177

348

Chapter 26: Biomolecules: Amino Acids, Peptides, and Proteins

monomer unit: α-amino acids biopolymer: peptide (< 50 amino acids)

protein (> 50 amino acids)

R CO2H

NH2H

!- Amino Acid

R = sidechain

Peptide or protein

NH

O

R3

HN

O

R2

NH

O

R1

HN

NH

O

R7

HN

O

R6

NH

O

R5

R4 HN

O

Amino acids are linked together through amide bonds (peptide bonds)

349

26.1 Structures of Amino AcidsAmino acids exist as a zwitterion: a dipolar ion having

both a formal positive and formal negative charge (overall charge neutral); internal salts

CO2H

R

H

H2N CO2

R

H

H3N_+

Amino acids are amphoteric: they can react as either an acid or a base. Ammonium ion acts as an acid, the carboxylate as a base

178

350

20 common amino acids19 are 1°-amines, one (proline) is a 2°-amine

19 amino acids are “chiral; one (glycine) is achiral (R=H)

The configuration “natural” amino acid is L19 are of the S stereochemistry, one (cysteine) is R

R CO2H

NH2H

R = sidechain

primary α-amino acid

N CO2_

H H

+

prolinesecondary α-amino acid

CHO

CH2OH

H OH

D-glyceraldehyde

CHO

CH2OH

HO H

L-glyceraldehyde

CO2H

CH3

H2N H

CO2H

CH2SH

H2N H

L-alanine L-cysteine

351

Neutral amino acids

(S)-(+)-Valine (Val, V)(S)-(–)-Tryptophan (Trp, W)

(S)-(–)-Proline (Pro, P)(S)-(–)-Phenylalanine (Phe, F)

COO–

NH3

H

N

H

COO–

NH3

COO–

NH3

COO–

NH3

NH3

COO–

COO–

(2S,3S)-(+)-Isoleucine (Ile, I)

(S)-(+)-Alanine (Ala, A)

(S)-(–)-Leucine (Leu, L)

NH3

COO–S

(S)-(–)-Methionine (Met, M)

(S)-(+)-Glutamine (Gln, Q)

Glycine (Gly, G)

NH3

COO–

H2N

O

NH3

COO–

(S)-(–)-Tyrosine (Tyr, Y)

(2S,3R)-(–)-Threonine (Thr, T)(S)-(–)-Serine (Ser, S)

COO–

NH3

COO–

NH3

COO–

NH3 NH3

COO–

NH3

COO–

O

H2NHS

HO

OH

HO

(R)-(–)-Cysteine (Cys, C)(S)-(–)-Asparagine (Asn, N)

pKa ~ 13 pKa ~ 13

pKa ~ 8.2

pKa ~ 10.1

COO–

NH3NH

179

352

Acidic amino acids

Basic amino acids

(S)-(–)-Histidine (His, H)

NH3

COO–COO–

NH3 NH3

COO–N

H

NN

H

H

(S)-(+)-Lysine (Lys, K) (S)-(+)-Arginine (Arg, R)

pKa ~ 10.5 pKa ~ 6.0 pKa ~ 12.5

H2N

NH H

H3N

-O

O NH3

COO–

(S)-(+)-Aspartic Acid (Asp, D)

COO–

NH3

O

-O

(S)-(+)-Glutamic Acid (Glu, E)

pKa ~ 3.6 pKa ~ 4.2

353

Humans can produce (biosynthesize) ten of the twenty aminoacids; the remaining ten must be obtained by diet andare called essential amino acids: tryptophan, lysine, methionine, phenylalanine, threonine, valine, leucineand isoleucine.

