Chapter 25 Amino Acids, Peptides, and Proteins

Chapter 25Chapter 25Amino Acids, Peptides, Amino Acids, Peptides,

and Proteinsand Proteins

25.125.1Classification of Amino AcidsClassification of Amino Acids

Fundamentals

While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3

+ and —CO2– respectively.

They are classified as , , , etc. amino acids according the carbon that bears the nitrogen.

Amino Acids NH3

+

CO2–

an -amino acid that is anintermediate in the biosynthesisof ethylene

+H3NCH2CH2CO2

–a -amino acid that is one ofthe structural units present incoenzyme A

+H3NCH2CH2CH2CO2

– a -amino acid involved inthe transmission of nerveimpulses

The 20 Key Amino Acids

More than 700 amino acids occur naturally, but 20 of them are especially important.

These 20 amino acids are the building blocks of proteins. All are -amino acids.

They differ in respect to the group attached to the carbon.

These 20 are listed in Table 25.1.

Table 25.1

The amino acids obtained by hydrolysis of proteins differ in respect to R (the side chain).

The properties of the amino acid vary as the structure of R varies.

C C

O

O–

R

H

H3N+

Table 25.1

C C

O

O–

R

H

H3N+

The major differences among the side chains concern:

Size and shapeElectronic characteristics

Table 25.1

General categories of -amino acids

nonpolar side chainspolar but nonionized side chainsacidic side chainsbasic side chains

Table 25.1

C C

O

O–

H

H

H3N+

Glycine is the simplest amino acid. It is the only one in the table that is achiral.

In all of the other amino acids in the table the carbon is a chirality center.

Glycine

(Gly or G)

C C

O

O–

CH3

H

H3N+

Table 25.1

Alanine

(Ala or A)

Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar and hydrophobic.

Table 25.1

C C

O

O–

CH(CH3)2

H

H3N+

Valine

(Val or V)

Table 25.1

C C

O

O–

CH2CH(CH3)2

H

H3N+

Leucine

(Leu or L)

Table 25.1

C C

O

O–

CH3CHCH2CH3

H

H3N+

Isoleucine

(Ile or I)

Table 25.1

C C

O

O–

CH3SCH2CH2

H

H3N+

Methionine

(Met or M)

The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat polarizable.

Table 25.1

Proline

C C

O

O–

CH2

H

H2N+

H2CCH2

(Pro or P)

Proline is the only amino acid that contains a secondary amine function. Its side chain is nonpolar and cyclic.

Table 25.1

Phenylalanine

C C

O

O–

CH2

H

H3N+

(Phe or F)

The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group.

Table 25.1

C C

O

O–

CH2

H

H3N+

N

H

Tryptophan

(Trp or W) The side chain in tryptophan (a nonpolar amino acid) is larger and more polarizable than the benzyl group of phenylalanine.

Table 25.1



Table 25.1

C C

O

O–

CH2OH

H

H3N+

Serine

(Ser or S)

The —CH2OH side chain in serine can be

involved in hydrogen bonding.

Table 25.1

C C

O

O–

CH3CHOH

H

H3N+

Threonine

(Thr or T)

The side chain in threonine can be involved in hydrogen bonding, but is somewhat more crowded than in serine.

Table 25.1

C C

O

O–

CH2SH

H

H3N+

Cysteine

(Cys or C)

The side chains of two remote cysteines can be joined by forming a covalent S—S bond.

Table 25.1

TyrosineC C

O

O–

CH2

H

H3N+

OH

(Tyr or Y) The side chain of tyrosine is similar to that of phenylalanine but can participate in hydrogen bonding.

Table 25.1

Asparagine

C C

O

O–

H

H3N+

H2NCCH2

O(Asn or N)

The side chains of asparagine and glutamine (next slide) terminate in amide functions that are polar and can engage in hydrogen bonding.

Table 25.1

Glutamine

C C

O

O–

H

H3N+

H2NCCH2CH2

O(Gln or Q)

Table 25.1



Table 25.1

Aspartic Acid

C C

O

O–

H

H3N+

OCCH2

O

–

(Asp or D)

Aspartic acid and glutamic acid (next slide) exist as their conjugate bases at biological pH. They are negatively charged and can form ionic bonds with positively charged species.

Table 25.1

Glutamic Acid

C C

O

O–

H

H3N+

OCCH2CH2

O

–

(Glu or E)

Table 25.1



Table 25.1

C C

O

O–

CH2CH2CH2CH2NH3

H

H3N+

Lysine+(Lys or K)

Lysine and arginine (next slide) exist as their conjugate acids at biological pH. They are positively charged and can form ionic bonds with negatively charged species.

