CHAPER 24AMINO ACIDS AND PROTEINS

24.1 INTRODUCTION

Of the three groups of biopolymers, protein have the most diverse function. Most of its molecular weights are much larger. Their shapscover a range from the globular protein to the helical coils of a α–keratin. But all proteins have common features.

Proteins are polyamides and their monomeric units are about20 different α-amino acids.

N

H

HCH

C

O

OH

R

N

H

CHC

O

R

N

H

CHC

O

R'

N

H

CHC

O

R''

N

H

CHC

O

R''''

N

H

CHC

O

R'''

Amide links

An -amino acid A portion of a protien molecular

Primary structure: the exact sequence of the different α-amino acids along the protein chain.

Second and tertiary structure: the folding of the polyamide chain which give rise to higher levels of complexity.

Although hydrolysis of natural occurring proteins may yield as manyas 22 different amino acids, the amino acids have an importantstructural feature in common.

24.2 AMINO ACIDS

24.2A STRUCTURES AND NAMES

H2N H

CO2H

R

R NAMEABBRE-VIATION pI

-H

-CH3

-CH(CH3)2

-CH2CH(CH3)2

-CH(CH3)CH2CH3

Glycine

Alanine

Valinee

Leucinee

Isoleucinee

Gly

Ala

Val

Leu

Ile

6.0

6.0

6.0

6.0

6.1

R NAMEABBRE-VIATION pI

H2C

-CH2CONH2

-CH2CH2CONH2

N

CH2

H

Phenylalaninee

Asparagine

Glutamine

Tryptophane

Phe

Asn

Gln

Trp

5.5

5.4

5.7

5.9

HNCH2

CH2

CH2CHHOOC

(complete structure)

-CH2OH

Proline

Serine

Pro

Ser

6.3

5.7

R NAMEABBRE-VIATION pI

-CH(CH3)OH

H2C OH

HNCH2

CH

CH2CHHOOC

(complete structure)

OH

Threoninee

Tyrosine

Hyroxyproline

Thr

Tyr

Hyp

6.5

5.7

6.3

-CH2SHH2C S

SH2C

-CH2CH2SCH3

-CH2COOH

Cysteine

Cystine

Methioninee

Aspartic acid

Cys

Cys-Cys

Met

Asp

5.0

5.1

5.8

3.0

The conversion of cysteine to cystine requires addition comment.it can be reversed by mild reducing agents.

R NAMEABBRE-VIATION pI

-CH2CH2CO2H

-CH2(CH2)3NH2

-CH2(CH2)2NHCNHNH2

N

N

H2C

H

Glutamic acid

Lysinee

Arginine

Histidien

Glu

Lys

Arg

His

3.2

9.8

10.8

7.6

2HO2CCHCH2SH

NH2

HO2CCHCH2S_SCH2CHCO2H

NH2 NH2

[0]

[H]

Cysteine(°ë ë×°±Ëá)

Cystine(ë×°±Ëá)

24.2B ESSENTIAL AMINO ACIDS

For adult humans there are eight essential amino acids. These aredesignated with the superscript e in above table.

24.2C AMINO ACIDS AS DIPOLAR IONS

Since amino acids contain both a basic group (-NH2) and an acidicgroup (-COOH) , they are amphoteric.

H3N+CHCO2H

R

H3N+CHCO2-

R

- H+

+ H+

- H+

+ H+

H2NCHCO2-

R

Cationic form (ÑôÀë×ÓÐÎʽ£©

Dipolar ion(Á½¼«Àë×Ó£©

Anionic form (ÒõÀë×ÓÐÎʽ£©

In strongly basic solutions all amino present as anions, in acidic solutions they are present as cations. At some intermediate Ph,called isoelectric point(pI) the concentration of the dipolar ion isat its maximum and the concentrations of the anions and cationsare equal.

