a-Amino Acids: Power Point

Fig. 4-1------------------------------Fig. 4-2

An amino acid is a carboxylic acid with an amino group on the a-carbon, the C next to -COOH

R-Group varies with different aa's. 20 different R-groups differentiate the 20 different common aa's, the aa's found in proteins.

Acid Base Properties: --COOH and --NH2 are weak acid (proton donor) and weak base (proton acceptor) respectively ===> they undergo dissociation and ionization; at neutral pH, the structure in H2O is shown above to the right. This form is Electrically neutral ===> there is no net charge; but it is very dipolar as there is a +1 charge on the amino end and a -1 charge on the carboxyl end. This is called a Zwitterion.

Titration curve for Alanine: Fig. 4-8

At intermediate pH's, a mixture of forms exists

If the R-group contains an acidic or basic group, the titration curve will have 3 equivalence points and 3 pKa's.

For Alanine: R = -CH3

a-COOH pKa1 = 2.34 (pKa of a -COOH varies from 1.71 to 2.63 in different aa's)

a-Amino pKa2 = 9.69 (pKa of a-Amino varies from 8.95 to 10.78 in different aa's)

Isoelectric pH or Isoelectric Point -- pI: the pH at which on average the amino acid or other molecule has a net charge of 0.

For alanine, this is halfway between the 2 pKa's: pI = 2.35 + (9.87 - 2.35)/2 = 6.11

For amino acids with non-ionizable R-groups, pI will have a similar value because their -COOH and -NH2 groups have similar pKa's.

For amino acids with ionizable R-groups, pI's will be very different and depend upon the pKa and charge of the R-group. For example, Glutamate's R-group contains a second -COOH with a pKa of 4.25; it's pI occurs at the first equivalence point after titrating the -COOH (pKa 2.19) but before titrating the R-group -COOH. Thus, pI for Glu = 2.10 + (4.07 - 2.10)/2 = 3.09. On the other hand, Lysine's R-group contains a second amino group (pKa = 10.53); its pI occurs at the second equivalence point after the a-amino group (pKa = 8.95) has been titrated and the -charge on -COO- balances the +charge on the R-group amino. pI = 9.06 + (10.54 - 9.06)/2 = 9.8.

Note: the pKa of -COOH is 2.34 in Ala (and is similar in other aa's) but the same functional group has a pKa of 4.76 in acetic acid. This is due to the presence of the + charge on the a-amino group which repells the H+.


Stereochemistry

Absolute Configuration: Called D and L -- The absolute configuration of optically active compounds (chiral molecules) is related chemically to optical isomers of glutaraldehyde.

(OH- to left) (OH- to right) drawn with the most oxidized carbon at the top

Figure 4-11

Non-superimposable mirror images are possible when a tetrahedral C has 4 different groups on it; this is said to be a Chiral carbon (chiral means hand, and left and right hands are enantiomers).

Pure enantiomers rotate the plane of plane polarized light. One isomer will it rotate clockwise (called + or d for dextrorotatory) and the other will rotate it counterclockwise (called - or l for levorotatory).

The amount of rotation depends upon:

[a]25°C = (observed rotation) / [(pathlength in decimeters) (concentration in gm/ml)]

Figure 4-11,12

 

Proteins contain only L-isomers of amino acids. D-amino acids are found rarely; bacterial cell walls and some antibiotics contain D-amino acids.

The absolute configuration (D or L) does not say whether plane polarized light is rotated clockwise or counter-clockwise; thus, and L-amino acid may be either d(+) or l(-).

Box 4-1

Organic chemists normally use a different nomenclature to specifiy the absolute config. of a chiral C.

Note: L-compounds aren't always S, but it turns out that all amino acids in proteins except Cys are S.

R-Groups: Amino acids are classified according to their properties which are determined largely by their
R-groups as nonpolar, uncharged polar, and charged (acidic or basic). See
Table 4-1.







Peptide Bond: How are proteins (polypeptides) made from amino acids? Fig. 4-3

The peptide bond is an amide bond; this is formed by condensation of an amine with a carboxyl group by the elimination of a molecule of water. The C-N bond in amides is very different from the C-N bond in amines:

a) it's shorter than the amine C-N bond

b) the amide N is a much weaker base than an amine N ---> doesn't protonate at physiological pH's

The amide C-N bond is shorter because it has Double Bond Character:

The extra pair of bonding electrons are shared (unequally) between the carbonyl O and the amide N so that the amide C-N bond has about 40% double bond character.

What does this mean structurally?? The 2nd bond in a double bond is a p-bond formed between p atomic orbitals. The bilobed structure of p orbitals prevents rotation about p-bonds.

The bonds to alpha-C's are single bonds ===> free rotation about these bonds is permitted restricted only by steric interference of groups (i.e. do things bump into one another?).

Here each planar peptide group is represented by a rectangle connecte to a-C's at its vertices.

Acid/Base Properties of Peptides -- are determined by the N-Terminal Amino Group , the C-Terminal Carboxyl Group, and by any ionizable R-groups present. The pKa's of the groups will, in general, be different for aa's incorporated into peptides than for free aa's because the N-terminal and C-terminal groups are further apart and don't affect the titration of one another as much. Thus, C-terminal carboxyl groups in peptides generally have higher pKa's than those in free aa's while the N-terminal amino groups in peptides generally have lower pKa's.


SEPARATION OF AMINO ACIDS:

Separations based upon charge:

Electrophoresis -- place aa's in an electric field (e.g. paper moistened with a buffer)

aa's with a net +charge (cations) migrate toward the cathode (-electrode)

aa's with a net -charge (anions) migrate toward the anode (+electrode)

The rate of migration is proportional to the (net charge)/mass and upon the electric field strength (volts) ===> higher net charge ===> faster migration ===> smaller molecules move faster (for same net charge)

Ion Exchange Chromatography:

Whether an aa is anionic or cationic depends upon the pH and upon the pKa's of the ionizable groups, including the pKa of the R-group. The strength of binding depends upon the charge of the molecule (e.g. aa). The ion exchanger has counter ions bound to it electrostatically (e.g. see Na+ below); these are displaced (exchanged) by the charged aa's which are applied to the column.

a) Amino acids are removed in order of increasing affnity for the exchanger by applying a salt gradient; the ions added to increase the [salt] of the buffer compete with the aa's for binding sites on the ion exchanger. The aa's bound least strongly are competed off first; fractions are collected in test tubes in regular intervals using a fraction collector. The [salt] can be increased either in steps (step gradient) by changing the buffer reservoir or one can continuously increase the [salt] linearly by using a simple mixing device consisting of two chambers connected at the bottom.

b) Amino acids can also be removed by changing the pH either stepwise or gradually. The net charges of various aa's will change at different pH's depending upon their functional groups. Can also use a combination of these two methods; e.g. see Lehninger Fig. 5-15 for separation of 20 aa's.

Paper Chromatography -- separates aa's by polarity; solubility in H2O bound to cellulose in the paper vs. solvent which travels along the paper by capilarity.