Table 25.1

C C

O

O–

CH2CH2CH2NHCNH2

H

H3N+

Arginine

+NH2

(Arg or R)

Table 25.1

Histidine C C

O

O–

H

H3N+

CH2 NHN

(His or H) Histidine is a basic amino acid, but less basic than lysine and arginine. Histidine can interact with metal ions and can help move protons from one site to another.

25.225.2Stereochemistry of Amino Stereochemistry of Amino

AcidsAcids

Configuration of -Amino Acids

Glycine is achiral. All of the other amino acids in proteins have the L-configuration at their carbon.

H3N+

H

R

CO2–

25.325.3Acid-Base Behavior of Amino Acid-Base Behavior of Amino

AcidsAcids

Recall

While their name implies that amino acids are compounds that contain an —NH2 group and a —CO2H group, these groups are actually present as —NH3

+ and —CO2– respectively.

How do we know this?

Properties of Glycine

The properties of glycine:

high melting point: (when heated to 233°C it decomposes before it melts)solubility: soluble in water; not soluble in nonpolar solvent

O

OHH2NCH2C••

••

••

•• ••

–••

O

OH3NCH2C ••

••

•• ••+

more consistent with this than this

Properties of Glycine

The properties of glycine:

high melting point: (when heated to 233°C it decomposes before it melts)solubility: soluble in water; not soluble in nonpolar solvent

–••

O

OH3NCH2C ••

••

•• ••+

more consistent with thiscalled a zwitterion or

dipolar ion

Acid-Base Properties of Glycine

The zwitterionic structure of glycine also follows from considering its acid-base properties.

A good way to think about this is to start with the structure of glycine in strongly acidic solution, say pH = 1.

At pH = 1, glycine exists in its protonated form (a monocation).

O

OHH3NCH2C+

••

••

•• ••


Now ask yourself "As the pH is raised, which is the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is it the proton of the carboxyl group?"

You can choose between them by estimating their respective pKas.

O

OHH3NCH2C+

••

••

•• ••

typical ammonium ion: pKa ~9

typical carboxylic acid: pKa ~5


The more acidic proton belongs to the CO2H group. It is the first one removed as the pH is raised.


O

OHH3NCH2C+

••

••

•• ••


Therefore, the more stable neutral form of glycine is the zwitterion.

O

OHH3NCH2C+

••

••

•• ••


–••

O

OH3NCH2C ••

••

•• ••+

The measured pKa of glycine is 2.34.

Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an electron-withdrawing, acid-strengthening substituent on the carbon.



O

OHH3NCH2C+

••

••

•• ••


–••

O

OH3NCH2C ••

••

•• ••+

The pKa for removal of this proton is 9.60.This value is about the same as that for NH4

+ (9.3).

HO–

–••

O

OH2NCH2C ••

••

•• ••••

A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased further.

Isoelectric Point (pI)

pKa = 2.34

pKa = 9.60

The pH at which the concentration of the zwitterion is a maximum is called the isoelectric point. Its numerical value is the average of the two pKas.

The pI of glycine is 5.97.

O

OHH3NCH2C+

••

••

•• ••

–••

O

OH3NCH2C ••

••

•• ••+

–••

O

OH2NCH2C ••

••

•• ••••

Acid-Base Properties of Amino Acids

One way in which amino acids differ is in respect to their acid-base properties. This is the basis for certain experimental methods for separating and identifying them.

Just as important, the difference in acid-base properties among various side chains affects the properties of the proteins that contain them.

Table 25.2 gives pKa and pI values for amino acids with neutral side chains.

