Amino Acids

Amino Acids and Peptides

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Amino Acids

ƒ

ƒAmino acid: Amino acid: a compound that contains both an amino group and a carboxyl group

–

– α α- -Amino acid Amino acid: : an amino acid in which the amino group is on the carbon adjacent to the carboxyl group

– although α-amino acids are commonly written in the unionized form, they are more properly written in the zwitterion zwitterion (internal salt) form

R- CH-COH N H2

R- CH-CO^- N H3+ unionized

form

zwitterion

O O

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Chirality of Amino Acids

ƒ With the exception of glycine, all protein-derived amino acids have at least one stereocenter (the α-carbon) and are chiral

– the vast majority of α-amino acids have the L- configuration at the α-carbon

H N H₃⁺ COO^-

CH₃

+H₃N H

COO^-

CH₃ D-Alanine L-Alanine

(Fischer projections)

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Chirality of Amino Acids

ƒ Comparison of the stereochemistry of alanine and glyceraldehyde (Fischer projection formulas)

H N H₃⁺ COO^-

CH₃

+H₃N H

COO^-

CH₃ D-Alanine L-Alanine

H OH

CHO

CH₂OH

HO H

CHO

CH₂OH D-Glyceraldehyde L-Glyceraldehyde

the naturally occurring form

the naturally occurring form

20 Protein-Derived AA

Nonpolar side chains (predominant form at pH 7.0)

H-

CH₃-

CH₃CH₂CH( CH₃) - ( CH₃)₂CH CH₂-

CH₃SCH₂CH₂-

N H ( CH₃)₂CH

glycine (gly, G)

alanine (ala, A)

valine (val, V)

leucine (leu, L)

isoleucine (ile, I)

methionine (met, M)

phenylalanine (phe, F)

tryptophan (trp, W)

N

H H

+ proline (Pro, P)

20 Protein-Derived AA

Polar side chains (predominant form at pH 7.0) asparagine (asn, N)

glutamine (glu, G)

serine (ser, S)

threonine (thr, T) H₂N CCH₂-

O

H₂N CCH₂CH₂- O

HOCH₂-

CH₃CH- OH

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20 Protein-Derived AA

Acidic side chains (predominant form at pH 7.0) aspartic acid (asp, D) glutamic acid (glu, E)

cysteine (cys, C) tyrosine (tyr, Y) -OCCH₂-

HSCH₂-

-OCCH₂CH₂-

HO CH₂-

O O

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20 Protein-Derived AA

Basic side chains (predominant form at pH 7.0) arginine (arg, R) histidine (his, H)

lysine (lys, K) H2N CN HCH2CH2CH2-

N H2+

H₃N CH₂CH₂CH₂CH₂- N

N H

CH2-

+

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20 Protein-Derived AA

ƒ Note these structural features

1. All 20 are α-amino acids

2. For 19 of the 20, the α-amino group is primary;

for proline, it is secondary

3. With the exception of glycine, the α-carbon of each is a stereocenter

4. Isoleucine and threonine contain a second stereocenter

5. The sulfhydryl group (pK

8.3) of cysteine, the imidazole group (pK

6.0) of histidine, and the phenolic hydroxyl (pK

10.1) of phenylalanine are

partially ionized at pH 7.0, but the ionic form is not

Uncommon Amino Acids

ƒ Each example is derived from a common amino acid by the modification shown in color

– hydroxylysine and hydroxyproline are found only in a few connective tissues such as collagen

– thyroxine is found only in the thyroid gland

+H3N

N H₃⁺ COO^- OH

Hydroxylysine

N COO^- HO

H H Hydroxyproline

N H₃⁺ COO^- O

I I

I HO

I Thyroxine +

Ionization of Amino Acids

+H₃N COOH pKa = 2.34

+H₃N COO^- H₂N COO^-

pK_a = 9.69

+1 charge 0 charge -1 charge

Isoelectric zwitterion

+H₃N COOH +2 charge

N N H H

pKa = 1.82

+H₃N COO^- +1 charge

N N H H

pKa = 6.04

+H₃N COO^- 0 charge

N N H

pK_a = 9.17 H₂N COO^- -1 charge

N N H

+ +

Titration of Amino Acids

Figure (a) Titration of alanine with NaOH

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Titration of Amino Acids

Figure (b) Titration of histidine with NaOH

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Acidity: α-COOH Groups

ƒ The average pKaof an α-carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pKa4.76)

– the greater acidity of the amino acid carboxyl group is due to the electron-withdrawing inductive effect of the -NH

group

The ammonium ion has an electron-withdrawing inductive effect

+ pK_a = 2.19

N H3 N H3 +

+

RCHCOO^-

RCHCOOH+H₂O H₃O⁺

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Acidity: α-NH

groups

ƒ The average value of pKafor an α-NH3+group is 9.47, compared with a value of 10.76 for a 2° alkylammonium ion

