COOH

Edmans Reaction

Edmans 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 -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 Lglyceraldehyde. 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. Fischers proposed structure was confirmed by J. M. Bijvoet in 1951 (by Xray 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.

Spectroscopic Properties
The UV spectra of the aromatic amino acids at pH 6. Beers Law: A = cl

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 = cl

Spectroscopic Properties

Proton NMR spectra of several amino acids.

Spectroscopic Properties
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.

Separation of Amino Acids

Gradient separation of common PTHamino acids

Separation of Amino Acids

Separation of 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

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

Charged Amino Acids

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

Polar Amino Acids

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

Special Amino Acids

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

End Chapter 4 Amino Acids