PROTEINS

lecture 4
Main topic:
• Aminoacids:
Proteinogenic aminoacids. Non-proteinogenic aminoacids
Structures
Properties and functions
• Peptides:
Peptide bond
Peptides structure
Peptides with functional role
PROTEINS
ROLE:

Structural

Functional :
Enzymes
Hormones
Coagulating factors
Antibodies
Transcription factors

Energy source - 15 – 25% of the daily need of energy

• Chemically, proteins are formed of amino acids, bound by
peptide bonds.
• The amino acid number in proteins is variable, from 40 –
40,000 that corresponds to molecular masses from 5,000 –
4,200,000 Da.
• Protein diversity in nature is based on the possibilities,
practically unlimited, of combination of those 21 proteinogenic
amino acids.
• Therefore, from 21 amino acids, 212 (441) different dipeptides
can be obtained, or 213 (9261) different tripeptides, or 214
(194 481) different tetrapeptides, etc.
• This diversity permits the existence of specific proteins for
each organism, organ, tissue, cell.
Amino acids (AA)

• contain both amino (-NH2) and carboxylic acid (-COOH) groups.

• contain saturated, unsaturated, aromatic or heterocyclic
chains.
• can be natural (synthesized by living organisms in biochemical
processes) and artificial, synthesized in lab in chemical
processes.
• About 300 amino acids occur in nature.
 21 AA in proteins.
free proteinogenic AA (in biological liquids, intra- and
extra cellular).
Plasma protein concentration 6-8 grams/100 ml,
Free amino acid plasma concentration is about 0.05
grams/100 ml

Some proteins also contain derivatives of amino acids,

resulted posttranslational. For example, hydroxyproline,
hydroxylysine, cystine.
8 AA are essential amino acids valine, leucine, isoleucin,
phenylalanine, tryptophan, threonine, lysine and
methionine.
2 AA, arginine and histidine, are semi-essential amino acids.
AA`s properties
• Optical properties
• Acido-basic properties
• Reactions of –COOH group:
• Reactions of the side chain
• Reactions of –NH3 group:
• Coloring reactions
ninhydrin (blue-violet) and fluorescamine (fluorescent)
•Reaction with active
metals
•Reaction with metallic
oxides
•Neutralization
• Esters formation
•Amide formation
• Reaction with PCl5 →
R-COCl
•Decarboxylations
•Reaction with acids or R-COCl
•Reaction with R-X (alkylation)
• Desamination
•Amide formation
•Formation of diazonium salts
•Formation of Schiff bases
 Table 3. Non-proteinogenic amino acids with functional role

Denomi- Scientific Structural formula Function

nation denomonation
component of coenzyme
β-amino-
β-alanine H2N–CH2–CH2–COOH A, structure of some
propionic ac.
dipeptides
γ-amino-
GABA H2N–CH2–CH2–CH2–COOH Neurotransmitter
butiric ac.
δ-amino-
4-ceto-5-amino- intermediar in heme
levulinic H2N–CH2–CO–CH2–CH2–COOH
pentanoic ac. biosynthesis
ac.
α-amino-δ-
H2N–CO–NH–(CH2)3–CH(NH2)– intermediar of urea
citrulline amidino-
COOH cycle
valerianic ac.

α,δ-diamino- intermediar of urea

ornithin H2N–(CH2)3–CH(NH2)–COOH
valerianic ac. cycle

homo- α-amino-γ- intermediar in certain

HS–(CH2)2–CH(NH2)–COOH
cysteine thiobutiric ac. amino acids metabolism

α-amino-
intermediar in certain
homoserine γ-hydroxy- HO–(CH2)2–CH(NH2)–COOH
amino acids metabolism
-butiric ac.
 Growing factor for
p-amino-
PABA H2N–C6H4–COOH bacteria, component
benzoic ac.
of folic acid

3,4-
intermediar of
dihydroxy- H O C H 2 C H ( N H 2) C O OH
DOPA catecholamines
phenylalani-
synthesis
ne H O

3,5,3'-triiodo-
T3 HO O C H2 C H C OOH thyroid hormone
thyronine NH2

I I

I I

3,5,3',5'- HO O C H2 C H C OOH
T4
tetra-iodo- NH2 thyroid hormone
(thyroxin) I
thyronine I
 Proteinogenic amino acids with functional role

Amino acids with aliphatic side chain R

CH2 C OOH
1. glycine (glicocol), Gly (G)
NH2 Side chain :
- does not react
CH3 CH C OOH - Gives hydrophobic bonds
(van der Waals)
2. alanine, Ala (A) NH2

CH3 CH CH C OOH

3. valine, Val (V) essential CH3 NH2

CH3 CH CH2 CH C OOH

CH3 NH2
4. Leucine, Leu (L) essential

CH3 CH2 CH CH C OOH

CH3 NH2
5. Isoleucine, Ile (I) essential
Amino acids with side chain R containing hydroxyl group (OH)