+ pK_a = 9.47

N H3

+ N H2

RCHCOO^-+ H2O RCHCOO^- H3O⁺

pK_a = 10.76 N H3

+ N H2

CH3CHCH3+ H₂O CH3CHCH3 + H₃O⁺

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Basicity: Guanidine Group

ƒ The side chain of arginine is a considerably stronger base than an aliphatic amine

– basicity of the guanido group is attributed to the large resonance stabilization of the protonated form relative to the neutral form

+

pK_a = 12.48 C

N H₂⁺ C

RN H N H₂⁺ N H₂

N H₂

RN H RN H C

N H₂ N H₂

C RN

N H₂ N H₂

+ H₃O⁺ H2O :

:

:

: :

: :

:

Basicity: Imidazole Group

ƒ The imidazole group on the side chain of histidine is a heterocyclic aromatic amine

+

this lone pair is not a part of the aromatic sextet; it is the proton acceptor

pKa 6.04 N

H

N H3 N + H

CH2CHCOO^-

N H3 N + H

CH2CHCOO^- N

+ H3O⁺ H₂O N

N H H

+

CH2CHCOO^- N H3

+

: :

: :

Ionization vs pH

ƒ Given the value of pKaof each functional group, we can calculate the ratio of each acid to its conjugate base as a function of pH

ƒ Consider the ionization of an α-COOH

– writing the acid ionization constant and rearranging terms gives

[α-COO H]

[α-COO^-] Ka = [ H₃O⁺]

= Ka [α-COO H]

[α-COO^-] [ H₃O⁺] or

pK_a = 2.00

α−COO^-

α−COOH+ H2O + H3O⁺

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Ionization vs pH

– substituting the value of K

(1 x 10

) for the hydrogen ion concentration at pH 7.0 (1.0 x 10

) gives

– at pH 7.0, the α-carboxyl group is virtually 100%

in the ionized or conjugate base form, and has a net charge of -1

– we can repeat this calculation at any pH and determine the ratio of [α COO

] to [α COOH] and

= K_a [α-COO H]

[α-COO^-] [ H₃O⁺]

= 1.00 x 10⁵ 1.00 x 10^-7

1.00 x 10^-2

=

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Ionization vs pH

ƒ We can also calculate the ratio of acid to conjugate base for anα-NH3+

group; for this calculation, assume a value 10.0 for pKa

– writing the acid ionization constant and rearranging gives

[α-NH3+] K_a

= [H₃O⁺] + pK_a = 10.00

α−NH₂

α−NH₃⁺ + H2O H3O⁺

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Ionization vs pH

– substituting values for K

of an α-NH

group and the hydrogen ion concentration at pH 7.0 gives

– at pH 7.0, the ratio of α-NH

to α-NH

is approximately 1 to 1000

– at this pH, an α-amino group is 99.9% in the acid or protonated form and has a charge of +1

[α-NH

] [α-NH

]

K

= [H

O

] = 1.00 x 10

1.00 x 10

= 1.00 x 10

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Henderson-Hasselbalch

ƒ We have calculated the ratio of acid to conjugate base for an α-carboxyl group and an α-amino group at pH 7.0

ƒ We can do this for any weak acid and its conjugate base at any pH using the Henderson-Hasselbalch equation

[weak acid]

[conjugate base]

log pH = pK_a +

Henderson-Hasselbalch

– using the Henderson-Hasselbalch equation, we can calculate the percent of charged or uncharged form present and the net charge on serine at pH 3.0, 7.0, and 10.0

+ +

pH 3.0 pH 7.0 pH 10.0

Net charge +1 N et charge 0 N et charge -1

100% 86% 99% 100% 88% 100%

CH

OH CH

OH CH

OH

H

N- CH -C- OH H

N- CH -C- O

H

N- CH -C- O

O O O

Isoelectric pH

ƒ

ƒIsoelectricIsoelectricpH, pIpH, pI::the pH at which the majority of molecules of a compound in solution have no net charge

– the pI for glycine, for example, falls midway between the pK

values for the carboxyl and amino groups

2 ( p Ka α−COOH + p K_a α−NH₃⁺)

= 21 (2.35 + 9.78) = 6.06

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Isoelectric pH (pI)

