Amino Acids and Proteins CHAPTER EIGHTEEN Amino Acids and Proteins Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Amino Acids Amino acid: A compound that contains both an amino group and a carboxyl group. a-amino acid: an amino acid in which the amino group is on the carbon adjacent to the carboxyl group. Although a-amino acids are commonly written as (a) the un-ionized form, they are more properly written as (b) the zwitterion (internal salt) form. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Chirality of Amino Acids With the exception of glycine, all protein- derived amino acids have at least one stereocenter (the a-carbon) and are chiral. The vast majority have the L-configuration at their a-carbon. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Protein-derived Amino Acids Nonpolar side chains (predominate form at pH 7.0) Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Protein-derived Amino Acids Polar side chains (predominant form at pH 7.0) Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Protein-derived Amino Acids Acidic and basic side chains (form at pH 7.0) Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Amino Acids Other common L-amino acids Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Acid Base Properties Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Acid-Base Properties Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Acidity of a-COOH Groups The average pKa of an a-carboxyl group is 2.19, which makes them considerably stronger acids than aliphatic carboxylic acids such as acetic acid (pKa 4.76). The greater acidity is accounted for by the electron- withdrawing inductive effect of the adjacent -NH3+. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Side Chain COOH Groups Due to the electron-withdrawing inductive effect of the a-NH3+ group, side chain ‑COOH groups are also stronger acids than acetic acid. The effect, however, decreases with distance from the ‑NH3+ group. compare: -COOH group of alanine (pKa 2.35) b-COOH group of aspartic acid (pKa 3.86) g-COOH group of glutamic acid (pKa 4.07) Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Acidity of a-NH3+ Groups The average value of pKa for an -NH3+ group is 9.47, compared with a average value of 10.76 for 1° alkyl ammonium ions. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Basicity of Guanidine Group The side chain of arginine contains a guanidine group. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

The Imidazole Groups of His The imidazole group is a heterocyclic aromatic amine. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Titration of Amino Acids Figure 18.3 Titration of glycine with NaOH. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Isoelectric Point (pI) Isoelectric point, pI: The pH at which the majority of amino acid molecules in solution have no net charge. The pI for glycine (6.06), for example, falls between the pKa values for the carboxyl and amino groups. The following tables give isoelectric points for the 20 protein-derived amino acids. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Isoelectric Point (pI) Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Isoelectric Point (pI) Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Electrophoresis Electrophoresis: The process of separating compounds on the basis of their electric charge. Electrophoresis of amino acids can be carried out using paper, starch, agar, certain plastics, and cellulose acetate as solid supports. In paper electrophoresis A paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between two electrode vessels. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Electrophoresis Figure 18.4 Electrophoresis of a mixture of amino acids. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Electrophoresis A sample of amino acids is applied as a spot on the paper strip. An electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own. Molecules with a high charge density move faster than those with low charge density. Molecules at their isoelectric point remain at the origin. After separation is complete, the strip is dried and developed to make the separated amino acids visible. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Electrophoresis The reagent most commonly used to detect amino acids is ninhydrin. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Polypeptides and Proteins In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by peptide bonds. Peptide bond: The special name given to the amide bond between the -carboxyl group of one amino acid and the -amino group of another. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Peptides Peptide: The name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain. Dipeptide: A molecule containing two amino acids joined by a peptide bond. Tripeptide: A molecule containing three amino acids joined by peptide bonds. Polypeptide: A macromolecule containing many amino acids joined by peptide bonds. Protein: A biological macromolecule of molecular weight 5000 g/mol or greater, consisting of one or more polypeptide chains. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Serylalanine (Ser-Ala) Figure 18.5 The peptide bond in serylalanine Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Writing Peptides By convention, peptides are written from the left to right , beginning with the free -NH3+ group and ending with the free –COO– group. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Primary Structure Primary structure: The sequence of amino acids in a polypeptide chain, read from the N‑terminal amino acid to the C‑terminal amino acid. Amino acid analysis Hydrolysis of the polypeptide, most commonly carried out using 6 M HCl at elevated temperature. Quantitative analysis of the hydrolysate by ion-exchange chromatography. