Amino Acids Student Edition 5/23/13 Version Pharm. 304 Biochemistry Fall 2014 Dr. Brad Chazotte 008 Campbell Hall Web Site: Original material only © B. Chazotte

Goals Learn all the amino acid side chain structures Learn the pKa of amino acid side chains Understand the structure of the peptide bond Understand the basics of a titration curve and the relationship between pH and variation of the charge on an amino acid with pH. Be able to calculate/determine an isoelectric point Be able to calculate the pH of an amino acid solution Understand the concepts of stereochemistry, chirality, enantinomers, etc, and the stereochemistry of amino acids

Amino Acid: General Structure Voet, Voet & Pratt 2013 Fig 4.2 α-carbon Side chain Typical pK a1 ~ 2.2 Typical pK a2 ~ 9.4

Condensation of Two Amino Acids Voet, Voet & Pratt 2013 Fig 4.3 condensation Peptide bond, amide linkage Amino or N- terminus carboxy or C- terminus

Peptide Bond Structure Matthews et al.1999 Figure 5.12 Delocalization of the  -electrons over the O-C-N bonds to give a partial double bond character to the C-N bond - Significance: effects the bond angles, i.e., structure

The Amino Acids

Standard Amino Acids: Properties & Conventions Table Lehninger 2006 Table 3.1

Voet, Voet & Pratt 2013 Table 4.1A-D Standard Amino Acids: Properties & Conventions Tables

Lehninger 2005 Figure 3.5a Nonpolar Aliphatic Amino Acid Structures L Alanine Voet, Voet & Pratt 2006 Figure 4.4a M I GA V L P

Lehninger 2000 Figuree 5.5b Aromatic Amino Acid Structures FYW H-bonding Voet, Voet & Pratt 2013 Figure 4.4a

Lehninger 2005 Figure 3.5c Polar, Uncharged Amino Acid Structures Voet, Voet & Pratt 2013 Figure 4.5a Voet, Voet & Pratt 2013 Figure 4.5b STC N Q

Cysteine disulfide bonds Voet, Voet & Pratt 2013 Fig 4.6

Lehninger 2005 Figuree 3.5d Positively Charged Amino Acid Structures Voet, Voet & Pratt 2013 Figure 4.7b K RH

Lehninger 2005 Figuree 3.5e Negatively Charged Amino Acid Structures Voet, Voet & Pratt 2006 Figure 4.7a DE

Glycine Titration Voet, Voet & Pratt 2006 Fig 4.8 pI = ½ (pK i + pK j ) where K i and K j are the ionization constants for the neutral species In text K r refers to side chain ionization

Lehninger 2000 Figure 5.9 Nonionic & Zwitterionic Amino Acid Forms

Isoelectric Point Calculation pI = ½ (pK i + pK j ) where K i and K j are the ionization constants for the neutral species In text K r refers to side chain ionization For mono amino & monocarboxylic K i =K 1, K j =K 2 Aspartic & glutamic acids K i =K 1, K j =K r Arg, His, Lys K i =K r, K j =K 2

Lehninger 2000 Figuree 5.12a Titration of Glutamate (a “Negatively” charged Amino Acid)

Lehninger 2000 Figure 5.11 Effect of Chemical Environment on pK a

Calculation of the pH of a 0.1 M Glycine-HCl solution I Calculate the pH of a 0.1 M solution of glycine hydrochloride. Glycine-HCl is essentially a diprotic acid. The carboxyl group is a much stronger acid pKa=2.34 than the charged amino group pKa=9.6 the pH of the solution is established almost exclusively by carboxyl ionization + H 3 N-CH 2 -COOH  + H 3 N-CH 2 -COO - + [H + ] “AA +1 ” “AA 0 ” [AA 0 ] [H + ] K a1 = [AA +1 ]

Calculation of the pH of a 0.1 M Glycine-HCl solution II Let y = M of AA +1 that ionizes  y = M of H + produced And y = M of AA 0 produced Then y = M of AA +1 remaining (y)(y) K a1 = (0.1 – y) = 4.57 x = antilog pK a 2.34

Calculation of the pH of a 0.1 M Glycine-HCl solution III 4.57 x – 4.57 x y = y 2 y x y -4.57x = 0 -b ± √ b 2 -4ac Need to solve the quadratic y = 2a Where a = 1 b = 4.57 x and c = x and solving for y [H+] = 1.92 x M pH = x / 1.00 x =19.2% glycine HCl ionized

