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Macromolecules in the cell. 513341 BIOCHEMISTRY I Chapter 2 Amino acids and Primary structure. Dr. PORNTIP CHAIMANEE Chemistry Department Faculty of Science Silpakorn University. Amino acids. 1. Amino acids structure 2. Asymmetry of amino acids

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513341 BIOCHEMISTRY I

Chapter 2

Amino acids and Primary structure

Dr. PORNTIP CHAIMANEE

Chemistry Department

Faculty of Science

Silpakorn University


Amino acids
Amino acids

1. Amino acids structure

2. Asymmetry of amino acids

3. Acid and Base properties

of amino acids



Three classes of amino acids
Three classes of amino acids

1. Non polar R groups

2. Uncharged polar R groups

3. Charges polar R group



D l fischer nomenclature

Stereochemistry of Amino Acids

D,L (Fischer)Nomenclature

• Horizontal substituents are pointing towards you

• Vertical substituents are pointing away

• Carbon in highest oxidation state is at the top

• D if the non-hydrogen group is to the right

• For glycine both horizontal groups are H - achiral

L-Alanine

D-Alanine


Stereochemistry of amino acids
Stereochemistry of Amino Acids

D,L (Fischer) Nomenclature

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

  • L-amino acids predominate in nature


Acid and base properties of amino acids

Amphoteric

@ All amino acids have three ionizable groups

except glycine

@ carboxyl at Ca with pKa between 1.8-2.8

@ amino group with pKa between 8.8-10.6

@ side chain R- groups


Acid base chemistry amino acids are weak polyprotic acids
Acid-Base ChemistryAmino acids are weak polyprotic acids

It has both anionic and cationic properties


Titration curve of glycine

The pH at which amino acid has no net charge is called the isoelectric point (pI)

Titration curve of glycine

Sodium hydroxide

glycine


Electrical properties and titration curve of glutamic acid

Monopositive Isoelectric Mononegative Dinegative

(Cation) (Zwitterion) (Zwitterion) (Anion)

Electrical properties and titration curve of Glutamic acid

Isoelectric point (pI) for amino acids with ionizable side chains:

Take average pKa for the two ionizations involving the neutral (net charge of zero) species.

pI of Glu = (2.2 + 4.3)/2 = 3.3


Sample calculation
Sample Calculation

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

  • Glu+ is dissociated 1/4 so Glu0is 1/4 and Glu+(what’s left) is 3/4.

  • Use the Henderson-Hassebalch Equation :

    pH = pKa of COOH+ log [A-]/[HA]

    pH = pKa of COOH + log [Glu+]/[Glu+]

    pH = 2 + log (0.25/0.75)

    pH = 2 + (-0.477)

    pH = 1.523


Electrical properties and titration curve of lysine

Dipositive Monopositive Isoelectric Mononegative

(Cation) (Zwitterion) (Zwitterion) (Anion)

Electrical properties and titration curve of Lysine

a

b

g

pKe-NH2

pK a-NH2

pK a-COOH

d

e

pI of Lys = (9.2 + 10.8)/2 = 10


Another sample calculation
Another Sample Calculation

At pH 9.5, what percentage of the alpha- and epsilon- amino groups of a 1 mM lysine solution are not protonated?

For each amino group: [NH2] + [NH3+] = 1 x 10-3

For the a-amino group

9.5 = 9.0 + log[NH2]/[NH3+]

3.16 = [NH2]/[NH3+]

So [NH3+] = 0.24mM and [NH2] = 0.76mM

76% is present as NH2

For the e-amino group

9.5 = 10.5 + log10[NH2]/[NH3+]

0.1 = [NH2]/[NH3+]

So [NH3+] = 0.91mM and [NH2] = 0.09mM

9% is present as NH2

At pH 9.5 the ratio of a-NH2 to the e-NH2 is 8.4:1

This is the kind of thing that’s useful to know if you want to do chemistry specifically at the a-amino group of a peptide

The Henderson-Hasselbalch equation allows calculation of the ratio of a weak acid and its conjugate base at any pH


Electrical properties and t itration curve of histidine

His0

His-

His2+

His+

Electrical properties and titration curve of histidine

pI of His = (6.0 + 9.17)/2


The pk values of the a carboxyl a amino groups and side chains found in the individual amino acids
The pK values of the a-carboxyl, a-amino groups and side chains found in the individual amino acids

pI

(isoelectric point)