H3CCHCO2H

CH3

H3CCHCO2-

CH3

H3CCHCO2-

CH3

Cationic form (pKa1 = 2.3£©

Dipolar ion(pKa2 = 9.7£©

Anionic form

OH- OH-

H+ H+

As the acidity reaches pH 2.3, one half of the cationic form will be converted to the dipolar ion. As the pH increase to2.3- 9.7 thepredominant form will be the dipolar ion. When pH rise to 9.7,the dipolar ion will be half-converted to the anionic form. As pH approached to 14,the anionic form becomes predominant form.

If the side chain of an amino acid contains an extra acidic or basic

group, then the equilibria are more complex.

The isoelectric point (pI) of an amino such as the alanine is the average of pKa1 and pKa2.

pI = 2.3 +9.72

= 6.0

24.3 LABORATORY SYNTHESIS OF α-AMINO ACIDS

A variety of methods have been developed for the laboratorysynthesis of α-amino acids. We shall describe here three generalmethods.

24.3A DIRECT AMMONOLYSIS OF AN α-HALO ACID

CH2CO2HR(1) X2, P4(2) H2O RCHCO2H

X

NH3 (excess) RCHCO2-

NH3+

This method is probably used least often because yields tend to be poor.

24.3B FROM POTASSIUM PHTHALIMIDE

This method is a modification of the Gabriel synthesis of amines.the yields are usually high and the products are easily purified.

N-K+

O

O

+ ClCH2CO2C2H5 N

O

O

CH2CO2C2H5

(1) KOH / H2O(2) HCl

COOH

COOH

+ C2H5OH

CH2CO2-

NH3+ +

Potassium phthalimide (±½ÁÚ¶þ¼×õ£ÑÇ°· ¼Ø£©

Ethyl chloroacetate (ÂÈ́ú ´×ËáÒÒõ¥£©

Glycine(°±»ùÒÒËá)

Phthalic acid (ÁÚ±½¶þ¼×Ëá)

24.3C THE STRECKER SYNTHESIS

Treating an aldehyde with ammonia and hydrogen cyanide produces an α-amino nitrile. Hydrolysis of the nitrile groupof the α-amino nitrile converts the latter to an α-amino acid.

RCHO + NH3 + HCN RCHCN

NH2

H3O+, heat

NH3+

RCHCO2-

¦Á-Amino nitrile ¦Á-Amino acid

Mechanism of the first step:

RCH + NH3 RCHNH3+

RCHNH2

O O- OH-H2O

RCH=NH RCHNH-

CNH+

RCHNH2

CN

24.3D RESOLUTION OF DL-AMINO ACIDS

One interesting method for resolving amino acids is based on the useof enzymes called deacylases.

RCHCO2-DL

NH3+

(CH3CO)2ORCHCO2HDL

NHCOCH3

deacylase

+H3N H

CO2-

R

H3COCHN H

CO2-

R

+

Easily separated

L-Amino acid (L-°±»ùËᣩ

D-N-Acylamino acid (D-N-õ£°±»ùËᣩ

CH3COOH

24.3E STEREOSELECTIVE SYNTHESIS OF AMINO ACIDS

Producing only the naturally occurring L-amino acid has been realized through the use of chiral hydrogenation catalysts from transition metals. One of which is called “(R)-prophos”.

CH2

(C6H5)2P

H

H3C

P(C6H5)2

(R)-Prophos

[Rh(NBD)2]ClO4 + (R)-prophos [Rh((R)-prophos)(NBD)]ClO4

Chiral rhodium complex (ÊÖÐÔîî ÅäºÏ Îï £©

+ NBD

Hydrolysis of N-acetyl group under this chiral rhodium complexyields L-alanine. Because the hydrogenation catalyst is chiral, it transfers its hydrogen atoms in a stereoselective way. This typeof reaction is called asymmetric synthesis.

