The Organic Chemistry of Amino Acids, Peptides, and Proteins Chapter 16 The Organic Chemistry of Amino Acids, Peptides, and Proteins

Amino Acids a-Amino carboxylic acids The “building blocks” from which proteins are made Note: except for glycine (R = H), all a-amino acids are chiral molecules There are 20 naturally occuring R-groups *

Amino Acids As with carbohydrates, it is traditional to use the D and L nomenclature with amino acids – based on the configuration of glyceraldehyde Naturally occurring amino acids generally have the same configuration as L-glyceraldehyde (S-configuration at the a-carbon): L-glyceraldehyde L-alanine

Amino Acids Acid–Base Properties Since amino acids have both an acidic functionality and a basic functionality, we should expect the following equilibrium: In fact, the equilibrium lies to the right all amino acids are charged at any pH! Such species that are overall neutral molecules but contain charged ends are called zwitterions

Amino Acids Acid–Base Properties Amino acids can react as either acids or bases:

glutamic acid – acidic R Amino Acids Acid–Base Properties Amino acids can also have side chains containing acidic or basic groups: Deprotonated at physiological pH Protonated at physiological pH glutamic acid – acidic R Lysine –basic R

Amino Acids Classification - Amino acids are classified on the basis of the structure of R Aliphatic side chains – hydrophobic Polar side chains – text classifies as HO-, S-, and amide contining – hydrophilic Acidic – hydrophilic Basic – hydrophilic Heterocyclic/Aromatic – hydrophilic or hydrophobic

Amino Acids Classification - Amino acids are classified on the basis of the structure of R Aliphatic side chains – hydrophobic

Amino Acids Classification - Amino acids are classified on the basis of the structure of R Polar side chains – text classifies as HO-, S-, and amide contining – hydrophilic

Amino Acids Classification - Amino acids are classified on the basis of the structure of R Acidic – hydrophilic Basic – hydrophilic

Amino Acids Classification - Amino acids are classified on the basis of the structure of R Heterocyclic/Aromatic – hydrophilic or hydrophobic

Acid-Base Properties

Acid-Base Properties

Acidity of a-COOH Groups The average pKa of an -carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pKa 4.76) The greater acidity is accounted for by the electron- withdrawing inductive effect of the adjacent -NH3+ group

Side Chain COOH Groups Due to the electron-withdrawing inductive effect of the - NH3+ group, side chain -COOH groups are also stronger than acetic acid the effect 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)

Acidity of -NH3+ Groups The average value of pKa for an -NH3+ group is 9.47, compared with a value of 10.76 for 1° alkylammonium ions

Basicity of Guanidine Group the side chain of arginine contains a guanidine group

The Imidazole Groups of His the imidazole group is a heterocyclic aromatic amine

Titration of Amino Acids Titration of glycine with NaOH

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, 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

Isoelectric Point (pI)

Isoelectric Point (pI)

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

Electrophoresis Electrophoresis of a mixture of amino acids

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

Electrophoresis a reagent commonly used to detect amino acid is ninhydrin

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

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

glycylalanine = gly-ala Peptides Peptides are amino acid polymers containing 2–50 individual units Peptides with >50 units are called proteins By convention, peptide structures are written with the N-terminal amino acid on the left and the C-terminal amino acid on the right. A dipeptide glycylalanine = gly-ala

Peptides glycylalanine = gly-ala glycine amino-terminal amino acid

Peptides glycylalanine = gly-ala alanine carboxy-terminal amino acid

glycylalanine = gly-ala Peptides glycylalanine = gly-ala peptide bond

glycylserylphenylalanylglycine gly-ser-phe-gly Peptides A tetrapeptide: N-terminus C-terminus glycylserylphenylalanylglycine gly-ser-phe-gly

Peptides The Peptide (Amide) Bond The amide nitrogen is sp2 hybridized and the lone pair is conjugated with the carbonyl group There is considerable C–N double-bond character Rotation about the C–N bond is difficult

Peptides The Peptide (Amide) Bond The amide groups are planar. The R-groups are on opposite sides of the plane. gly-ser-phe-gly

Peptides The Peptide (Amide) Bond The amide groups are planar. The R-groups are on opposite sides of the plane. gly-ser-phe-gly

Peptides The only other type of covalent bond between amino acids in proteins and peptides is the disulfide linkage between two cysteine units:

Peptides Note: Thiols are readily oxidized to disulfides. Disulfides are readily reduced to thiols.

