Amino acids peptides and proteins
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Amino Acids, Peptides, and Proteins. Proteins Occur in every living organism Are of many different types Have many different biological functions Keratin of skin and fingernails Fibroin of silk and spider webs

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Amino Acids, Peptides, and Proteins

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Amino Acids, Peptides, and Proteins


  • Occur in every living organism

  • Are of many different types

  • Have many different biological functions

    • Keratin of skin and fingernails

    • Fibroin of silk and spider webs

    • Estimated 50,000 to 70,000 enzymes that catalyze the biological functions of the human body

  • Made up of many amino acids linked together

Amino Acids, Peptides, and Proteins

Amino acids are difunctional

  • Contain both a basic amino group and an acidic carboxyl group

  • Can join together into long chains by forming bonds between the –NH2 of one amino acid and the –CO2H of another

    • Peptides are chains with fewer than 50 amino acids

    • Proteins are large chains of amino acids

19.1 Structures of Amino Acids

Amino acids exist in aqueous solution primarily in the form of a dipolar ion, or zwitterion


  • German zwitter, meaning “hybrid”

  • A neutral dipolar molecule in which the positive and negative charges are not adjacent

Amino acid zwitterions

Are internal salts

Have large dipole moments

Are soluble in water but are insoluble in hydrocarbons

Are crystalline substances with relatively high melting points

Are amphiprotic

Can react as acids

Reaction takes place in an aqueous base solution

Loses a proton to form an anion

Can react as bases

Reaction takes place in an aqueous acid solution

Accepts a proton to yield a cation

Structures of Amino Acids

Structures of Amino Acids

Structures of Amino Acids

Structures of Amino Acids

Structures of Amino Acids

Structures of Amino Acids

All 20 amino acids found in proteins are a-amino acids

  • The amino group in each is a substituent on the a carbon atom – the one next to the carbonyl group

  • 19 of the amino acids are primary amines, RNH2

    • Differ in the side chain, the substituent attached to the a carbon

    • Proline, a secondary amine, is the only amino acid whose nitrogen and a carbon atoms are part of the ring

Structures of Amino Acids

Selenocysteine and pyrrolysine are amino acids found in some organisms

Structures of Amino Acids

More than 700 nonprotein amino acids are found in nature

  • g-Aminobutyric acid (GABA) is found in the brain and acts as a neurotransmitter

  • Homocysteine is found in blood and is linked to coronary heart disease

  • Thyroxine is found in the thyroid gland, where it acts as a hormone

Structures of Amino Acids

a Carbons of amino acids are chirality centers, except for glycine, H2NCH2CO2H

  • Nature uses only one enantiomer to build proteins

    • Often referred to as L amino acids

  • The nonnaturally occurring enantiomers are called the D amino acids

Structures of Amino Acids

The 20 common amino acids can be acidic, basic, or neutral, depending on their side chains

  • 2 (aspartic acid and glutamic acid) of the 20 have an extra carboxylic acid function in their side chains

  • 3 (lysine, arginine, and histidine) have basic amino groups in their side chains

  • 15 have neutral side chains

    • Cysteine, a thiol, and tyrosine, a phenol, have weakly acidic side chains that can be deprotonated in a sufficiently basic solution

Structures of Amino Acids

At physiological pH 7.3 within cells:

  • The side-chain carboxyl groups of aspartic acid and glutamic acid are deprotonated

  • The basic side-chain nitrogens of lysine and arginine are protonated

  • Histidine contains a heterocyclic imidazole ring in its side chain and is not basic enough to be protonated

    • Only the pyridine-like nitrogen is basic

    • The pyrrole-like nitrogen is nonbasic because its lone pair of electrons is part of the 6p electron aromatic imidazole ring

19.2Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

Using the Henderson-Hasselbalch equation the ratio of [A-] to [HA] in the solution can be calculated

Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

Species present in a 1.00 M solution of alanine at pH = 9.00

  • From Table 19.1

    • [+H3NCH(CH3)CO2H] has pKa(a-CO2H) = 2.34

    • [+H3NCH(CH3)CO2-] has pKa(a-NH3+) = 9.69

Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points



Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

Solving the two simultaneous equations gives

[HA] = 0.83 and [A-] = 0.17

Therefore, in a 1.00 M solution at pH = 9.00

83% of alanine molecules are neutral zwitterionic


17% of alanine molecules are deprotonated [+H3NCH(CH3)CO2H]

Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

Similar calculations at other pH values leads to titration curve

Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

In acid solution, an amino acid is protonated and exists primarily as a cation

In basic solution, an amino acid is deprotonated and exists primarily as an anion

  • Isoelectric point, pI

    • The pH at which the number of positive charges and the number of negative charges on a protein or an amino acid are equal

    • Depends on the structure of the amino acid

      • Values given in Table 19.1

Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

The pIof any amino acid is the average of the two acid-dissociation constants that involve the neutral zwitterion

  • The 4 amino acids with either a strongly or weakly acidic chain, pIis the average of the two lowest pKa values

  • The 13 amino acids with a neutral side chain, pI is the average of pKa1 and pKa2

  • The 3 amino acids with a basic side chain, pI is the average of the two highest pKa values

Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

Proteins have an overall pI because of the acidic or basic amino acids they may contain

  • Lysozyme has a preponderance of basic amino acids and thus has a high isoelectric point (pI = 11.0)

  • Pepsin has a preponderance of acidic amino acids and a low isoelectric point (pI ~ 1.0)

    The solubilities and properties of proteins with different pI ’s are strongly affected by the pH of the medium

  • Solubility in water is usually lowest at the isoelectric point where the protein has no net charge, and higher above and below the pI, where the protein is charged

Amino Acids and the Henderson-Hasselbalch Equation: Isoelectric Points

Electrophoresis uses isoelectric points to separate a mixture of proteins into its pure constituents

  • A mixture of proteins is placed near the center of a strip of paper or gel

  • The paper or gel is moistened with an aqueous buffer of a given pH

  • Electrodes are connected to the ends of the strip and an electric potential is applied

  • Proteins separate according to charges (determined by the pH of the buffer strip)

    • Proteins with negative charges migrate slowly toward the positive electrode

    • Proteins with positive charges migrate slowly toward the negative electrode

      Different proteins migrate at different rates depending upon their pI and the pH of the buffer

19.3 Synthesis of Amino Acids

The Amidomalonate Synthesis

  • Used for synthesizing a-amino acids:

    Amidomalonate synthesis of amino acids is an extension of the malonic ester synthesis

  • Conversion of diethyl acetamidomalonate into an enolate ion by treatment with a base

  • SN2 alkylation with a primary alkyl halide

  • Hydrolysis of both the amide group and the esters occurs when the alkylated product is warmed with aqueous acid

  • Decarboxylation takes place to yield an a-amino acid

    Preparation of aspartic acid from ethyl bromoacetate, BrCH2CO2Et

  • Reaction yields a racemate

Synthesis of Amino Acids

Reductive Amination of a-Keto Acids

  • Another method of synthesizing a-amino acids

  • Reduces an a-keto acid with ammonia and a reducing agent

    • Preparation of alanine by treatment of pyruvic acid with ammonia in the presence of NaBH4

      • Reaction proceeds through formation of an intermediate imine that is then reduced

      • Reaction yields a racemate

Synthesis of Amino Acids

Enantioselective Synthesis

  • One of the two methods used to obtain enantiomerically pure amino acids

    • More efficient than resolving the racemate into its pure enantiomers

  • Prepares only the desired S enantiomer

  • Idea of the synthesis is to find a chiral reaction catalyst that will temporarily hold a substrate molecule in an unsymmetrical environment

    • While in the unsymmetrical environment, the substrate may be more open to reaction on one side than on another, leading to an excess of one enantiomeric product over another