Table 25.2 Amino Acids with Neutral Side Chains

C C

O

O–

H

H

H3N+

GlycinepKa1 = 2.34pKa2 = 9.60pI = 5.97


AlaninepKa1 = 2.34pKa2 = 9.69pI = 6.00

C C

O

O–

CH3

H

H3N+


ValinepKa1 = 2.32pKa2 = 9.62pI = 5.96

C C

O

O–

CH(CH3)2

H

H3N+


LeucinepKa1 = 2.36pKa2 = 9.60pI = 5.98

C C

O

O–

CH2CH(CH3)2

H

H3N+


IsoleucinepKa1 = 2.36pKa2 = 9.60pI = 6.02

C C

O

O–

CH3CHCH2CH3

H

H3N+


MethioninepKa1 = 2.28pKa2 = 9.21pI = 5.74

C C

O

O–

CH3SCH2CH2

H

H3N+


ProlinepKa1 = 1.99pKa2 = 10.60pI = 6.30

C C

O

O–

CH2

H

H2N+

H2CCH2


PhenylalaninepKa1 = 1.83pKa2 = 9.13pI = 5.48

C C

O

O–

CH2

H

H3N+


TryptophanpKa1 = 2.83pKa2 = 9.39pI = 5.89

C C

O

O–

CH2

H

H3N+

N

H


AsparaginepKa1 = 2.02pKa2 = 8.80pI = 5.41

C C

O

O–

H

H3N+

H2NCCH2

O


GlutaminepKa1 = 2.17pKa2 = 9.13pI = 5.65

C C

O

O–

H

H3N+

H2NCCH2CH2

O


SerinepKa1 = 2.21pKa2 = 9.15pI = 5.68

C C

O

O–

CH2OH

H

H3N+


ThreoninepKa1 = 2.09pKa2 = 9.10pI = 5.60

C C

O

O–

CH3CHOH

H

H3N+


TyrosinepKa1 = 2.20pKa2 = 9.11pI = 5.66

C C

O

O–

CH2

H

H3N+

OH

Table 25.3 Amino Acids with Ionizable Side Chains

Aspartic acidpKa1 = 1.88pKa2 = 9.60 pKa* = 3.65 pI = 2.77

For amino acids with acidic side chains, pI is the average of pKa1 and pKa*.

C C

O

O–

H

H3N+

OCCH2

O

–


Glutamic acidpKa1 = 2.19pKa2 = 9.67 pKa* = 4.25pI = 3.22

C C

O

O–

H

H3N+

OCCH2CH2

O

–


Lysine

pKa1 = 2.18pKa2 = 8.95 pKa* = 10.53pI = 9.74

For amino acids with basic side chains, pI is the average of pKa2 and pKa*.

C C

O

O–

CH2CH2CH2CH2NH3

H

H3N+

+


Arginine

pKa1 = 2.17pKa2 = 9.04pKa* = 12.48pI = 10.76

C C

O

O–

CH2CH2CH2NHCNH2

H

H3N+

+NH2


Histidine

pKa1 = 1.82pKa2 = 6.00pKa* = 9.17 pI = 7.59

C C

O

O–

H

H3N+

CH2 NHN

25.425.4Synthesis of Amino AcidsSynthesis of Amino Acids

From -Halo Carboxylic Acids

CH3CHCOH

Br

O

2NH3+H2O

CH3CHCO

NH3

O

+

–

(65-70%)

+ NH4Br

Strecker Synthesis

NH4Cl

NaCNCH3CH

O

CH3CHC

NH2

N

CH3CHCO

NH3

O

+

– (52-60%)

1. H2O, HCl, heat

2. HO–

Using Diethyl Acetamidomalonate

CC

COCH2CH3

H

O O

CH3CH2O

CH3CNH

O

Can be used in the same manner as diethyl malonate (Section 20.11).

Example

1. NaOCH2CH3

2. C6H5CH2Cl

O O

CH3CH2OCCCOCH2CH3

HCH3CNH

O

O O

CH3CH2OCCCOCH2CH3

CH2C6H5CH3CNH

O

(90%)

Example

HBr, H2O, heat

O O

HOCCCOH

CH2C6H5H3N+

O

HCCOH

CH2C6H5H3N+(65%)

–CO2

O O

CH3CH2OCCCOCH2CH3

CH2C6H5CH3CNH

O

25.525.5Reactions of Amino AcidsReactions of Amino Acids

Acylation of Amino Group

The amino nitrogen of an amino acid can be converted to an amide with the customary acylating agents.

O

H3NCH2CO–+

+ CH3COCCH3

O O

CH3CNHCH2COH

O O(89-92%)

Esterification of Carboxyl Group

The carboxyl group of an amino acid can be converted to an ester. The following illustrates Fischer esterification of alanine.

+ CH3CH2OH

HCl

O

H3NCHCO–+

CH3

(90-95%)

O

H3NCHCOCH2CH3

+

CH3

–Cl

Ninhydrin Test

Amino acids are detected by the formation of a purple color on treatment with ninhydrin.

OH

O

O

OH+

O

H3NCHCO–+

R

O O

O

N

O

–

O

RCH + CO2 + H2O +

25.625.6Some Biochemical ReactionsSome Biochemical Reactions

of Amino Acidsof Amino Acids

Biosynthesis of L-Glutamic Acid

This reaction is the biochemical analog of reductive amination (Section 21.10).

HO2CCH2CH2CCO2H

O

NH3+

enzymes andreducing coenzymes

HO2CCH2CH2CHCO2

–

NH3+

Transamination via L-Glutamic Acid

L-Glutamic acid acts as a source of the amine group in the biochemical conversion of -ketoacids to other amino acids. In the example to beshown, pyruvic acid is converted to L-alanine.

HO2CCH2CH2CHCO2

–

NH3+

+ CH3CCO2H

O

Transamination via L-Glutamic Acid

HO2CCH2CH2CHCO2

–

NH3+

+ CH3CCO2H

O

enzymes

HO2CCH2CH2CCO2H

O

+ CH3CHCO2

–

NH3+

Mechanism

HO2CCH2CH2CHCO2

–

NH3+

+

The first step is imine formation between theamino group of L-glutamic acid and a coenzyme called pyridoxal phosphate (PLP).