Peptides Disulfide links serve to connect polypeptide chains:

Peptides … or can form a macrocycle: bovine oxytocin Copyright © 2010 Pearson Education, Inc. … or can form a macrocycle: bovine oxytocin

Peptides Conotoxin G I A peptide neurotoxin isolated from the venom of the fish- hunting cone snail Conus geographicus. It is a paralytic toxin that blocks nicotinic cholinoreceptors.

Peptides Conotoxin G I

Structure Determination of Peptides Amino Acid Analysis. Find out which amino acids and how many make up the peptide Terminal Residue Analysis. Find out what’s on the ends N-Terminal Analysis Edman degradation C-Terminal Analysis Carboxypeptidase

Structure Determination of Peptides Partial Hydrolysis (enzymatic) Hydrolyze the peptide into smaller fragments. Trypsin - Cleaves at lys and arg Chymotrypsin - Cleaves at phe, tyr, and trp Pepsin - Cleaves at phe, tyr, trp, leu, asp, glu Cyanogen bromide (not enzymatic) - Cleaves at met Determine the sequence of the fragments. Successive Edman degradations.

gly2ala4leu4phe3trp1lys2met2ser1arg1 Sample Problem Give the sequence for a 20 amino acid polypeptide with amino acid analysis: gly2ala4leu4phe3trp1lys2met2ser1arg1 Step 1: Terminal residue analysis: N-terminus = ala C-terminus = phe

Sample Problem Step 2: Trypsin hydrolysis (cleaves at lys and arg) gave four fragments: trpphearg alaleuglymetlys leuglyleuleuphe alaalasermetalaphelys

Sample Problem Step 2: Trypsin hydrolysis (cleaves at lys and arg) gave four fragments: trpphearg alaleuglymetlys leuglyleuleuphe alaalasermetalaphelys Fragment III represents the last five amino acids (trypsin does not cleave at phenylalanine).

Sample Problem Step 2: Trypsin hydrolysis (cleaves at lys and arg) gave four fragments: trpphearg alaleuglymetlys leuglyleuleuphe alaalasermetalaphelys Either fragment II or IV must be N-terminus (cannot know which).

Sample Problem Step 3: Cyanogen bromide cleavage gave three fragments: alaleuglymet alaphelysleuglyleuleuphe lystrppheargalaalasermet

Sample Problem Step 3: Cyanogen bromide cleavage gave three fragments: alaleuglymet alaphelysleuglyleuleuphe lystrppheargalaalasermet Fragment 2 must be the C-terminus.

Sample Problem Step 3: Cyanogen bromide cleavage gave three fragments: alaleuglymet alaphelysleuglyleuleuphe lystrppheargalaalasermet Fragment 1 must be the N-terminus.

Sample Problem Step 4: We can now assemble the peptide:

Sample Problem Step 4: We can now assemble the peptide:

Proteins Protein function depends on structure. Depends on various amino acids. Primary Structure: The amino acid sequence.

Proteins Secondary Structure: The “local” hydrogen- bonding scheme. a-Helix Hydrogen bonds

Proteins Secondary Structure: The “local” hydrogen-bonding scheme. a-Helix

Interchain hydrogen bonds Proteins Secondary Structure: The “local” hydrogen-bonding scheme. b-Sheet Interchain hydrogen bonds

Proteins Tertiary Structure: How the protein, with all of its regions of secondary structure (a-helix, b-sheet) has folded over upon itself Interaction between R-groups is important All intermolecular forces we have studied are at play

Proteins Tertiary Structure: Chemical bonds between cysteines Disulfide bonds

Hydrophobic interactions Hydrophilic interactions Proteins Tertiary Structure: Interaction between R-groups Hydrophobic interactions Hydrogen bonding Ionic bonding (“salt bridge”) Hydrophilic interactions

Proteins Tertiary Structure: How the protein, with all of its regions of secondary structure (a-helix, b-sheet) has folded over upon itself. Crystal Structure Of Human Rab9 Gtpase Courtesy of Protein Data Bank

Proteins Tertiary Structure: How the protein, with all of its regions of secondary structure (a-helix, b-sheet) has folded over upon itself. a-Helix regions

Proteins Tertiary Structure: How the protein, with all of its regions of secondary structure (a-helix, b-sheet) has folded over upon itself. b-Sheet regions

Proteins Quaternary Structure: How protein subunits aggregate into a larger functional unit. Hemoglobin has two  and two  subunits that fit together to form the whole hemoglobin molecule with four hemes and their associated iron atoms to transport O2 and CO2 Human deoxy hemoglobin Courtesy of Protein Data Bank