26.2 Isoelectric points pI: pH at which the amino acid exists largely in a neutral, zwitterionic form (influenced by the nature of the sidechain)

CO2H

R

H

H2N CO2

R

H

H3N_+

CO2H

R

H

H3N+H3O

+

pKa1

CO2

R

H

H2N

HO_

pKa2

low pH

high pH

_

180

354

pI =pKax + pKay

2

pI =pKa1 + pKa2

2

pI =pKa1 + pKa3

2

pI =pKa2 + pKa3

2

pI = 9.7

pI = 2.7

CO2

CH3

H

H3NCO2H

CH3

H

H3N

low pH

CO2

CH3

H

H2N+ +

high pH

pKa1(2.3)

pKa2(9.7)

pI = 6.0

CO2H

(CH2)4

H

H3N

NH3

pKa2(9.0)

CO2

H

H3N

(CH2)4

NH3

CO2

H

H2N

(CH2)4

NH3

pKa3(10.5)

pKa1(2.2)

CO2

H

H2N

(CH2)4

NH2

low pH high pH

CO2H

CH2

H

H3N

CO2H

pKa3(3.6)

CO2

H

H3N

CH2

CO2H

CO2

H

H3N

CH2

CO2

pKa2(9.6)

pKa1(1.9)

CO2

H

H2N

CH2

CO2

low pH high pH

355

Henderson-Hasselbalch equation: allows the calculation of the relative amounts of the protonated, neutral, and deprotonated forms at a given pH from the pKa values of the amino acid (please read)

Electrophoresis: separation of polar compounds based on theirmobility through a solid support. The separation is based on charge (pI) or molecular mass.

+ _

_ _ _ _ + + + +

+ _

181

356

26.3 Synthesis of Amino AcidsFrom Chapter 24: HVZ reaction followed by SN2 reaction

with NH3 or ammonia equivalents

Reductive amination

R-CH2-CO2H

R C CO2H

Br

R C CO2H

NH2

R C CO2H

N3

Br2, PBr3

NH3

NaN3

H2, Pd/C

R C CO2H

N OOKOH, H2O

N

O

O

KH

H

H

C CO2HR

O NH3

H2, Pd/C-or-

NaB(CN)H3

C CO2HR

NH2

H

357

RCH2 CO2EtC

CO2EtHN EtO Na

RCH2X

H3O

- CO2 RCH2 CO2HC

HH2N

O

RCH2 CO2EtC

CO2EtHN

O

Amidomalonate Synthesis

26.4 Enantioselective Synthesis of Amino AcidsThe syntheses in the previous section give racemic products!!

R-CH2-CO2H

Br2, PBr3

RCH Br

O

Br Br

R CH

CO2H

Br

R

H O

Br

Br Br

Br Br

Br

R

HCO2H

Br

R

HCO2H

R

S

50:50mixture of

enantiomers

182

358

Resolution: separation of enantiomers

RCH2 CO2HC

NH2H

N

N

H

H

(-)-sparteine(chiral base)

RCH2 CO2

C

NH2H

N

N

H

HH

N

N

H

HH

RCH2 CO2

C

HH2N

+

Diastereomeric salts(separate)

H3O H3O

RCH2 CO2

C

NH3H

RCH2 CO2

C

HH3N

racemic

Resolutions are inherently inefficient

359

Enantioselective Synthesis of Amino Acids (please read)

R

H CO2H

NHAc

H H

H H

R NHAc

H H

H CO2H

R NHAc

H H

H CO2HR

S

Rh(I) L*, H2

O

O

CO2Me

HO

OH

CO2

NH3

L-DOPA

O

O

CO2Me

HN

O

PhHN

O

Ph

H3O+

Chiral hydrogenation catalysts can differentiate the faces of the C=C double bond leading a products that is highly enriched in one enantiomer.

183

360

26.5 Peptides and ProteinsProteins and peptides are polymers made up of amino acid units

(residues) that are linked together through the formationof an amide bonds (peptide bonds) from the amino group of one residue and the carboxylate of a second residue

By convention, peptide sequences are written left to right from the N-terminus to the C-terminus

H2N CO2H

Alanine

H2N CO2H

HO

Serine

+ - H2O

H2N

HN

O

CO2H

OH

Ala - Ser(A - S)- H2O

H2N

HN

O

CO2H

HO

Ser - Ala(S - A)