N

-O3POH2C OH

CH3

H O

PLP

Mechanism

HO2CCH2CH2CHCO2

–

NH3+

+

N

-O3POH2C OH

CH3

H O

N

-O3POH2C OH

CH3

H N

HO2CCH2CH2CHCO2––

H

Formation of the imine is followed by proton removal at one carbon and protonation of another carbon.

N

-O3POH2C OH

CH3

H N

HO2CCH2CH2CCO2–

N

-O3POH2C OH

CH3

H NHH

HO2CCH2CH2CCO2–

H

N

-O3POH2C OH

CH3

H N

HO2CCH2CH2CCO2–

Hydrolysis of the imine function gives-ketoglutarate and pyridoxamine phosphate.

N

-O3POH2C OH

CH3

H NHH

HO2CCH2CH2CCO2–

HO2CCH2CH2CCO2

O

–+

H2O

N

-O3POH2C OH

CH3

H NH2H

N

-O3POH2C OH

CH3

H NHH

HO2CCH2CH2CCO2–

+CH3CCO2H

O

The pyridoxamine can do the same sequence of steps in reverse with pyruvate to generate alanine and regenerate PLP.

N

-O3POH2C OH

CH3

H NH2H

+CH3CCO2H

O

CH3CHCO2

–

NH3+

N

-O3POH2C OH

CH3

H NH2H

N

-O3POH2C OH

CH3

H O

L-Tyrosine is biosynthesized from L-phenylalanine.A key step is epoxidation of the aromatic ring to give an arene oxide intermediate.

Biosynthesis of L-Tyrosine

CH2CHCO2

–

NH3+


CH2CHCO2

–

NH3+

O2, enzyme

CH2CHCO2

–

NH3+

O


CH2CHCO2

–

NH3+

O

enzyme

CH2CHCO2

–

NH3+

HO


Conversion to L-tyrosine is one of the major metabolic pathways of L-phenylalanine.

Individuals who lack the enzymes necessary to convert L-phenylalanine to L-tyrosine can suffer from PKU disease. In PKU disease, L-phenylalanine is diverted to a pathway leading to phenylpyruvic acid, which is toxic.

Newborns are routinely tested for PKU disease. Treatment consists of reducing their dietary intake of phenylalanine-rich proteins.

Decarboxylation

Decarboxylation is a common reaction of -amino acids. An example is the conversion of L-histidine to histamine. Antihistamines act by blocking the action of histamine.

CH2CHCO2

–

NH3+NH

N

Decarboxylation

CH2CHCO2

–

NH3+NH

N

–CO2, enzymes

CH2CH2 NH2

NH

N

Neurotransmitters

The chemistry of the brain and central nervous system is affected by neurotransmitters.

Several important neurotransmitters are biosynthesized from L-tyrosine.

OH

CO2–

HHH

H3N+

L-Tyrosine

H3N

Neurotransmitters

The common name of this compound is L-DOPA. It occurs naturally in the brain. It is widely prescribed to reduce the symptoms of Parkinsonism.

OH

CO2–

HHH

+

L-3,4-Dihydroxyphenylalanine

HO

Neurotransmitters

Dopamine is formed by decarboxylation of L-DOPA.

OH

H

HHH

H2N

HO

Dopamine

Neurotransmitters

OH

H

HHOH

H2N

HO

Norepinephrine

Neurotransmitters

OH

H

HHOH

CH3NH

HO

Epinephrine

25.725.7PeptidesPeptides

Peptides

Peptides are compounds in which an amide bond links the amino group of one -amino acid and the carboxyl group of another.

An amide bond of this type is often referred to as a peptide bond.

Alanine and Glycine

CH3

O

C+

H

C O–

H3N

O

C

H

H

CH3N+

O–

Alanylglycine

CH3

O

CH3N+

H

C

O

CN

H

H

C O–

H

Two -amino acids are joined by a peptide bond in alanylglycine. It is a dipeptide.

Alanylglycine

Ala—Gly

AG

N-terminus C-terminusCH3

O

CH3N+

H

C

O

CN

H

H

C O–

H

Alanylglycine and glycylalanine are constitutional isomers

H

O

CH3N+

H

C

O

CN

H

CH3

C O–

H

AlanylglycineAla—Gly

AG

GlycylalanineGly—Ala

GA

CH3

O

CH3N+

H

C

O

CN

H

H

C O–

H

Alanylglycine

The peptide bond is characterized by a planar geometry.

CH3

O

CH3N+

H

C

O

CN

H

H

C O–

H

Higher Peptides

Peptides are classified according to the number of amino acids linked together.

dipeptides, tripeptides, tetrapeptides, etc.

Leucine enkephalin is an example of a pentapeptide.