CH2=C_CO2H

NHCOCH3

[Rh((R)-prophos)(H2)(solvent)2]+

H2

COOH

H3COCHN

H3C

H

N-Acetyl-L-alanine(N-ÒÒõ£»ù-L-±û°· Ëá)

(1) OH-, H2O, heat,(2) H3O+ CO2

-

+H3N

H3C

H

L-alanine(L-±û°· Ëá)

2-Acetylaminopropenoic acid (2-ÒÒõ£°·»ù±ûÏ©Ëᣩ

24.4 ANALYSIS OF AMINO ACID MIXTURES

Enzymes can cause α-amino acids to polymerize through the elimination of water:

+H3NHC

R

C

O

O- +H3NHC

R'

C

O

O-+H2O +H3N

HC

R

C

O

HNHC

R

C

O

O-

A Dipeptide (¶þëÄ£©

The –CO-NH- linkage between the amino acids is called a peptidebond. Amino acid when joined in this way, are called amino acidresidues. The polymers that contains 2,3, a few, or many amino acidresidues are called dipeptides, tripeptides, oligopeptides, and polypeptides, respectively.

Polypeptides are linear polymers. The free group and thefree group are called the N-terminal and the C-terminal Residues respectively.

NH3+

CO2-

HNHC

R¡¡̄®

C

O

O-+H3NHC

R

C

O

HNHC

R¡®

C

O

*

n

N-Terminal C-Terminal

The automatic amino acid analyzers are based on the use of insoluble polymers containing sulfonate groups, called cation-exchange resins.

CH

CH2

CH

CH2

CH2

CH2

HC

SO3-

SO3-

SO3-

SO3-

RCHCO2H

NH3+

R'CHCO2H

NH3+

R''CHCO2H

NH3+

R'''CHCO2H

NH3+

If the mixture of amino acids pass through a column which is washed with a buffered solution at a given pH, The individual amino acids will move down the column at different rates and ultimately separated.

24.5 AMINO ACID SEQUENCE OF POLYPEPTIDES AND PROTEINS

The different amino acid sequences of a protein which compose of 20 different amino acids in a single chain of 100 residues are Amazing large. They are 1.27 X 10130

The methods of determining the amino acid sequence include Terminal residue analysis, partial hydrolysis and so on.

24.5A TERMINAL RESIDUE ANALYSIS

One very useful method for determining the N-terminal amino acidresidue, called the Sanger method, is based on the use of 2,4-dinitrofluorobenzene (DNFB).

O2N

NO2

F + NH2CHCO-NHCHCO

R R'

etcHCO3

-

(-HF)

O2N

NO2

NHCHCO-NHCHCO

R R'

etcH3O+

2,4-Dinitrofluorobenzene (DNFB) (2,4-¶þÏõ »ù·ú ±½£©

Polypeptide (¶àëÄ£©

Labeled polypeptide (ÓбêÖ¾µÄ¶àëÄ£©

O2N

NO2

NHCHCO2H

R

+ H3N+CHCO2-

R'

Labeled N-terminal amino acid(ÓбêÖ¾µÄN-¶Ë°±»ùËᣩ

Mixture of amino acids (»ì ºÏ °±»ùËᣩ

Separated and identify

A second method of N-terminal analysis is the Edman degradation.This method offers an advantage over the Sanger method in that it Moves the N-terminal residue and leaves the remainder of the peptideChain intact.

+ NH2CHCO-NHCHCO

R R'

etcN=C=S OH-, pH 9

NHCSNHCHCO_NHCHCO

R R'

etc H+

NC

NCH

H

R

O

rearrangement heat

N

CCH

NHC

S

OR

H

Unstable intermediate

+

R'

H3N+CHCOH

Polypeptide with one less than amino acid residue

Phenylthiohydantoin

C-terminal residues can identified through the use of digestive enzymes called carboxypeptidases. These enzymes specificallycatalyze the hydrolysis of the amide bond of the amino acid residue containing a free –COOH group, liberating it as a free amino acid.