C-terminus

N-terminus

C-terminus

N-terminus

NH

O

R3

HN

O

R2

NH

O

R1

HN

NH

O

R7

HN

O

R6

NH

O

R5

R4 HN

O

backbone

361

26.6 Covalent Bonding in PeptidesI. The amide bond

II. Disulfide bonds: the thiol groups of cysteine can be oxidized to form disulfides (Cys-S-S-Cys)

C=N double bond characterdue to this resonance structure

restricts rotationsresistant to hydrolysis

R1

HN

N

O R2 HN

O

amide bond

R1

HN

N

O R2 HN

O

_

+

H H

SHHO2C

NH22

1/2 O2 H2O

SHO2C

NH2

SCO2H

NH2

NH

O

R2

HN

O

R1

NH

HN

O

R5

NH

O

R4

HN

O

NH

OHN

O

R6

NH

HN

O

R10

NH

O

R9

R8 HN

O

SH

NH

O

R2

HN

O

R1

NH

HN

O

R5

NH

O

R4

HN

O

NH

OHN

O

R9

NH

HN

O

R13

NH

O

R12

R11 HN

O

S

SHS 1/2 O2

184

362

Epidermal Growth Factor (EGF):53 amino acid, 3 disulfide linkages

Cys14-Cys31Cys6-Cys20

Cys33-Cys42

1986 Nobel Prize in Medicine:Stanley Cohen Rita Levi-Montalcini

363

26.7 Structure Determination of Peptides: Amino Acid Analysis

Primary (1°) structure of a peptide or protein is the amino acid sequence

Amino acid analyzer- automated instrument to determine the amino acid content of a peptide or protein

peptide-or-

protein

[H] reduce anydisulfidebonds

Enzymaticdigestion

R CO2

NH3 individualamino acids-or-

H3O+, Δ

1972 Nobel Prize in ChemistryWilliam Stein Stanford Moore

liquidchromatography

derivatize w/ninhydrin

Detected w/UV-vis

Different amino acids have different chromatographicmobilities (retention times)

185

364

Reaction of primary amines with ninhydrin

Intense purple color

R CO2

NH3

O

O

O+-H2O

O

O

N

R

O

O H

O

O

N

R

H2O

- RCHO

O

O

NH2

- CO2

O

O

OO

O

N

O

O

Amino Acid Analysis Chromatogram

365

26.8 Peptide Sequencing: The Edman Degradationchemical method for the sequential cleavage andidentification of the amino acids of a peptide, one at a time starting from the N-terminus.

Reagent: Ph-N=C=S, phenylisothiocyanate (PITC)

S

C

NPh

+H2N

O

R1 HN CO2

pH 9.0

NH

O

R1 HN CO2

NH

S

Ph

H+

H+

HN

S

OH

HN CO2

N

R1

PhH+

HN

S OH2N CO2

N

R1

Ph

H+

HN

N OS

R1

Ph

+

N-phenylthiohydantoin:separated by liquid chromatography (based of the R group) and detected by UV-vis

-1 peptide with a new N-terminal amino acid (repeat degradation cycle)

186

366

Peptide sequencing by Edman degradation: cycle the pH to control the cleavage of the N-terminal

amino acid by PITC. Monitor the appearance of the new N-phenylthiohydantoin

for each cycle. Good for peptides up to ~ 25 amino acids long. Longer peptides and proteins must be cut into smaller

fragments before Edman sequencing.

Enzymatic cleavage of peptides and proteins at defined sites

• trypsin: cleaves at the C-terminal side of basic residues, Arg, Lys but not His

NH

O

HN

O

NH

R3

O

HN

R1O

H3NCO2

NH3

NH

O

HN

O

H3N

R3

O

HN

R1O

H3NCO2

NH3

O +trypsin

H2O

367

• chymotrypsin: cleaves at the C-terminal side of aromatic residuesPhe, Tyr, Trp

NH

O

HN

O

NH

R3

O

HN

R1O

H3NCO2 N

HO

HN

O

H3N

R3

O

HN

R1O

H3NCO2

O +chymotrypsin

H2O

trypsin

H2N–Val–Phe–Leu–Met–Tyr–Pro–Gly–Trp–Cys–Glu–Asp–Ile–Lys–Ser–Arg–His-CO2H

H2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-CO2HH2N-His-CO2HH2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-CO2HH2N-Ser-Arg-CO2H