Synthesis of Amino Acids

William Knowles discovered that a-amino acids can be prepared enantioselectively by hydrogenation of a Z enamido acid with a chiral rhodium hydrogenation catalyst

  • (S)-Phenylalanine, is prepared in 98.7% purity contaminated by only 1.3% if the (R) enantiomer when a chiral rhodium catalyst is used

  • Knowles received the 2001 Nobel Prize in Chemistry

Synthesis of Amino Acids

Most effective catalysts for enantioselective amino acid synthesis are coordination complexes of rhodium(I) with cyclooctadiene (COD) and a chiral diphosphine such as (R,R)-1,2-bis(o-anisylphenylphosphine)ethane, the DiPAMP ligand

19.4 Peptides and Proteins

Peptides and proteins are amino acid polymers


  • An amino in a protein chain

  • Joined together by amide bonds, or peptide bonds

    An amino group from one residue forms an amide bond with the carboxyl group of a second residue

  • Alanylserine is the dipeptide that results when an amide bond is formed between the alanine carboxyl and the serine amino group

Peptides and Proteins

Two dipeptides can result from reaction between alanine and serine, depending on which carboxyl group reacts with which amino group

  • If the alanine amino group reacts with the serine carboxyl, serylalanine results

Peptides and Proteins


  • The protein’s backbone is continuous chain of atoms running the length of a protein or other polymer

    • The repetitive sequence of –N–CH–CO–atoms


  • Convention for writing peptides:

    • N-terminal amino acid on the left

      • Peptides with the free –NH2 group

    • C-terminal amino acid on the right

      • Peptides with the free –CO2H group

    • The name of the peptide is indicated using abbreviations

      • Alanylserine is abbreviated Ala-Ser or A-S

      • Serylalanine is abbreviated Ser-Ala or S-A

Peptides and Proteins

Amide Bonds

  • Amide nitrogens are nonbasic because their unshared electron pair is delocalized by interaction with the carbonyl group

    • The overlap of the nitrogen p orbital with the p orbital of the carbonyl group imparts a certain amount of double-bond character to the C-N bond and restricts rotation around it

  • The amide bond is planar

  • The N-H is oriented 180º to the C=O

Peptides and Proteins

A disulfide linkage, RS-SR

  • A covalent bond in peptides

  • Formed between two cysteine residues

  • Links chains of peptides together

  • Creates loops within a single chain of peptides

    • Vasopressin, an antidiuretic hormone found in the pituitary gland

Note: the C-terminal

end of vasopressin occurs

as the primary amide, -CONH2

19.5 Amino Acid Analysis of Peptides

Determination of structure of a protein or peptide

  • What amino acids are present?

  • How much of each is present?

  • In what sequence do the amino acids occur?

    Amino acid analyzer is an automated instrument that provides the answers to the first two questions

  • The peptide is broken into its constituent amino acids

    • Reducing all disulfide bonds

    • Capping the –SH groups of cysteine residues by SN2 reaction with iodoacetic acid

    • Hydrolyzing the amide bonds by heating with aqueous 6 M HCl at 110 ºC for 24 hours

  • The resultant amino acid mixture is analyzed by:

    • High-pressure liquid chromatography (HPLC)

    • Ion-exchange chromatography

Amino Acid Analysis of Peptides

  • As each amino acid exits (elutes) from the end of the ion-exchange chromatography column, it reacts with a solution of ninhydrin, giving an intense purple color

  • The color is detected by a spectrometer and a plot of elution time versus spectrometer absorbance is obtained

The identities of the amino acids in a peptide can be determined simply by noting the various elution times

The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin

The results of amino acid analysis of a standard equimolar mixture of 17a-amino acids

Amino acid analysis requires about 100 picomoles (2-3 mg) of sample for a protein containing about 200 residues

Amino Acid Analysis of Peptides

19.6Peptide Sequencing: The Edman Degradation

With the known identities and amounts of amino acids, the peptide is sequenced to find out in what order the amino acids are linked together