24.5B PARTIAL HYDROLYSIS

Break the polypeptide chain into small fragments, then examine the structure of these smaller fragments to determine the original polypeptide. For example:

We are given a pentapeptide known to contain valine(two residues), lucine, Histidine, and phenylalanine. Then the molecular formular:

Val2, Leu, His, Phe

By using DNFB and carboxypeptidase we discover that valine and leucine are the N-terminal and C-terminal, respectively.

Val ( Val, His, Phe) Leu

We then subject the pentapeptide to partial acid hydrolysis and obtainthe following dipeptides.

Val·His + His·Val + Val·Phe + Phe·Leu

The points of overlap of the dipeptides tell us that the originalPentapeptide must have been the following:

Val· His· Val· Phe· Leu

24.6 PRIMARY ATRUCTURES OF POLYPEPTIDES AND PROTEINS

The covalent structure of a protein or polypeptide is called primarystructure. Chemists have had remarkable success in determiningthe primary structure.

24.6A OXYTOCIN AND VASOPRESSIN

Oxytocin and vasopressin are two rather polypeptides with strikingly similar structures. But these two polypeptides havequite different physiological effects.

Oxytocin occurs only in the female of a species and stimulates uterine contraction during childbirth. Vasopressin occurs in maleand female. Its major function is as an antidiuretic.

24.6B INSULIN

Insulin, a hormone secreted by the pancreas, regulates glucose metabolism.

Bocine insulin has a total of 51 amino acid residues in two poly-peptide chains, called A and B chains. These chains are joined by two disulfide linkage.

Human insulin differs from bovine insulin at only three amino acids residues. Insulin from most mammals has a similar structure.

24.6C OTHER POLYPEPTIDES AND PROTEINS

Successful sequential analyses have now been achieved with hundreds of other polypeptides and proteins including the following:(1) Bovine ribonuclease.(2) human hemoglobin.(3) bovine trypsinogen and chymotrypsinogen.(4) gamma globulin.

24.7 POLYPEPTIDE AND PROTEIN SYNTHESIS

We must first “activate” the carboxyl group of an acid by converting it to an anhydride or acid chloride and then allow it react with an amine. But when both the acid group and the amino group are presentin the same molecular, the problem becomes more complicate.

24.7A PROTECTING GROUPS

We must “protect” the amino group by converting it to some othergroup of low nucleophilicity-one that will not react with a reactive acyl derivative. Then remove the protecting group.

The reagents are benzyl chloroformate and di-tert-butyl carbonate:

C6H5CH2OCOCL

Benzyl chloroformate (ÜлùÂȼ×Ëáõ¥£©

(CH3)3COCOOC(CH3)3

Di-tert-butyl carbonate (¶þÊ嶡 »ù̼ËáÑΣ©

Both reagents react with the following amino group to form derivatives that are unreactive toward further acylation.

CH2OCOCl + H2NR OH-25¡æ

CH2OCONHR + Cl-

CH3 + CO2 +H2NRCH2Br + CO2 +H2NR

HBr

acetic acid (cold) H2 / Pd

(CH3)3COCOC(CH3)3

O

+ H2NR base25¡æ

(CH3)3COCNHR

O

+ (CH3)3CHO

tert-Butyloxycarbonyl or boc group

(CH3)2C=CH2 + CO2 +H2NR

Benzyl chloroformateBenzyloxycarbonyl or Z group

HCl or CF3CO2Hacetic acid, 25¡æ

Remove of the benzyl group with hydrogen and a catalyst dependson the fact that benzyl-oxygen bonds are weaker and are subject to hydrogenolysis at low temperatures.

H2 / Pd 25¡æC6H5CH2OCR

O

C6H5CH3 HOCR

O

+

24.7B ACTIVATION OF THE CARBOXYL GROUP

A much better method is to convert the carboxyl group of the “protected” amino acid to a mixed anhydride using ethyl chloro-Formate.