Leucine Enkephalin

Tyr—Gly—Gly—Phe—LeuYGGFL

Oxytocin

Oxytocin is a cyclic nonapeptide.

Instead of having its amino acids linked in an extended chain, two cysteine residues are joined by an S—S bond.

N-terminus

C-terminusIle—Gln—Asn

Tyr

Cys S S

Cys—Pro—Leu—GlyNH2

1

2

34 5

6 7 8 9

Oxytocin

S—S bond

An S—S bond between two cysteines isoften referred to as a disulfide bridge.

25.825.8Introduction to Peptide Introduction to Peptide Structure DeterminationStructure Determination

Primary Structure

The primary structure is the amino acid sequence plus any disulfide links.

Classical Strategy (Sanger)

1. Determine what amino acids are present and their molar ratios.

2. Cleave the peptide into smaller fragments, and determine the amino acid composition of these smaller fragments.

3. Identify the N-terminus and C-terminus in the parent peptide and in each fragment.

4. Organize the information so that the sequences of small fragments can be overlapped to reveal the full sequence.

25.925.9Amino Acid AnalysisAmino Acid Analysis

Amino Acid Analysis

Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids.

The mixture is separated by ion-exchange chromatography, which depends on the differences in pI among the various amino acids.

Amino acids are detected using ninhydrin.

Automated method; requires only 10-5 to 10-7 g of peptide.

25.1025.10Partial Hydrolysis of PeptidesPartial Hydrolysis of Peptides

Partial Hydrolysis of Peptides and Proteins

Acid-hydrolysis of the peptide cleaves all of the peptide bonds.

Cleaving some, but not all, of the peptide bonds gives smaller fragments.

These smaller fragments are then separated and the amino acids present in each fragment determined.

Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis.

Partial Hydrolysis of Peptides and Proteins

The enzymes that catalyze the hydrolysis of peptide bonds are called peptidases, proteases, or proteolytic enzymes.

Trypsin

Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine.

NHCHC

O

R'

NHCHC

O

R"

NHCHC

O

R

lysine or arginine

Chymotrypsin

Chymotrypsin is selective for cleaving the peptidebond to the carboxyl group of amino acids withan aromatic side chain.

NHCHC

O

R'

NHCHC

O

R"

NHCHC

O

R

phenylalanine, tyrosine, tryptophan

Carboxypeptidase

proteinH3NCHC

O

R

+NHCHCO

O

R

–C

O

Carboxypeptidase is selective for cleavingthe peptide bond to the C-terminal amino acid.

25.1125.11End Group AnalysisEnd Group Analysis

End Group Analysis

Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left.

We need to know what the N-terminal and C-terminal amino acids are.

The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis.

Several chemical methods have been developed for identifying the N-terminus. They depend on the fact that the amino N at the terminus is more nucleophilic than any of the amide nitrogens.

Sanger's Method

The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene.

1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution (Chapter 12).

FO2N

NO2

Sanger's Method

1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid.

FO2N

NO2

NHCH2C NHCHCO

CH3

NHCHC

CH2C6H5

H2NCHC

O OOO

CH(CH3)2

–+

O2N

NO2

NHCH2C NHCHCO

CH3

NHCHC

CH2C6H5

NHCHC

O OOO

CH(CH3)2

–

Sanger's Method

Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group.

O2N

NO2

NHCH2C NHCHCO

CH3

NHCHC

CH2C6H5

NHCHC

O OOO

CH(CH3)2

–

H3O+

O

O2N

NO2

NHCHCOH

CH(CH3)2

H3NCHCO–

CH3

+H3NCH2CO–

O O

+

O

H3NCHCO–

CH2C6H5

++ +

+

25.1225.12InsulinInsulin

Insulin

Insulin is a polypeptide with 51 amino acids.

It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids).

The following describes how the amino acid sequence of the B chain was determined.

The B Chain of Bovine Insulin

Phenylalanine (F) is the N terminus.

Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL VGAL VCGERGF YTPKA


FVNQHLCGSHL

VGAL

VCGERGF

YTPKA




Overlaps between the above peptide sequences were found in four additional peptides:

SHLVLVGAALTTLVC


FVNQHLCGSHL

SHLVLVGA

VGAL

ALY

YLVCVCGERGF

YTPKA




Overlaps between the above peptide sequences were found in four additional peptides:

SHLVLVGAALTTLVC

Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence.


FVNQHLCGSHL

SHLVLVGA

VGAL

ALY

YLVCVCGERGF

GFFYTPK

YTPKA


FVNQHLCGSHL

SHLVLVGA

VGAL

ALY

YLVCVCGERGF

GFFYTPK

YTPKA

FVNQHLCGSHLVGALYLVCGERGFFYTPKA

Insulin

The sequence of the A chain was determined using the same strategy.

