Chapter 4 Amino Acids

General Properties

• There are 20 standard amino acids. With exception of proline, all have primary amino group and a carboxylic acid bonded to same carbon.

• Carboxyl group and amino group all have pKa values around 2.2 and 9.4 respectively

• Note that at physiological pH of 7, amino acids are charged as shown to left. They can act as either acid or base.

• Also referred to as zwitterions• This imparts special properties e.g. some have melting points of 300 C,

just like salts. Also very soluble in water and not organics.

Peptide Bonds• Peptide bond formed from

elimination of water from 2 amino acids resulting in CO-NH linkage.

• Can have di-, tri-, oligo- and poly-peptides.

• Proteins composed of linear polymer of amino acids.

• Large number of proteins can exist. A small protein of 100 amino acids can have 20100 or 1.27x10130 possible unique polypeptide chains.

Classification of amino acids• Side chains of amino acids are responsible

for many of the unique properties of proteins

• 3 major classes of sides chains– hydrophobic– charged– polar– can also count 4 classes if Gly considered in own class

• The 20 different amino acids are commonly abbreviated with 1 or 3-letter codes

Hydrophobic Amino Acids 1

Hydrophobic Amino Acids 2

Charged Amino Acids

Polar Amino Acids 1

Polar Amino Acids 2

Hydrophobic Amino Acids 1

Disulfide-linked Cys residues• Two adjacent cysteine

residues can be oxidized to form a disulfide bond

• disulfide bonds are usually found in extracellular and not intracellular proteins– inside of cell is a

reducing environment

• disulfide bonds can stabilize protein structure by providing crosslink

Significance of Side Chains

• Select number can participate in “chemistry” of enzyme active sites. Those with charged or polar groups provide interesting catalytic groups.

• Function of nonpolar amino acids? A protein folding issue. Can think of protein as hydrophobic core made of different Lego pieces. These pieces have to fit together in compact fashion.

Acid-Base Properties of Amino Acids• Titration curve for glycine

– Note that one starts with all groups in acid form.

– Note how many equivalents are added

– Note that at 0.5 and 1.5 equivalents, pH is equal to pK of group being titrated.

– Note pH which gives zero charge is the isoelectric point. Calculated as (pK1+ pK2)/2

– Note where the buffering capacity is best

More Titration Curves

Environment and pKas• pKa are influenced by environment. • To predict influence of environment, examine the charge of

the acid and the conjugate base. E.g. His side chain has a cationic acid and neutral base whereas Asp has neutral acid and anionic base.

• Do groups near the acid stabilize the acid or the base. E.g. if negative charge near a His residue, it would stabilize the positive charge of the acid, thereby making it a weaker acid and increasing the pK. Local environment can be:– Positively charged– Negatively charged– Hydrophobic

• Based on above, explain why pK of a-carboxyl and a-amino group is lower than expected.

Misc. about proteins and nomenclature• Proteins have complex titration curves.

Many ionizable side chains whose pKs can be shifted by local environment

• Isoelectric point of proteins can be calculated based on amino acid sequence or experimentally determined withisoelectric focusing.

• Assignment: determine the pI of ahexapeptide: Gly-Glu-Asp-His-Lys-Ala

Optical Activity

• All amino acids except for gly rotate polarized light

• Optically active molecules have asymmetry such that mirror images are not superimposable

• Terms: asymmetic centers, chiral centers, enantiomers.

• Enantiomers physically and chemically indistinguishable by most techniques

Systems for describing enantiomers

• Direction which a cpd rotates polarized light. Dextrorotator (rt) also (+) or levorotatory (lf) also (-). Determined by polarimeter.

• Fischer convention is related to glyceraldehyde and uses D or L.

• All amino acids from proteins have L stereochemical configuration.

•The RS system depends on atomic numbers. Lowers is placed behind chiral center. Number then from highest to lowest. R is clockwise and S is counter clockwise.

Diastereomers

L-threonine is 2S-3R, name the other diasteromers

Spectroscopy of amino acids

Only the aromatic amino acids absorb light in the UV region

Determination of extinction coefficient for a protein

• Variety of methods available, this one is useful when the sequence is known

• Edelhoch et al. [Biochemistry (1967) 6, 1948-1954]• From known sequence, determine Trp and Tyr content• With purified protein of interest, determine the absorbance at 280

nm (or maximum near this wavelength) in buffered solution• Take sample of known absorbance, dissolve in 6 M guanidine

hydrochloride in 0.02 M phosphate, pH 6.5. Record absorbance at288 and 280.

• Use 2 simultaneous equations to determine the number of Trp andTyr:– Abs. (288nm)= NTrp4815 + MTyr385– Abs. (280) = NTrp5690 + MTyr1280

• Calculate concentration of Trp and Tyr. Divide by number of Trpand Tyr per enzyme to obtain concentration of enzyme in solution.

Prochiral

• Prochiral: replacement of one of the substituents turns achiral center into a chiral one. Enzymes, like you, can recognize and distinguish between pro R and pro S atoms.

More prochiral• Planar molecules can also have

prochiral nature. Acetaldehyde has a re face and si face. Reduction of one side versus the other yields a different stereoisomer (if using deuterium or tritium).

• Enzymes (dehydrogenases) have different preferences for re or si face of substrates

• For example, if the aldehyde is reduced with deuterium based on the re or si side, the two different enantiomer products are formed.

CO

H

H3C

CCH3

H

O

re facesi face

C OH

H

H3CC

CH3

H

HOD D

Chirality and life• Ordinary synthesis of chiral molecules

produces racemic mixtures. Ordinary methods do not show stereochemical preference.

• A fact of life: biosynthesis of substances having asymmetric centers almost produce pure stereoisomers.

• Using this criteria, examination of amino acids in meterorites always show racemic mixture. Thus not based on life.

• Not known why life has shown this preference.