CH2 CH C OOH
6. Serine, Ser (S)
OH NH2

CH3 CH CH C OOH
7. Threonine, Thr (T) essential
OH NH2

19. Tyrosine, Tyr (Y) HO CH 2 CH COOH

NH 2

Side chain:
- Forms ether bond with carbohydrates
- Forms ester bonds with carboxylic acids, inorganic acids (phosphoric acid)
- Does not ionise
CH
at pH
OH
of the medium CH2 O PO 3 H 2
2
- Forms hydrogen bonds
CH NH2 + ATP CH NH2 + ADP
( H 3 PO 4 )
(+ H 2 O )
C O OH C OOH
Amino acids with side chain R containing sulfur (S)
8. cysteine, Cys (C)
CH2 CH C OOH

SH NH2
- forms –S-S- bridges ⇒ reducing character
⇒ stabilises protein structure (i.e. insulin)

CH2 SH CH2 S S CH2

- 2H
2 CH NH2 CH NH2 CH NH2
+ 2H
C O OH C OO H C OO H
cysteine cystine

9. Methionine, Met (M) essential

CH3 S ( C H 2) 2 CH C OO H

NH2

- General donor of methyl group (-CH3)

Amino acids with side chain R containing selenium (Se)

10. seleno-cysteine, Se-Cys (Se-C)

H
H2C C COOH

SeH NH2

Amino acids with side chain R containing an acid group

11. Aspartic acid, Asp (D)
H O OC CH2 CH C OO H

12. Glutamic acid, Glu (E) NH2

H OOC CH2 CH2 CH C OOH

NH2
Side chain forms:
- Negativ ion at pH of medium
- Hydrogen and ionic bonds
- Ester bond with alcohols
- Amide bond with amine
Amino acids with side chain R containing an amide group
13. asparagine, Asn, (N)
H 2 N OC CH2 CH C OOH

NH2

14. glutamine, Gln (Q) H 2N O C CH2 CH2 CH C OOH

NH2

Side chain:
- Gives hydrogen bonds
- Amides are neutral
- Can hydrolyse to give carbolxyl group
- Forms N-glycosidic bond with carbohydrates (Asn)
Amino acids with side chain R containing a basic group

15. lysine, Lys (K) essential CH2 ( C H 2) 3 CH C OOH

NH2 NH2

NH2 C NH ( C H 2) 3 CH C OOH
16. arginine, Arg (R) semiessential
NH NH2

CH2 CH COOH
17. histidine, His (H) semiessential N NH NH2

Side chain:
- Forms positive ions at pH of medium
- Has basic character
- Forms hydrogen bonds
- Forms amide bond (Lys)
Amino acids with side chain R containing an aromatic ring
18. Phenylalanine, Phe (F) essential

CH2 CH COOH
NH2
(19. Tyrosine, Tyr (Y) )

20. Tryptophane, Trp (W) essential CH2 CH C OOH

NH2
N

Imino-acid
C OOH
21. Proline, pro (P) N

Side chain:
- Hydrophobic
- Forms van der Waals bonds
 AMINO ACIDS PROPERTIES

Physical Properties
• solid substances, with high melting points (>200°C) and decompose before melting.
• may be dissolved in water (more or less) but are low soluble in non polar solvents
(ether, chlorophorm, benzene, etc.).
• They have a higher solubility in light acidic or basic solutions.
Optical properties
• all proteinogenic amino acids have at least one chiral centre (asymmetric carbon), the
Cα atom (except glycine)
• Isoleucine and threonine have an additional asymmetric carbon, the Cβ atom.
• D-type appear only occasionally, especially to some microorganisms, where they have
specific roles.
HO O O OH
C C

H 2N C H H C NH2

R R

L – amino acid D – amino acid

en.wikipedia.org
 en.wikipedia.org

The polarized light plane – optical

rotation
• The condition for optical isomerism – the molecular asymmetry
• At least one asymmetrical carbon (chiral carbon C* )
• Increased number of C* -s ⟶ increased number of isomers
• n C* ⟶ 2n enantiomers
Acid-base properties
• amphoter electrolyte, derived from the concomitant presence of at least
one –COOH group (weak acid) and a –NH2 group (weak base). A proton
transfer can take place between the two groups:

C OOH C OO -
R CH R CH
NH2 N H 3+

• protonated, or acidic forms R-COOH and R-NH3+.

• R-COO- and R-NH2 are the conjugate bases (proton acceptors) of the
corresponding acids.
• pH of blood plasma or intracellular space (7.4 and 7.1)
- carboxyl groups exist almost entirely as carboxylate ions - R-COO-.
- Most of the amino groups are predominantly in the associated
(protonated) form R-NH3+.
• AA behave as buffers - can neutralize low quantities of acids or bases added to their
solution.
+ H+ - H+
R CH C OOH R CH C OOH R CH C OO -
pH < 7 pH > 7
N H 3+ NH2 NH2

• AA with additional acidic or basic groups (Glu, Asp, Lys, Arg, Hys) have a
supplementary
-
ionization to that functional group.
C OO C OOH C OOH C OOH