  • Methods of peptide sequencing:

    • Mass spectrometry using electrospray ionization (ESI)

    • Mass spectrometry using matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer

    • Edman degradation

      • Cleave one amino acid at a time from an end of the peptide chain

      • Cleaved amino acid is separated and identified

      • Repeated until entire peptide sequence is known

Peptide Sequencing: The Edman Degradation

  • Automated protein sequencers

    • Allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by-products interferes with the results

    • Provide information from as little as 1 to 5 picomoles of sample – less than 0.1 mg

Peptide Sequencing: The Edman Degradation

  • Edman degradation for N-terminal analysis peptides

    • Involves treatment of a peptide with phenyl isothiocyanate (PITC), C6H5–N=C=S, followed by treatment with trifluoroacetic acid

    • The phenylthiohydantoin (PTH) is identified chromatographically by comparison of its elution times with the known elution times of PTH derivatives of all 20 common amino acids

Peptide Sequencing: The Edman Degradation

Partial hydrolysis of a peptide can carried out either chemically, using aqueous acid, or enzymatically

  • Acidic hydrolysis is unselective and leads to mixtures of small fragments

  • Enzymatic hydrolysis is quite specific

    • Trypsin catalyzes hydrolysis at the carboxyl side of the basic amino acids arginine and lysine

    • Chymotrypsin cleaves only at the carboxyl side of the aryl-substituted amino acids phenylalanine, tyrosine, and tryptophan

19.7 Peptide Synthesis

During the course of a peptide synthesis, many different amide bonds must be formed in a specific order

  • The solution of the specificity problem is to protect some functional groups rendering them unreactive while leaving exposed only those functional groups wanted for reaction

  • To synthesize Ala-Leu, by coupling alanine with leucine

    • Protect the –NH2 group of alanine and the –CO2H group of leucine to render them unreactive

    • Form the desired amide bond

    • Remove the protecting groups

Peptide Synthesis

Amino- and carboxyl-protecting groups

  • Carboxyl groups are often protected by converting them into methyl or benzyl esters

    • Both groups are easily introduced by standard methods of ester formation

    • Both groups are easily removed by mild hydrolysis with aqueous NaOH

    • Benzyl esters can also be cleaved by catalytic hydrogenolysis of the weak benzylic C-O bond (RCO2–CH2Ph + H2 PhCH3)

Peptide Synthesis

  • Amino groups are often protected as their tert-butoxycarbonyl amide, or Boc, derivatives

    • Protecting group is introduced by reaction of the amino acid with di-tert-butyl dicarbonate in a nucleophile acyl substitution reaction

    • Protecting group is removed by brief treatment with a strong organic acid such as trifluoroacetic acid, CF3CO2H

Peptide Synthesis

Five steps are needed to synthesize a dipeptide such

as Ala-Leu using the Boc protecting group

Merrifield solid-phase method simplifies the synthesis of a large peptide chain

Peptide synthesis is carried out with the growing amino acid chain covalently bonded to small beads of polymer resin

In the original Merrifield procedure, polystyrene resin was used

1 of every 100 or so benzene rings contained a chloromethyl (-CH2Cl) group

A Boc-protected C-terminal amino acid was bonded to the resin through an ester bond formed by SN2 reaction

Peptide Synthesis

Peptide Synthesis

  • With the first amino acid bonded to resin, a repeating series of four steps is carried out to build a peptide

Peptide Synthesis

Peptide Synthesis

The most commonly used resins at present are the Wang resin or the PAM (phenylacetamidomethyl) resin

  • The most commonly used N-protecting group is the fluorenylmethyloxycarbonyl, or Fmoc group

Peptide Synthesis

Robotic, computer-controlled peptide synthesis used to automatically repeat the coupling, washing, and deprotection steps with different amino acids

  • Each step occurs in high yield

  • The peptide intermediates are never removed from the insoluble polymer until the final step

