Biomolecules: Amino Acids, Peptides and Proteins

LecturerMr. S.H.K Mazando

SBT112 1st semester double credit course

Amino Acids, Peptides and Proteins

• Amino acids contain at least an amine and carboxylic acid functional group

• Amino acids can be joined via amide bonds to give peptides

• Proteins are polypeptides of 50 or more amino acids

• Proteins are a fundamental biological component for skin, hair, muscles, connective tissues, many enzymes etc.

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- = α amino acid, for example, glycine.

1, 2- = β amino acid, for example, β-alanine.

1,3- = γ amino acid, for example, γ-aminobutyric acid (GABA)

α-Amino Acids • The 20 naturally occurring α-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.

The generic structure of an α-amino acid

• These 20 α-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)

Amino acids with non-polar side chains• The hydrophobic side chains are chemically

unreactive and tend to aggregate rather than be exposed to the aqueous environment, so they tend to found on the interior of proteins.

• They exhibit the hydrophobic effect - the aggregation of non-polar systems in an aqueous environment

Amino acids with non-polar side chains

GlycineGlc (G)

AlanineAla (A)

ValineVal (V)

LeucineLeu (L)

IsoleucineIle (I)

MethionineMet (M)

ProlinePro (P)

PhenylalaninePhe (F)

TryptophanTrp (W)

Amino acids with polar side chains. • These are side chains can be involved in hydrogen

bonding interactions. • Cysteine is important because of its ability to form

disulfide bonds.

AsparagineAsn (N)

GlutamineGln (Q)

SerineSer (S)

ThreonineThr (T)

TyrosineTyr (Y)

CysteineCys (C)

Amino acids with acidic side chains.

• These carboxylate group will be -ve at physiological pH.

• Often involved at the active sites of enzymes, in hydrogen bonding interactions and in acid/base type reactivity.

Aspartic acidAsp (D)

Glutamic acidGlu (E)

Amino acids with basic side chains• Often involved at the active sites of enzymes,

in hydrogen bonding interactions and in acid/base type reactivity (e.g. histidine)

HistidineHis (H)

ArginineArg (R)

LysineLys (K)

Amino Acid Stereochemistry • Fischer projections are commonly used to

represent amino acids. • Recall that Fischer projections are typically drawn

with the longest chain oriented vertically and with the more highly oxidised C at the top.

alanineS-(-)-glyceraldehyde

or L-glyceraldehyde

• For the 20 α-amino acids that occur naturally in proteins, if we focus on the α-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 D-amino acids also occur naturally.

Structure and pKa of Amino Acids • 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-]

How this equation help us• It tells us that when the pH = pKa then log [HA] / [A-] =

0 therefore [HA] = [A-] i.e. equal amounts of the two forms, the acid and the conjugate base.

• If we make the solution more acidic, i.e. lower the pH, so pH < pKa, then log [HA] / [A-] has to be > 0 so [HA] > [A-]. This makes sense as it tells us that a stronger acid will cause the formation of HA, the protonated form.

• If instead we make the solution more basic, ie raise the pH, so pH > pKa and log [HA] / [A-] has to be < 0 so [HA] < [A-]. This makes sense as it tells us that a stronger base will cause the formation of A- , the deprotonated form.

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....

neutral side chains • These amino acids are characterised by two pKas :

pKa1 and pKa2 for the carboxylic acid and the amine respectively. The isoelectronic point will be halfway between, or the average of, these two pKas, i.e. pI = 1/2 (pKa1 + pKa2).

• at very acidic pH (below pKa1) the amino acid will have an overall +ve charge and at very basic pH (above pKa2 ) the amino acid will have an overall -ve charge. For the simplest amino acid, glycine, pKa1= 2.34 and pKa2 = 9.6, pI = 5.97.

acidic side chains • The pI will be at a lower pH because the acidic

side chain introduces an "extra" negative charge.• So the neutral form exists under more acidic

conditions when the extra -ve has been neutralised.

• for aspartic acid shown below, the neutral form is dominant between pH 1.88 and 3.65, pI is halfway between these two values, i.e. pI = 1/2 (pKa1 + pKa3), so pI = 2.77

basic side chains • The pI will be at a higher pH because the basic

side chain introduces an "extra" positive charge. • So the neutral form exists under more basic

conditions when the extra +ve has been neutralised. For example, for histidine, which was discussed on the previous slide, the neutral form is dominant between pH 6.00 and 9.17, pI is halfway between these two values, i.e. pI = 1/2 (pKa2 + pKa3), so pI = 7.59.

Titration curve of an amino acid

Reactions of Amino Acids • Amino acids contain two functional groups:

amines and carboxylic acids. • So amino acids undergo the reactions

characteristic of those functional groups:• The α-carboxyl and α-amino groups of all amino

acids exhibit similar chemical reactivity• The side chains, however, exhibit specific

chemical reactivities, depending on the nature of the functional groups.

• 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

Carboxylic Acid Esterification• 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.

R COH

O

+ H+

carboxylic acid

Fischer Esterification Reaction Mechanism (nucleophilic acyl substitution)

R COH

OH

R C

OH

OH

+R'

O

alcohol(weak nucleophile)

H

R

C

O

OH

R' O

H

H

R

C

O

OH

R' O

H

H

tetrahedral intermediate

R

C

O

OH

R' O H

H

R

C

O

OH

R' O

H

H

+

R

C

OH

R' O O H

H

R C

O

R'OO H

H

H

+

(H3O+)ester

proton transfergood leavinggroup

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 Ninhydrin Reaction• common alpha-amino acids, except proline,

undergo a unique reaction with the triketohydrindene hydrate known as ninhydrin.

• ninhydrin, is a strong oxidizing agent and causes the oxidative deamination of the a-amino function.

• The products of the reaction are the resulting aldehyde, ammonia, carbon dioxide, and hydrindantin, a reduced derivative of ninhydrin .

•The ammonia produced in this way can react with the hydrindantin and another molecule of ninhydrin to yield a purple product ( Ruhemann’s Purple) that can be quantified spectrophotometrically at 570 nm.• The appearance of CO2 can also be monitored.

• α-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 α-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.)

Specific Reactions of Amino Acid Side Chains

• 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?