Establishing the disulfide links between cysteine residues completed the primary structure.

Primary Structure of Bovine Insulin

N terminus of A chain

N terminus of B chain

C terminus of B chain

C terminus of A chain

C

S

S5

5

15

10

15

20

20

2530

S

10

S SSF

F F

F

V N Q H L

C C

C

C

VV

VVG

G

G

S

S

S

H L L

L

G A

A

AC

L Y

Y

E

E L E

R

Y

YI Q

K P T

QN

N

C

25.1325.13The Edman Degradation and The Edman Degradation and

Automated Sequencing of Automated Sequencing of PeptidesPeptides

Edman Degradation

1. Method for determining N-terminal amino acid.

2. Can be done sequentially one residue at a time on the same sample. Usually one can determine the first 20 or so amino acids from the N-terminus by this method.

3. 10-10 g of sample is sufficient.

4. Has been automated.

Edman Degradation

The key reagent in the Edman degradation is phenyl isothiocyanate.

N C S

Edman Degradation

Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid.

peptideH3NCHC

O

R

+NHC6H5N C S +

Edman Degradation

peptideC6H5NHCNHCHC

O

R

NH

S

peptideH3NCHC

O

R

+NHC6H5N C S +

Edman Degradation

The product is a phenylthiocarbamoyl (PTC)derivative.

The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the peptide.

peptideC6H5NHCNHCHC

O

R

NH

S

Edman Degradation

HCl

peptideH3N+

+C6H5NH C

SC

N CH

R

O

peptideC6H5NHCNHCHC

O

R

NH

S

Edman Degradation

The product is a thiazolone. Under theconditions of its formation, the thiazolonerearranges to a phenylthiohydantoin (PTH)derivative.

peptideH3N+

+C6H5NH C

SC

N CH

R

O

Edman Degradation

CCN

HN CH

R

OS

C6H5The PTH derivative is isolated and identified. The remainder of the peptide is subjected to a second Edman degradation.

peptideH3N+

+C6H5NH C

SC

N CH

R

O

25.1425.14The Strategy of Peptide SynthesisThe Strategy of Peptide Synthesis

General Considerations

Making peptide bonds between amino acids is not difficult.

The challenge is connecting amino acids in the correct sequence.

Random peptide bond formation in a mixture of phenylalanine and glycine, for example, will give four dipeptides.

Phe—Phe Gly—Gly Phe—Gly Gly—Phe

General Strategy

1. Limit the number of possibilities by "protecting" the nitrogen of one amino acid and the carboxyl group of the other.

N-Protectedphenylalanine

C-Protectedglycine

NHCHCOH

CH2C6H5

O

X H2NCH2C

O

Y

General Strategy

2. Couple the two protected amino acids.

NHCH2C

O

YNHCHC

CH2C6H5

O

X

NHCHCOH

CH2C6H5

O

X H2NCH2C

O

Y

General Strategy

3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus.

NHCH2CO

O

H3NCHC

CH2C6H5

O+ –

Phe-Gly

NHCH2C

O

YNHCHC

CH2C6H5

O

X

25.1525.15Amino Group ProtectionAmino Group Protection

Amino groups are normally protected by converting them to amides.

Benzyloxycarbonyl (C6H5CH2O—) is a common protecting group. It is abbreviated as Z.

Z-protection is carried out by treating an amino acid with benzyloxycarbonyl chloride.

Protect Amino Groups as Amides

Protect Amino Groups as Amides CH2OCCl

O

+ H3NCHCO

CH2C6H5

O–+

1. NaOH, H2O

2. H+

NHCHCOH

CH2C6H5

O CH2OC

O

(82-87%)

Protect Amino Groups as Amides

NHCHCOH

CH2C6H5

O CH2OC

O

is abbreviated as:

ZNHCHCOH

CH2C6H5

O

or Z-Phe

An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by:

a) hydrogenolysis

b) cleavage with HBr in acetic acid

Removing Z-Protection

Hydrogenolysis of Z-Protecting Group

NHCHCNHCH2CO2CH2CH3

CH2C6H5

O CH2OC

O

H2, Pd

H2NCHCNHCH2CO2CH2CH3

CH2C6H5

O CH3 CO2

(100%)

HBr Cleavage of Z-Protecting Group

NHCHCNHCH2CO2CH2CH3

CH2C6H5

O CH2OC

O

HBr


CH2C6H5

O CH2Br CO2

(82%)

+

Br–

The tert-Butoxycarbonyl Protecting Group

NHCHCOH

CH2C6H5

O

(CH3)3COC

O

is abbreviated as:

BocNHCHCOH

CH2C6H5

O

or Boc-Phe

HBr Cleavage of Boc-Protecting Group

NHCHCNHCH2CO2CH2CH3

CH2C6H5

O

(CH3)3COC

O

HBr


CH2C6H5

O

CO2

(86%)

+

Br–CH2C

H3C

H3C

25.1625.16Carboxyl Group ProtectionCarboxyl Group Protection

Carboxyl groups are normally protected as esters.