6.2

5.9

5.9

5.7

5.9

2.8

3.2

7.6

5.0

5.6

9.7

10.7


Reactions of amino acids
Reactions of Amino Acids

• Amino groups form Schiff ‘s bases and amides

• Carboxyl groups form amides & esters

• Some side chains show unique reactivities

– Cys residues can form disulfides and can be easily alkylated

– Cys His Asp Glu common metal-binding ligands



Chemical properties of amino acids

NINHYDRIN REACTION

a - amino acid + Ninhydrin

Violet solution

+

3


Spectroscopic properties
Spectroscopic properties

UV Absorbance Spectra of Aromatic Amino Acids

• All amino acids absorb in infrared region

• Only Phe, Tyr, and Trp absorb UV

• Absorbance at 280 nm is a good diagnostic device for amino acids


Biological active amino acid derivatives
Biological active amino acid derivatives

If the acid is removed , it is converted to Dopamine

Glutamic acid

COOH

Dihydroxyphenylalanine

GABA

COOH

Histidine

Thyroxine

Histamine


Formation of a peptide
Formation of a Peptide

Peptides

• Short polymers of amino acids

• Each unit is called a residue

• 2 residues - dipeptide

• 3 residues - tripeptide

• 12-20 residues - oligopeptide

• many - polypeptide

Glycylalanine


Properties of peptide
Propertiesofpeptide

Direction of peptide chain

amino or

N-terminus

carboxyl or C-terminus

Peptides

• Short polymers of amino acids

• Each unit is called a residue

• 2 residues - dipeptide

• 3 residues - tripeptide

• 12-20 residues - oligopeptide

• many - polypeptide


Primary structure or sequence

SEQUENCE

The single- and three- letters codes for amino terminal of a primary sequence


Many peptides are biologically active
Many peptides are biologically active

Two amino acid difference at position 3 and 8 !!!!!

Oxytocin

-stimulates uterine

contractions during labor

Vasopressin

-Antidiuretic

*adjust water reabsorbed

by the kidney



Primary structure or sequence

The single- and three- letters codes for amino terminal of a primary sequence


Primary Structure of Bovine Insulin

Hormone insulin that regulates sugar levels in blood.

First protein to be fully sequenced (by Fred Sanger in 1953). For this, he won his first Nobel Prize.

Insulin compose with two chains (A and B)

Amino acid sequence of human insulin is very similar to that from cows and pigs (only amino acids at 8,9,10 (A) and 30 (B) differ )


1 o structure sequencing
1o structure sequencing



Ion exchange chromatography
Ion-Exchange Chromatography

Separation of amino acid mixtures by chromatography(Ion exchange or High-performance liquid chromatography)

Amino acids

Amino acids


Identification of n and c terminal residue
Identification of N and C-Terminal Residue

  • N-terminal analysis:

  • FDNB

  • Edman degradation using phenylisothiocyanate and derivatives are phenylthiohydantions (PTH)

  • C-terminal analysis

  • (1)Enzymatic analysis (C-terminus-specific exopeptidase ,carboxypeptidase).

  • – Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys

  • – Carboxypeptidase B (hog pancreas) only works on Arg and Lys

  • (2) done by hydrazinolysis (reaction with anhydrous hydrazine in presence of mildly acidic ion exchange resin)

NO2

(trifluoroacetic acid)

(PTC)

(PTH)

and identified by HPLC



Fragmentation of the chains1
Fragmentation of the chains

Enzymatic fragmentation– trypsin, chymotrypsin, clostripain, staphylococcal protease


Fragmentation of the chains2
Fragmentation of the chains

Chemical Fragmentation :Cyanogen bromide

CNBr acts only on methionine residues

CNBr is useful because proteins usually have only a few Met residues


Reconstructing the sequence
Reconstructing the Sequence

• Use two or more fragmentation agents in separate fragmentation experiments

• Sequence all the peptides produced (usually by Edman degradation)

• Compare and align overlapping peptide sequences to learn the sequence of the original polypeptide chain

Compare cleavage by trypsin and staphylococcal protease on a typical peptide:

• Trypsin cleavage:

A-E-F-S-G-I-T-P-K L-V-G-K

• Staphylococcal protease:

F-S-G-I-T-P-K L-V-G-K-A-E

• The correct overlap of fragments:

L-V-G-K A-E-F-S-G-I-T-P-K

L-V-G-K-A-E F-S-G-I-T-P-K

• Correct sequence:

L-V-G-K-A-E-F-S-G-I-T-P-K





A molecular clock
A molecular clock

  • Plot the number of changes in amino-acids between the same protein in different species (such as cytochrome C) against the time since the species diverged

  • Gives a straight line - so evolution of a protein sequence proceeds at a constant rate and therefore can be used as a clock

  • Calibration of the clock for specific protein families would ensure the dating of biological events not present in the fossil record and would imply that changes are non-adaptive due to their independence of the selective constraints


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