Lecturer Mr. S.H.K Mazando SBT112 1st semester double credit course
Amino Acids
As the name implies, amino acids contain an amine and a carboxylic acid functional group. These functional groups dominate their physical and chemical properties. There are over 700 naturally occurring amino acids. Amino acids are classified according to the relative position of the functional groups.
1, 1- = amino acid, for example, glycine. 1, 2- = amino acid, for example, -alanine. 1,3- = amino acid, for example, -aminobutyric acid (GABA)
-Amino Acids
The 20 naturally occurring -amino acids used by cells to synthesise proteins can be generally represented by the generic formula shown below. This means the main difference between the various amino acids lies in the structure of the "R" group.
These 20 -amino acids can be sub-classified according to how the properties of other functional groups in the "R" group influence the system.
non-polar side chains (e.g. alkyl groups) polar (e.g. amides, alcohols) acidic (e.g. carboxylic acids, phenols) basic (e.g. amines)
Alanine
Ala (A)
alanine
S-(-)-glyceraldehyde or L-glyceraldehyde
For the 20 -amino acids that occur naturally in proteins, if we focus on the -center, a chirality center, and draw the Fischer projection putting the -CO2H group up, then the ammonium group, NH3+, will be on the left, making it like L-glyceraldehyde where the -OH is on the left Although only the L-amino acid series are incorporated into natural proteins, the Damino acids also occur naturally.
Even the simplest amino acids have both an acidic functional group, the carboxylic acid, and a basic functional group the amine. Compounds that can behave as both acids and bases are said to be amphoteric. The equations that define acidity and basicity are: Remember that the lower the pKa, the stronger the acid
From these expressions it is possible to derive the important Henderson-Hasselbalch equation : pKa = pH + log [HA] / [A-]
Implications:
Typical simple carboxylic acids, RCO2H, have a pKa of about 5, and typical simple ammonium ions RNH3+ have a pKa of about 9. Therefore, since the acid is the stronger acid (lower pKa) the amino acid will exist in the zwitterionic form where the acid has protonated the amine in neutral aqueous solution (or normal physiological pH).
This information will let you decide which structure of an acid or base will dominate at a particular pH.
To the left are the processes for the amino acid Histidine which has an extra basic group. It has three acidic groups of pKa's 1.82 (carboxylic acid), 6.04 (pyrrole NH) and 9.17 (ammonium NH). Histidine can exist in the four forms shown, depending on the solution pH, from acidic pH (top) to basic pH. (bottom). Starting from the top, we can imagine that as we add base, the most acidic proton is removed first (COOH), then the pyrrole NH then finally the amino NH. These take us through each of the forms in turn. At pH < 1.82, A is the dominant form. In the range 1.82 < pH < 6.02 B is the dominant form. In the range 6.02 < pH < 9.17 C is the dominant form, and when pH > 9.17, D is the major form in solution.
Isoelectronic point, pI
The isoelectronic point or isoionic point is the pH at which the amino acid does not migrate in an electric field. This means it is the pH at which the amino acid is neutral, i.e. the zwitterion form is dominant. The pI is given by the average of the pKas that involve the zwitterion, i.e. that give the boundaries to its existence. There are 3 cases to consider....
Whereas all of these reactivities are important in the study and analysis of isolated amino acids, it is the characteristic behaviour of the side chain that governs the reactivity of amino acids incorporated into proteins. The carboxyl groups of amino acids undergoes reactions with ammonia and primary amines to yield unsubstituted and substituted amides, respectively Esters and acid chlorides are also readily formed. Esterification proceeds in the presence of the appropriate alcohol and a strong acid
Esterification of the carboxylic acid is usually conducted under acidic conditions, as shown in the two equations written below. Under such conditions, amine functions are converted to their ammonium salts and carboxylic acids are not dissociated. The initial product is a stable ammonium salt. The amino ester formed by neutralization of this salt is unstable, due to acylation of the amine by the ester function.
+ H+
C OH
R' O H
O O
R C R' O
O O H
R C R' O
O H
O R H C R' O O H
H O R C O R' H O H (H3O+)
ester
Step 1: An acid/base reaction. Protonation of the carbonyl makes it more electrophilic. Step 2: The alcohol O functions as the nucleophile attacking the electrophilic C in the C=O, with the electrons moving towards the oxonium ion, creating the tetrahedral intermediate. Step 3: An acid/base reaction. Deprotonate the alcoholic oxygen. Step 4: An acid/base reaction. Need to make an -OH leave, it doesn't matter which one, so convert it into a good leaving group by protonation. Step 5: Use the electrons of an adjacent oxygen to help "push out" the leaving group, a neutral water molecule. Step 6: An acid/base reaction. Deprotonation of the oxonium ion reveals the carbonyl in the ester product.
Amine Acylation
In order to convert the amine function of an amino acid into an amide, the pH of the solution must be raised to 10 or higher so that free amine nucleophiles are present in the reaction system. Carboxylic acids are all converted to carboxylate anions at such a high pH, and do not interfere with amine acylation reactions. The following two reactions are illustrative. In the first, an acid chloride serves as the acylating reagent. The second reaction employs an anhydride-like reagent for the acylation.
This is a particularly useful procedure in peptide synthesis, thanks to the ease with which the t-butylcarbonyl (t-BOC) group can be removed at a later stage. Since amides are only weakly basic ( pKa~ -1), the resulting amino acid derivatives do not display zwitterionic character, and may be converted to a variety of carboxylic acid derivatives.
The ammonia produced in this way can react with the hydrindantin and another molecule of ninhydrin to yield a purple product ( Ruhemanns Purple) that can be quantified spectrophotometrically at 570 nm. The appearance of CO2 can also be monitored.
-Imino acids, such as proline and hydroxyproline, give bright yellow ninhydrin products with absorption maxima at 440 nm, allowing these to be distinguished from the amino acids. Because amino acids are one of the components of human skin secretions, the ninhydrin reaction was once used extensively by law enforcement and forensic personnel for fingerprint detection (Fingerprints as old as 15 years can be successfully identified using the ninhydrin reaction.)
In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. The mild oxidant iodine reacts selectively with certain amino acid side groups. These include the phenolic ring in tyrosine, and the heterocyclic rings in tryptophan and histidine, which all yield products of electrophilic iodination. In addition, the sulfur groups in cysteine and methionine are also oxidized by iodine.
Ellman's reagent is used for the modification of free thiols in proteins. It rapidly forms a disulfide bond with the thiol and releases a thiolate ion which is coloured. The maximal absorbance of this thiolate is at 412 nm. It's presence can be plotted against a standard curve to determine the total amount of free thiols in proteins.
Cysteine is a thiol, and like most thiols it is oxidatively dimerized to a disulfide, which is sometimes listed as a distinct amino acid under the name cystine. Disulfide bonds of this kind are found in many peptides and proteins. For example, the two peptide chains that constitute insulin are held together by two disulfide links.
Qu 4:
The amino acid histidine has two potentially basic N sites in the side chain, which one is more basic and why?