Deprotection of methyl and ethyl esters isby hydrolysis in base.

Benzyl esters can be cleaved byhydrogenolysis.

Protect Carboxyl Groups as Esters

Hydrogenolysis of Benzyl Esters

NHCHCNHCH2COCH2C6H5

CH2C6H5

O

C6H5CH2OC

O O

H2, Pd

H3NCHCNHCH2CO

CH2C6H5

O

C6H5CH3 CO2

(87%)

+ –CH3C6H5

25.1725.17Peptide Bond FormationPeptide Bond Formation

The two major methods are:

1. coupling of suitably protected amino acids using N,N'-dicyclohexylcarbodiimide (DCCI)

2. via an active ester of the N-terminal amino acid.

Forming Peptide Bonds

DCCI-Promoted Coupling

ZNHCHCOH

CH2C6H5

O

+ H2NCH2COCH2CH3

O

DCCI, chloroform

ZNHCHC

CH2C6H5

O

NHCH2COCH2CH3

O

(83%)

Mechanism of DCCI-Promoted Coupling

ZNHCHCOH

CH2C6H5

O

+ C6H11N C NC6H11

CH2C6H5

O

C6H11N C

C6H11N

H

OCCHNHZ


CH2C6H5

O

C6H11N C

C6H11N

H

OCCHNHZ

The species formed by addition of the Z-protected amino acid to DCCI is similar in structure to an acid anhydride and acts as an acylating agent.

Attack by the amine function of the carboxyl-protected amino acid on the carbonyl group leads to nucleophilic acyl substitution.


H2NCH2COCH2CH3

O

C6H11N C

C6H11NH

H

O + ZNHCHC

CH2C6H5

O

NHCH2COCH2CH3

O

CH2C6H5

O

C6H11N C

C6H11N

H

OCCHNHZ

A p-nitrophenyl ester is an example of an "active ester."

p-Nitrophenyl is a better leaving group than methyl or ethyl, and p-nitrophenyl esters are more reactive in nucleophilic acyl substitution.

The Active Ester Method

The Active Ester Method

ZNHCHCO

CH2C6H5

O

+ H2NCH2COCH2CH3

O NO2

chloroform

ZNHCHC

CH2C6H5

O

NHCH2COCH2CH3

O

(78%)

+ HO

NO2

25.1825.18Solid-Phase Peptide Synthesis:Solid-Phase Peptide Synthesis:

The Merrifield MethodThe Merrifield Method

Solid-Phase Peptide Synthesis

In solid-phase synthesis, the starting material is bonded to an inert solid support.

Reactants are added in solution.

Reaction occurs at the interface between the solid and the solution. Because the starting material is bonded to the solid, any product from the starting material remains bonded as well.

Purification involves simply washing the byproducts from the solid support.

The Solid Support

The solid support is a copolymer of styrene and divinylbenzene. It is represented above as if it were polystyrene. Cross-linking with divinylbenzene simply provides a more rigid polymer.

CH2 CH2 CH2 CH2CH CH CH CH

The Solid Support

Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings.


The Solid Support

The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2).


CH2Cl

The Solid Support


CH2Cl

The chloromethylated resin is treated with the Boc-protected C-terminal amino acid. Nucleophilic substitution occurs, and the Boc-protected amino acid is bound to the resin as an ester.

The Merrifield Procedure


CH2Cl

BocNHCHCO

R

O–


BocNHCHCO

R

O


CH2

Next, the Boc protecting group is removed with HCl.


H2NCHCO

R


CH2

O

DCCI-promoted coupling adds the second amino acid.


NHCHCO

R

OCH2

CH2 CH2 CH2 CH2CH CH CH CH BocNHCHC

R'

O

Remove the Boc protecting group.


CH2


NHCHCO

R

O

H2NCHC

R'

O

Add the next amino acid and repeat.


Remove the peptide from the resin with HBr in CF3CO2H.


CH2

NHCHCO

R

O

NHCHC

R'

O

C

O+

H3N peptide

The Merrifield Procedure CH2 CH2 CH2 CH2CH CH CH CH

CH2Br

NHCHCO

R

O

NHCHC

R'

O

C

O+

H3N peptide–

The Merrifield Method

Merrifield automated his solid-phase method.

Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in 68% yield.

Synthesized ribonuclease (124 amino acids) in 1969.

369 reactions; 11,391 steps

Nobel Prize in chemistry: 1984

25.1925.19Secondary StructuresSecondary Structures

of Peptides and Proteinsof Peptides and Proteins

Levels of Protein Structure

Primary structure = the amino acid sequence plus disulfide links.