Z_NHCHC_OH

R

O(1) (C2H5)3N(2)ClCO2C2H5

Z_NHCH_C_O_C_OC2H5

OO

RMixed anhydride (»ì ºÏ ôû£©

The mixed anhydride can be used to acylate another amino acid and form a peptide linkage.

Z_NHCH_C_O_C_OC2H5

OO

R

H3N+CHR'CO2-

Z_NHCH_C_NHCHCO2H

O

R R'

+ CO2 +C2H5OH

Dicyclohexylcarbodiimide can also be used to activate the carboxyl group of an amino acid.

24.7C PEPTIDE SYNTHESIS

The principle involve here can,of course, be extended to the synthesis of much longer polypeptide chains.

CH3CHCO2-

NH3+

+ C6H5CH2OC

O

Cl CH3CH CO2H

NH

C

OCH2C6H5

O

OH-25¡æ

(1) (C2H5)3N(2) ClCO2C2H5

AlaZ-Ala

benzyl chloroformate (Âȱ½¼×Ëáõ¥)

CH3CHCOOCOOC2H5HNH

C

OCH2C6H5

O

(CH3)2CHCH2CHCO2-

NH3+

CO2 + C2H5OH

CH3CH C

O

NHCHCH2O2H

CH2

CHH3C CH3

NH

C O

OCH2C6H5

H2 / Pd CH3CH C

O

NHCHCO2-

CH2

CHH3C CH3

NH3+

+ CH3 + CO2

CH2Cl + HOCCHNHCOC(CH3)3

O

R

O

base

CH2OCCHNHCOC(CH3)3

O

R

O

CF3CO2H / CH2Cl2

CH2OCCHNH2

O

RHOCOCHR'NHCOOC(CH3)3

dicyclohexylcarbodiimide

Step 1

Step 2

Step 3

Step 4

Step 5

Attaches C-terminal(protect) amino acidresidue to resin

Purifies resin withattached residue bywashing

Removes protecting group

Purifies by washing

Adds nest (protect)amino acid residue

The Merrifield method for automated synthesis:

24.7D AUTOMATED PEPTIDE SYNTHESIS

24.8 SECONDARY AND TERTIARY STUCTURES OF PROTEINS

24.8A SECONDARY STRUCTURE

CH2OCCHNHC

O

R

O

R'

O

CHNHCOC(CH3)3

CF3CO2H / CH2Cl2

Step 6

Step 7

Purifies by washing

Removes protecting group

Repetitions of step 4-7

HBr / CF3CO2H

CH2Br

O

R

O

HOCCHNHC

R'

O

CHNHCCHNH

R''

+

Final step

etc

Detaches completedpolypeptide

The secondary structure of a protein is defined by the local Confor-mation of its polypeptide backbone. These local conformation havecome to be specified terms of regular folding patterns.

Polypeptide chain of a natural protein can interact with itself two major ways: through formation of a β-pleated sheet and an α helix.

NC

CN

CC

NC

CN

H O H OR

R RH H

CC

NC

CN

CC

NC

O H O H

H HR R

H R

H

OHO

H O Hcrowding crowding

Fully extended polypeptide chains could conceivably form a flat-

sheet structure (above).

Slight rotation of bonds can transform a flat-sheet structure intothe β-pleated sheet or β configuration.

The α helix structure is a right-handed helix with 3.6amino acid residues per turn in naturally occurring. It is the predominant structure of the polypeptide.α

Helices and pleated sheets account for only about one half of the average globular protein. The remaining polypeptide segmentshave what is called a coil or loop conformation.

24.8B TERTIARY STRUCTURE

The tertiary structure of a protein is its three-dimensional shape that arises from further foldings of its polypeptide chains, foldings superimposed on the coils of the α helixes.

