Chapter4 Amino Acid

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 CONH 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 with isoelectric focusing. • Assignment: determine the pI of a hexapeptide: 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 at 288 and 280. • Use 2 simultaneous equations to determine the number of Trp and Tyr: – Abs. (288nm)= NTrp4815 + MTyr385 – Abs. (280) = NTrp5690 + MTyr1280

• Calculate concentration of Trp and Tyr. Divide by number of Trp and 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 H C H3C

O re face

H C H3C

D

• Planar molecules can also have prochiral nature. C Acetaldehyde has a re face CH3 O and si face. Reduction of one si face side versus the other yields a different stereoisomer (if using deuterium or tritium). • Enzymes (dehydrogenases) have different preferences H for re or si face of substrates C • For example, if the aldehyde HO CH3 is reduced with deuterium D based on the re or si side, the two different enantiomer products are formed. H

OH

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.