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Cyanogen Bromide Figure 18.7 Cleavage by BrCN of a peptide bond formed by the carboxyl group of methionine. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Enzyme-Catalyzed Cleavage A group of protein-cleaving enzymes can be used to catalyze the hydrolysis of specific peptide bonds. Among them are the following: Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Edman Degradation Edman degradation: Cleaves the N-terminal amino acid of a polypeptide chain. Figure 18.8 Cleavage of the N-terminal amino acid as a substituted phenylthiohydantoin. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Primary Structure Example 18.6 Deduce the 1° structure of a pentapeptide from this experimental information. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Geometry of a Peptide Bond The four atoms of a peptide bond and the two alpha carbons bonded to it lie in a plane with bond angles of 120° about C and N. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Geometry of a Peptide Bond To account for this geometry, Linus Pauling proposed that a peptide bond is most accurately represented as a hybrid of two contributing structures. The hybrid has considerable C–N double bond character and rotation about the peptide bond is restricted. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Geometry of a Peptide Bond Two configurations are possible for a planar peptide bond. Virtually all peptide bonds in naturally occurring proteins studied to date have the trans configuration. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Secondary Structure Secondary structure: The ordered arrangements (conformations) of amino acids in localized regions of a polypeptide or protein. To determine from model building which conformations would be of greatest stability, Pauling and Corey assumed that All six atoms of each peptide bond lie in the same plane and have the trans configuration. There is hydrogen bonding between the N-H group of one peptide bond and a C=O group of another peptide bond as shown in the next screen. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Secondary Structure Figure 18.10 Hydrogen bonding between amide groups. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Secondary Structure On the basis of model building, Pauling and Corey proposed that two types of secondary structure should be particularly stable. the a-helix the antiparallel -pleated sheet -Helix: A type of secondary structure in which a section of polypeptide chain coils into a spiral, most commonly a right-handed spiral. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. An -Helix Figure 18.11 An a-helix. Polyalanine, cylindrical bonds model, viewed along its length. Sites of Hydrogen bonding are visible. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. An -Helix Polyalanine looking down the axis of the helix. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. An a-Helix Structural features of the -helix There are 3.6 amino acids per turn of the helix. Each peptide bond trans and planar. N-H groups of all peptide bonds point in the same direction, which is roughly parallel to the axis of the helix. C=O groups of all peptide bonds point in the opposite direction, and also roughly parallel to the axis of the helix. The C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it. All R- groups point outward from the helix. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. -Pleated Sheet The antiparallel -pleated sheet consists of adjacent polypeptide chains running in opposite directions. Each peptide bond is planar and has the trans configuration. The C=O and N-H groups of peptide bonds from adjacent chains point toward each other and are in the same plane so that hydrogen bonding between them is possible. All R- groups on any one chain alternate, first above, then below the plane of the sheet, etc. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. -Pleated Sheet Figure 18.12 b-Pleated sheet conformation with three polypeptide chains running in opposite directions. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Tertiary Structure Tertiary structure: The three-dimensional arrangement in space of all atoms in a single polypeptide chain. Disulfide bonds between the side chains of cysteine play an important role in maintaining 3° structure. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Primary structure Figure 18.13 Human insulin. An A chain of 21 amino acids and a B chain of 30 amino acids. The two chains are connected by two interchain disulfide bonds. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Myoglobin Figure 18.15 Myoglobin consists of single polypeptide chain of 153 amino acids plus a single heme unit. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Heme Figure 18.14 The structure of heme found in myoglobin and hemoglobin. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Quaternary Structure Quaternary structure: The arrangement of polypeptide chains into a noncovalently bonded aggregation. The major factor stabilizing the aggregation of polypeptide subunits is the hydrophobic effect. Hydrophobic effect: The tendency of nonpolar groups to cluster together in such a way as to be shielded from contact with an aqueous environment. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Quaternary Structure Figure 18.16 Hemoglobin consists of two a chains and two b chains. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Quaternary Structure If two polypeptide chains, for example, each have one hydrophobic patch, each patch can be shielded from contact with water if the chains form a dimer. Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.

Amino Acids and Proteins End Chapter 18 Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.