24.9 INTRODUCTION TO ENZYMES

Enzymes have the ability to bring about vast increases in the ratesof reaction. Enzymes also show remarkable specificity for their reactants and for their products.

The enzyme and the substrate combine to form an enzyme-substratecomplex.

Enzyme + Substrate enzyme-substrate complex

Enzyme + Product

Almost all enzymes are proteins. Reactions catalyzed by enzymesare completely stereospecific, and this specificity comes from the way enzymes bind their substrates.

Some enzymes will accept only one compound as its substrate, others will accept a range of compounds with similar groups.

Inhibitor: a compound that can alter the activity of an enzyme.competitive inhibitor: a compound that competes directly with the substrate for the active site.

Some enzymes require the presence of a cofactor. Others may require the presence of an organic molecule called a coenzyme.

Many of the water-soluble vitamins are the precursors of coenzymes.

N

C

O

OH

Niacin(ÄáÑÇÐÂ)

HO

O

HN

OH

O

OH

CH3

CH3

Pantothenic Acid (·º Ëá)

24.10 LYSOZYME: MODE OF ACTION OF AN ENZYME

Lysozyme is made up of 129 amino acid residues. Three shortsegments of the chain between residues 5-15, 24-34, 88-96 havethe structure of an α helix; the residues between 41-45, and 50-54 form pleated sheets; and a hairpin turn occurs at residues 46-49. The remaining polypeptide segments of lysozyme have a coil orloop conformation.

Lysozyme’s substrate is a polysaccharide of amino sugar that makesup part of the bacterial cell wall.

24.11 SERINE PROTEASES

Serine proteases: the digestive enzymes secreted by the pancreas into the small intestines to catalyze the hydrolysis of peptide bonds.

The digestive enzymes includes chymotrypsin, trypsin, and elastin.

The catalytic triad of chymotrypsin cause cleavage of a peptidebond by acylation of the serine residue 195 of chymotrysin. Near the active site is a hydrophobic binding site that accommodates nonpolar side chains of the protein.

H2C C

O

O-H N

N

CH2

H OCH2

N CO

R

H

R'

102

57

195

AspHis

Ser

Hydrophobic pocket binding site

H2C C

O

O-H N

N+

CH2

OCH2

N CO-

R

H

R'

102

57

195

AspHis

H

Ser

Tetrahedralintermediate

H2C C

O

O-H N

N

CH2

H

102

57AspHis

NH

R'

OCH2

CO

R

195

Ser

Acetylatedserineresifue

H2C C

O

O-H N

N+

CH2

OCH2

O C O-

RH

102

57

195

AspHis

H

Ser

Tetrahedralintermediate

H2C C

O

O-H N

N

CH2

H

102

57AspHis

OH

OCH2

CO

R

195

Ser

+

Regeneration of the active site of chymotrypsin. Water causes hydrolysis of the acyl-serine bond.

Compounds such as diisopropylphosphofluoridate (DIPF) thatirreversibly inhibit serine proteases. It has been shown that theydo this by reacting only with Ser 195.

CH2OH195Ser + P

O

O

O

F

CH(CH3)2

CH(CH3)2

CH2195Ser P

O

O

O

O

CH(CH3)2

CH(CH3)2

(DIPF) DIP-Enzyme

24.12 HEMOGLOBIN: A CONJUGATED PROTEIN

Hemoglobin: a protein can carry oxygen.

N Fe

N

N

N

C

CCH

CH3C

H

HOOCH2CH2C

HOOCH2CH2C

H H

H

CH3

CH3

CH=CH2

CH=CH2H3C

The iron of the heme group is in the 2+ oxidation state and it formsa coordinate bond to a nitrogen of the imidazole group of histidineof the polypeptide chain. This leaves one valence of the ferrous ioncombine with oxygen as follows:

When the heme combing with oxygen the ferrous ion does notbecome readily oxidized to the ferric state.

N

Fe

N

N N

O2

N

A portion of oxygenated hemoglobin