Secondary structure = conformational relationship between nearest neighbor amino acids.

helixpleated sheet

Levels of Protein Structure

Planar geometry of peptide bondAnti conformation of main chainHydrogen bonds between N—H and O=C

The -helix and pleated sheet are both characterized by:

Pleated Sheet

Shown is a sheet of protein chains composed of alternating glycine and alanine residues.

Adjacent chains are antiparallel.

Hydrogen bonds between chains.

van der Waals forces produce pleated effect.

Pleated Sheet

Sheet is most commonly seen with amino acids having small side chains (glycine, alanine, serine).

80% of fibroin (main protein in silk) is repeating sequence of —Gly—Ser—Gly—Ala—Gly—Ala—.

Sheet is flexible, but resists stretching.

Helix

Shown is an helix of a protein in which all of the amino acids are L-alanine.

Helix is right-handed with 3.6 amino acids per turn.

Hydrogen bonds are within a single chain.

Protein of muscle (myosin) and wool (-keratin) contain large regions of -helix. Chain can be stretched.

25.2025.20Tertiary StructureTertiary Structure

of Polypeptides and Proteinsof Polypeptides and Proteins

Tertiary Structure

Refers to overall shape (how the chain is folded).

Fibrous proteins (hair, tendons, wool) have elongated shapes.

Globular proteins are approximately spherical.

Most enzymes are globular proteins.

An example is carboxypeptidase.

Carboxypeptidase

Carboxypeptidase is an enzyme that catalyzes the hydrolysis of proteins at their C-terminus.

It is a metalloenzyme containing Zn2+ at its active site.

An amino acid with a positively charged side chain (Arg-145) is near the active site.

Carboxypeptidase

Disulfide bond

N-terminus

C-terminus

Zn2+

Arg-145

Tube model Ribbon model

What Happens at the Active Site?

H3N peptide

O

NHCHC+

C

•• ••

R

O

O

–

H2N

H2N

C Arg-145+


H3N peptide

O

NHCHC+

C

•• ••

R

O

O

–

H2N

H2N

C Arg-145+

The peptide or protein is bound at the active site by electrostatic attraction between its negatively charged carboxylate ion and arginine-145.


H3N peptide

O

NHCHC+

C

•• ••

R

O

O

–

H2N

H2N

C Arg-145+

ZnZn2+2+

Zn2+ acts as a Lewis acid toward the carbonyl oxygen, increasing the positive character of the carbonyl carbon.


H3N peptide

O

NHCHC+

C

•• ••

R

O

O

–

H2N

H2N

C Arg-145+

ZnZn2+2+

Water attacks the carbonyl carbon. Nucleophilic acyl substitution occurs.

O•• ••

H

H


ZnZn2+2+

H2N

C Arg-145+

H3N peptide

O+

C

•• ••

O ••••

••

–

H3NCHC

R

O

O

–

H2N

+

25.2125.21CoenzymesCoenzymes

Coenzymes

The range of chemical reactions that amino acid side chains can participate in is relatively limited.

Acid-base (transfer and accept protons)Nucleophilic acyl substitution

Many other biological processes, such as oxidation-reduction, require coenzymes, cofactors, or prosthetic groups in order to occur.

Coenzymes

NADH, coenzyme A and coenzyme B12 are examples of coenzymes.

Heme is another example.

Heme N

N N

N

Fe

H3C

H3C CH3

CH3

CH2CH2CO2H

CH CH2

H2C CH

HO2CCH2CH2

Molecule surrounding iron is a type of porphyrin.

Myoglobin

N-terminusN-terminus

C-terminusC-terminus Heme

Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen transport).

25.2225.22Protein Quaternary Structure:Protein Quaternary Structure:

HemoglobinHemoglobin

Protein Quaternary Structure

Some proteins are assemblies of two or more chains. The way in which these chains are organized is called the quaternary structure.

Hemoglobin, for example, consists of 4 subunits.

There are 2 chains (identical) and 2 chains (also identical).

Each subunit contains one heme and each protein is about the size of myoglobin.

25.2325.23G-Coupled Protein ReceptorsG-Coupled Protein Receptors

G-Coupled Protein Receptors

GCPRs (the “G” stands for guanine in “guanine nucleotide-binding proteins”) occur throughout the body and function as “molecular switches” that regulate many physiological processes.

GCPRs span the cell membrane and when they bind their specific ligand (a small organic molecule, lipid, peptide, ion, etc.), they undergo a conformational change, which results in the transduction of a signal across the membrane.

The Core of Modern Medicine

GCPRs are the target for many therapeutic agents in the treatment of cancer, cardiac malfunction, inflammation, pain, obesity, diabetes and disorders of the central nervous system.

Documents

Chapter 25 Amino Acids, Peptides, and Proteins