CH2 + H+ CH2 + H+ CH2 + H+ CH2

CH NH2 CH N H 3+ CH N H 3+ CH N H 3+
- H+ - H+ - H+
C OO - C OO - C OO - C OOH

pK α pK pK
- N H 3 + = 9,82 R-COOH
= 3,86 α -COOH = 2,09

• pKa = - logKa (weak acids)

Isoelectric pH (pI)
• pI is the pH at which an amino acid bears no net charge and hence does not move in a direct
current electrical field.
• pI is the pH midway between pK values on either side of the isoelectric species.
• For an amino acid with only 2 dissociating groups:
pKα −COOH + pKα − NH +
pI = 3

• For an amino acid with 2 carboxyl groups (and one amino):

pK α−COOH + pK R −COOH
pI =
2

• For an amino acid with 2 basicpK

groups (and one acidic):
+ pK
α − NH3+ R − NH 3+
pI =
2
• The pI values for the 21 proteinogenic amino acids range in the domain 4 - 10.5.
• at pH = pI ⇒ no electric net charge,
• it will not migrate in a direct current electrical field.
• the solubility of the amino acid is the lowest
• At pH < pI ⇒ positiv charge,
• it will migrate to the negative pol (cathode) in a direct current electrical field.
• At pH > pI ⇒ negative charge,
• it will migrate to the positive pole (anode) in a direct current electrical field.
• Titration curves show the neutralization of these acids by adding a base and the
change in pH during the titration.
Considering the acid-base properties, amino acids can be divided in four groups:
1. Amino acids with hydrophobic, nonpolar R radical: Ala, Val, Leu, Ile, Pro, Phe,
Trp, Met.
2. Amino acids with polar R, but without an electrical charge (at physiological
pH): Ser, Thr, Tyr, Cys, Asn, Gln, Gly.
3. Amino acids with negatively charged R (-) at the physiological pH: Asp, Glu.
4. Amino acids with positively charged R (+) at the physiological pH: Lis, Arg, His.
Chemical properties of amino acids
• The carboxyl and amino groups of amino acids exhibit all the expected reactions of
these functions, e.g. salt formation, esterification, acylation, condensation,
decarboxylation.
• The properties of individual amino acids are dictated by the nature of their α-R groups.
Glycine, the smallest amino acid, can fit into regions of 3-dimensional structure of proteins
inaccessible to other amino acids and occurs in regions where peptides bend sharply.
Alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan have an aliphatic
R which is hydrophobic, a property that has important consequences for the ordering of
water molecules in the immediate neighborhood of the protein. These amino acids
typically occur primarily in the interior of cytosolic proteins.
Aspartic acid, glutamic acid, lysine, histidine, arginine, due to their acidic or basic R groups,
perform key roles in stabilizing specific protein conformations via formation of salt
bonds. These amino acids are found in the “charge relay” systems that transmit charges
across considerable distances during enzymatic catalysis.
Histidine occupies a unique and important place in enzymatic catalysis, since the pK of its
imidazole proton permits it, at pH = 7.0 to function as either a base or an acid catalyst.
Serine and cysteine have a primary alcohol, respectively thioalcohol (-SH) group which are
excellent nucleophyles and may function as such during enzymatic catalysis.
Cysteine has redox properties due to its -SH group.
 Serine and tyrosine, due to their -OH group, function in regulation of the activity of
certain enzymes, whose catalytic activity depends upon the phosphorylation state
of specific seryl or tyrosyl residues
• The hydroxylated amino acids Ser and Thr determine, in the polypeptide structures,
specific reactive possibilities, with significance in the relation enzyme – substrate, in
binding to carbohydrate structures, forming glycoproteins.
The most important property of amino acids is the peptide bond formation.

– H 2O
H 2N CH C OH H N CH C OOH H 2N CH C NH CH C OOH
O H +H2O
R1 R2 O
R1 R2

• The equilibrium constant of that reaction strongly favours the peptide bond
hydrolysis. To synthesize the peptide bond, firstly the carboxyl group must be
activated. Chemically, it can be realized by transformation into a acidic chloride.
• Biologically, the activation implies the initial condensation with ATP.
Color reactions of amino acids

Fluorescamine

Ninhydrin
Separatory techniques for amino acids
• Chromatography: In this technique molecules are partitioned between a
stationary and a mobile phase. Separation depends on the relative
tendencies of molecules in a mixture to associate more strongly with one or
the other phase.
• Paper chromatography is a normal partition chromatography largely used for
the separation of amino acids. Thin-layer chromatography is also used.
• Ion-exchange chromatography (today an automated technique) uses columns,
which contain the Na+ form of a sulfonated polystyrene resin. The amino acids
will bind to the resin via cation exchange with Na+. Columns are then eluted
with sodium citrate under programmed conditions of pH and temperature.
Eluted material is reacted with ninhydrin reagent and color densities are
monitored in a flow-through colorimeter.
• High voltage electrophoresis: Amino acids net charge depends on the pH
of the surrounding medium. According to their charge, mass and form
(shape) they will be separated in a direct current field. For amino acids,
paper sheets or thin layers of powdered cellulose are most frequently used
as supports.