α−NH₃⁺ α−COOH

6.02 5.41 5.65 5.97 6.02 6.02 5.74 5.48 6.30 5.68 6.53 5.89 5.97 pK_a of pK_a of

pK_a of

pI

---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ----

valine 2.32 9.62

tryptophan 2.38 9.39

10.43 2.63 threonine

serine 2.21 9.15

10.60 1.99 proline

phenylalanine 1.83 9.13

9.21 2.28

methionine

9.68 2.36

leucine

isoleucine 2.36 9.68

glycine 2.34 9.60

9.13 2.17

glutamine

8.80 2.02

asparagine

9.69 2.34

alanine

Side Chain Nonpolar &

polar side chains

Table 3.2 pKa and pI of α-amino acids

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Isoelectric pH (pI)

α−NH₃⁺ α−COOH

α−NH₃⁺ α−COOH

10.76 2.98

5.02 3.08

7.64 9.74 5.63 pK_a of pK_a of pK_a of

pI

10.07 9.11 2.20 tyrosine

lysine 2.18 8.95 10.53

6.10 9.18 1.77 histidine

glutamic acid 2.10 9.47 4.07

8.00 10.25 2.05 cysteine

aspartic acid 2.10 9.82 3.86

arginine 2.01 9.04 12.48

Side Chain Acidic

Side Chains

pK_a of pK_a of pK_a of

Side Chain Basic

Side Chains pI

Table 3.2 (cont'd)

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Electrophoresis

ƒ

ƒElectrophoresisElectrophoresis::the process of separating compounds on the basis of their electric charge

– electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose acetate as solid supports

– in paper electrophoresis, a paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between two electrode vessels

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Electrophoresis（Process)

– a sample of amino acids is applied as a spot (the origin) on the solid support strip

– an electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own

– molecules with a high charge density move faster than those with a low charge density

– molecules at their isoelectric point remain at the origin

– after separation is complete, the strip is dried and developed to make the separated amino acids

Polypeptides

ƒ In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds to which he gave the name peptide bonds

ƒƒPeptide bondPeptide bond::the special name given to the amide bond between the α- carboxyl group of one amino acid and the α-amino group of another

Serylalanine (Ser-Ala)

+

Alanine (Ala) Serine (Ser)

H₃N O^- H HOH₂C

O H₃N

O^- H CH3

O

H3N N

H HOH₂C

O H CH3 O

O^- H +

+ +

peptide bond

Serylalanine (Ser-Ala)

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Peptides

– – peptide peptide: the name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain

– – dipeptide dipeptide: a molecule containing two amino acids joined by a peptide bond

–

– tripeptide tripeptide: a molecule containing three amino acids joined by peptide bonds

– – polypeptide polypeptide: a macromolecule containing many amino acids joined by peptide bonds

–

– protein protein: a biological macromolecule of molecular

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Geometry of Peptide Bond

– the four atoms of a peptide bond and the two alpha carbons joined to it lie in a plane with bond angles of 120° about C and N

– to account for this geometry, Linus Pauling proposed that a peptide bond is most accurately represented as a hybrid of two contributing structures

– the hybrid has considerable C-N double bond character and rotation about the peptide bond is restricted

(2) C_α

C_α

N H C Cα

C_α O O

C N

H

+ -

(1)

:

:

:

: : :

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Writing Peptides

ƒ By convention, peptides are written from the left, beginning with the free -NH3+group and ending with the free -COO^-group

– the repeat pattern, starting from the N-terminal amino acid, is N ---> α-carbon ---> carbonyl carbon etc.

+ H3N

OH NH

O H

N O^-

O

O C-terminal

amino acid

N-terminal amino acid

Ser-Met-Asn O N H₂ S

peptide bonds

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Some Small Peptides

β-Alanyl-L-histidine (Carnosine) L-Aspartyl-L-phenylalanine

methyl ester (Aspartame) H3N -CH- C-N H-CH -C-OCH3

O

CH₂ CH₂

C₆H₅ COO^-

+

H3N -CH2- CH2-C-N H- CH-COO^- O

CH₂

N N H

+

Glutathione

Glutathione, GSH (reduced form)

Glutathione, GS-SG (oxidized form) 2e^- oxidation 2e^- reduction

A disulfide bond

O O

H N N H₃⁺ -O

SH N

H O

O O^-

O O

H N N H₃⁺ -O

S N

H O

O O^-

O O

N N H₃⁺ H -O

S H N

O

O O^-

Enkephalins

Tyr-Gly-Gly-Phe-Leu = Y-G-G-F-L Leucine enkephalin

Tyr-Gly-Gly-Phe-Met = Y-G-G-F-M Methionine enkephalin

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Oxytocin & Vasopressin

H₃N -Cy s- Ty r -Ile

S Gln

S

Pr o-Le u- Gly -C- NH2 O +

Oxytocin Cy s-A sn

H3N -Cy s- Ty r -Phe

S Gln

S

Pr o-A r g- Gly- C-N H2 O +

Cy s-A sn

Vasopressin

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End