  • Using this procedure, up to 30 mg of a peptide with 20 amino acids can be routinely prepared

19.8 Protein Structure

Proteins are classified according to their three-dimensional shape

  • Fibrous proteins

    • Consist of polypeptide chains arranged side by side in long filaments

    • Collagen in tendons and connective tissue

    • Myosin in muscle tissue

    • Tough and insoluble in water

    • Are used in nature as structural materials

  • Globular proteins

    • Usually coiled into compact, roughly spherical shapes

    • Generally soluble in water and are mobile within cells

    • Most of the 3000 or so enzymes are globular proteins

Protein Structure

Four levels for the structure of proteins

  • The primary structure of a protein is simply the amino acid sequence

  • The secondary structure of a protein describes how segments of the peptide backbone orient into a regular pattern

  • The tertiary structure describes how the entire protein molecule coils into an overall three-dimensional shape

  • The quaternary structure describes how different protein molecules come together to yield large aggregate structures

    Primary structure is determined by sequencing the protein

    Secondary, tertiary, and quaternary structures are determined by X-ray crystallography

  • Not possible to predict computationally how a given protein sequence will fold

Protein Structure

a Helix and the b-pleated sheet are the most common secondary structures

a Helix

  • A right-handed coil of the protein backbone

  • Each turn of the helix contains 3.6 amino acid residues, with a distance between the coils of 540 pm, or 5.4 Å

  • The structure is stabilized by hydrogen bonds between amide N-H groups and C=O groups four residues away, with an N-H…O distance of 2.8 Å

  • Almost all globular proteins contain many helical segments

    • Myoglobin is small globular protein containing 153 amino acid residues in a single chain

Protein Structure

(a) a Helix secondary (b) Myoglobin

structure of proteins

Protein Structure

b-pleated sheet

  • The peptide chain is extended

  • The hydrogen bonds occur between residues in adjacent chains

  • The neighboring chains run either in the same direction (parallel) or in opposite directions (antiparallel)

    • The antiparallel arrangement is more common and energetically more stable

      • Concanavalin A consists of two identical chains of 237 residues with extensive regions of antiparallel b sheets

Protein Structure

(a) The b-pleated sheet secondary structure of proteins

(b) The structure of concanavalin A

Protein Structure

The forces that determine the tertiary structures of a protein are the same forces that act on all molecules, regardless of size, to provide maximum stability

  • Hydrophilic (water loving) interactions of the polar side chains of acidic or basic amino acids

    • Acidic or basic amino acids with charged side chains congregate on the exterior of the protein, where they can be solvated by water

    • Amino acids with neutral, nonpolar side chains congregate on the hydrocarbon-like interior of a protein molecule away from the aqueous medium

Protein Structure

Other factors for stabilization of a protein’s tertiary structure

  • Formation of disulfide bridges between cysteine residues

  • The formation of hydrogen bonds between nearby amino acid residues

  • The presence of ionic attractions, called salt bridges, between positively and negatively charged sites on various amino acid side chains within the protein

Protein Structure

Tertiary structure of a globular protein

  • Held together by weak intramolecular attractions

    • A change in temperature or pH is often enough to disrupt the structures and causes denaturation of the protein

      • Denaturation occurs under mild conditions so that the primary structure remains intact but the tertiary structure unfolds from a specific globular shape to a randomly looped chain

Protein Structure


  • It is accompanied by changes in both physical and biological properties

    • Solubility is drastically decreased

  • Most enzymes lose all catalytic activity

  • Most denaturation is irreversible

    • In some cases spontaneous renaturation of an unfolded protein to its stable tertiary structure occurs and is accompanied by a full recovery of biological functions

19.9 Enzymes and Coenzymes


  • Usually a large protein

  • A substance that acts as a catalyst for a biological reaction

  • Does not affect the equilibrium constant of a reaction

  • Acts only to lower the activation energy for a reaction to make the reaction proceed more rapidly