Amino Acid Nomenclature Lehninger 2000 Figure 5.2

Lehninger 2000 Figure 5.14 Peptide Nomenclature: A Pentapeptide “Serylglycyltyrosylalanylleucine” (Ser-Gly-Tyr-Ala-Leu)

Amino Acids & Stereochemistry

Optical Activity & Chiral Centers All amino acids recovered from proteins are optically active with the exception of glycine. Optical activity can be defined as the ability to rotate plane polarized light. Optically active molecules have an asymmetric configuration, i.e. they are not superimposable on their mirror image. The asymmetric center is called the chiral center. The  -carbon of amino acids is a chiral center. Caution: Whether a compound rotates plane polarized light to the left or the right does not provide information about the absolute configuration (arrangement of atoms in space).

Configuration Sequence Rules about an Asymmetric Carbon Cahn-Ingold-Prelog System Rule 1. If the four atoms attached to the asymmetric carbon are all different, priority depends on atomic number, with the atom of higher atomic number getting priority. If two atoms are isotopes of the same element, the atom of the higher mass number has the priority Rule 2. If the relative priority of two groups cannot be decided by Rule 1, it shall be determined by a similar comparison of the atoms next in the groups (and so on, if necessary, working out from the asymmetric carbon). Morrison & Boyd, 1966 Chapter 3; Voet & Voet 2003 Chapter 4;Matthew et al Fig 9.6 Designation: R – Rectus (right) the order of the groups about the asymmetric centers is clockwise S – Sinister (left) the order of the groups about the asymmetric center is counter clockwise

Lehninger Biochemistry 2000 Fig 9.2 Enantiomers (Mirror Images)

Chiral (Asymmetric) Carbons, Optical Activity & Stereoisomers Optically active molecules rotate plane polarized light. D -dextrorotary (right, clockwise). L -levorotary (left, counterclockwise). [ Note: Designation uses small capital letters] Optically active molecules have an asymmetry such that they are not superimposable on their mirror image. This situation is characteristic of substances that contain tetrahedral carbons having four different substituents. Stereoisomers compounds that have the same molecular formula but differ in the configuration of their atoms in space, about one of more of their chiral centers. Enantiomers are stereoisomers, molecules, that are not superimposable on their mirror images. Such molecule are physically and chemically indistinguishable by most techniques, except probing by plan polarized light

Fisher Convention for Viewing Carbohydrates Barker 1971 Chaper 5 RULES 1.The carbon chain is vertical with the lowest numbered carbon at the top. 2.The numbering usually follows the convention that the most oxidized end of the molecule has the lowest number. This “system” relates the configuration of the groups about an asymmetric center to that of glyceraldehyde. Glyceraldehyde has one asymmetric center. In a Fisher projection on paper: Horizontal bonds extend above the plane of the paper. Vertical bonds extend below the plane of the paper

Fischer Convention (Projections) Voet, Voet & Pratt 2013 Fig 4.11 COOH H 3 N + -C-H R L -  -amino acid

Stereochemical Facts for Amino Acids L -glyceraldehyde and L -  -amino acids are said to have the same relative configuration. All amino acids derived from protein are in the L stereochemical configuration. (Remember that the L or D designation for an amino acid does not indicate its ability to rotate plan polarized light.

Chirality and Pharmaceuticals In contrast to most chemical syntheses which produce a racemic product mixture, biosynthetic processes almost always produce pure stereoisomers. Due to the fact that most biomolecules are chiral a single enantiomeric molecule will likely bind to only one enantiomeric form of another molecule. Pharmaceutics: Many pharmaceuticals that are chemically synthesized are racemic mixtures where only one enantiomer is active, e.g. ibuprofen – an anti-inflammatory. In other cases a harmful enantiomer must be removed to prevent side effects such as with thalidomide, a mild sedative, whose other enantiomer caused severe birth defects.

Nonstandard Amino Acids Other nonstandard amino acids such as O-Phosphoserine, 4- hydroxyproline, 3-methyl histidine, etc. are part of proteins however: “In almost all cases, the unusual amino acids results from the specific modification of an amino acid residue after the polypeptide chain has been synthesized.” D -amino acids are found in short polypeptides of bacterial cell walls which renders the walls less susceptible to peptidases. (Many D amino acids are specifically joined together by bacterial enzymes and not by standard protein synthesis) Also some D -amino acids are components of bacterially produced antibiotics such as valinomycin, gramacidin A, and actinomycin D Voet, Voet & Pratt 2013 Chapter 4 p.88

Important non-Protein AA Matthews et al.1999 Table 5.2

End of Lectures