    • Glycosidase enzymes that hydrolyze polysaccharides increase the reaction rate by a factor of more than 1017, changing the time required for the reaction from millions of years to milliseconds

  • Unlike chemical catalysts, enzymes are usually specific in their actions

    • Often, an enzyme will catalyze only a single reaction of a single compound, the enzyme’s substrate

Enzymes and Coenzymes

Different enzymes have different specificities

  • Some are specific for a single substrate

    • Amylase catalyzes only the hydrolysis of starch to yield glucose in the human digestive tract

  • Others operate on a range of substrates

    • Papain, a globular protein of 212 amino acids isolated from papaya fruit, catalyzes the hydrolysis of many kinds of peptide bonds

      • Useful as a meat tenderizer

Enzymes function through a pathway that involves initial formation of an enzyme-substrate complex E • S, a multistep chemical conversion of the enzyme-bound substrate into enzyme-bound product E • P, and final release of product from the complex

Turnover number

The overall reaction rate constant for conversion of the E • S complex to products E + P

It represents the number of substrate molecules the enzyme turns over into product per unit time, 103 per second is typical

Enzymes and Coenzymes

E + S E • S E • P E + P

Factors affecting the rate of reaction acceleration by enzymes

The ability of the enzyme to stabilize and thus lower the energy of the transition state(s)

Its ability to bind and

thereby stabilize the

transition state

The enzyme binds the

transition structure as

much as 1012 times

more tightly than it

binds the substrate

or products

Enzymes and Coenzymes

Enzymes are classified into six categories depending on the kind of reaction they catalyze

The systematic name of an enzyme, ending with –ase

The first part identifies the enzyme’s substrate

The second part identifies its class

Hexokinase is a transferase that catalyzes the transfer of a phosphate group from ATP to a hexose sugar

Enzymes and Coenzymes

Enzymes and Coenzymes

Most enzymes also contain a small protein called the cofactor


  • Can be an inorganic ion, such as Zn2+

  • Can be a small organic molecule called a coenzyme


  • A reactant that undergoes chemical change during the reaction and requires an additional step to return to its initial state

  • Many are derived from vitamins

    • Examples found in Table 19.3:

      • Coenzyme A from pantothenate (vitamin B3),

      • NAD+ from niacin

      • FAD from riboflavin (vitamin B2)

      • Tetrahydrofolate from folic acid

      • Pyridoxal phosphate from pyridoxine (vitamin B6)

      • Thiamin diphosphate from thiamin (vitamin B1)

Enzymes and Coenzymes

Enzymes and Coenzymes

Enzymes and Coenzymes

Enzymes and Coenzymes

19.10 How Do Enzymes Work? Citrate Synthase


  • Bring reactant molecules together

  • Hold molecules in orientation necessary for reaction

  • Provide any necessary acidic or basic sites to catalyze specific steps

    • Citrate synthase

      • An enzyme that catalyzes the aldol-like addition of acetyl CoA to oxaloacetate to give citrate

      • The reaction is the first step in the citric acid cycle, in which acetyl groups produced by degradation of food molecules are metabolized to yield CO2 and H2O

How Do Enzymes Work? Citrate Synthase

Citrate synthase

  • A globular protein of 433 amino acids with a deep cleft lined by an array of functional groups that can bind to oxaloacetate

    • Upon binding oxaloacetate, the original cleft closes and another opens up, which is also lined with functional groups including a histidine at position 274 and an aspartic acid at position 375, to bind acetyl CoA

    • The two reactants are held by the enzyme in close proximity and with a suitable orientation for reaction

How Do Enzymes Work? Citrate Synthase

X-ray crystal structure of citrate synthase

  • Space-filling model

  • Ribbon model emphasizing a-helical segments

    • Enzyme is dimeric

      (c) Close-up of active site

How Do Enzymes Work? Citrate Synthase

Mechanism of the addition of acetyl CoA to oxaloacetate to give (S)-citryl CoA, catalyzed by citrate synthase

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