H2N-Val-Phe-CO2HH2N-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2HH2N-Val-Phe-Leu-Met-Tyr-CO2HH2N-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2HH2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-CO2HH2N-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2H

chymotrypsin

187

368

Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-ArgVal-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys Ser-Arg

His

Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-HisVal-Phe-Leu-Met-Tyr-Pro-Gly-Trp Cys-Glu-Asp-Ile-Lys-Ser-Arg-HisVal-Phe-Leu-Met-TyrVal-Phe

Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His

If given the sequence of the enzyme digest fragements, allignthe sequences of the fragments to get the sequence ofthe full peptide

Trypsin:

Chymotrypsin:

369

EPIDERMAL GROWTH FACTOR (EGF): 53 amino acids

H2N-ASN1•SER2•TYR3•PRO4•GLY5•CYS6•PRO7•SER8•SER9•TYR10•ASP11•GLY12•TYR13•CYS14•LEU15•ASN16•GLY17•GLY18•VAL19•CYS20•MET21•HIS22•ILE23•GLU24•SER25•LEU26•ASP27•SER28•TYR29•THR30•CYS31•ASN32•CYS33•VAL34•ILE35•GLY36•TYR37•SER38•GLY39•ASP40•ARG41•CYS42•GLN43•THR44•ARG45•ASP46•LEU47•ARG48•TRP49•TRP50•GLU51•LEU52•ARG53-CO2H

Trypsin Chymotrypsin Cyanogen Bromide (BrCN)

Cys14-Cys31Cys6-Cys20

Cys33-Cys42

26.9 Peptide Sequencing: C-Terminal ResidueDetermination (please read)

188

370

Peptide sequencing by mass spectrometry (Lagniappe)Peptides can be sequenced rapidly by tandem mass spectrometry.

Peptide and proteins are product of gene expression

The Central Dogma (F. Crick):

DNA mRNA Protein (genome) (proteome)

Can we understand . . .. . . the biological functions of proteins . . . . . . the relationship between protein function / expression

and disease . . . . . . the relationship between protein modification, either

biochemically or by toxicants and alter protein function / expression and disease . . .

. . . the biological target of drugs or toxicants . . .

. . . by profiling the proteins in a cell or tissue?

371

Mass spectrometry is a gas phase technique. Peptides (and proteins) are charged, polar, high molecular weight molecules (ions). How can peptides and proteins becoaxed into the gas phase?

Electrospray ionization (ESI): analyte is introduced into the massspectrometer as an aerosol.

+++++

+++

++++

+++++

++++

+++

++++

+++++

+

+

- +

+

to the mass analyzer

liquid chromatographyor capillary electrophoresis(separate the analytes)

+++++

+++

++++

+++++

++++

+++

++++

+++++

+

+ +++++

- +

+ ++++++

+++

++++

+++++

++++

+++

++++

+++++

+

+ + ++++

- +

+++++

++

+++

++++

+++++

++++

+++

++++

+++++

+

+ +++++

- +

+ ++++++

+++

++++

+++++

++++

+++

++++

+++++

+

+ +++ ++

- +

+ ++++++

+++

++++

+++++

++++

+++

++++

+++++

+

+ +++ ++

+

+

++

+

- +

+ ++

Coulombicfission

+++++

+++

++++

+++++

++++

+++

++++

+++++

+

+

+

+

++

+

- +

++

+++++

+++

++++

+++++

++++

+++

++++

+++++

+

+

- +

+

+

+

+

+

++

+++++

+++

++++

+++++

++++

+++

++++

+++++

+

+

- +

+

+

+

+

+

+

+++++

+++

++++

+++++

++++

+++

++++

+++++

+

+

- +

+

+

+

+

+

+

+

+

+

+

+

++++ ++

+

+

++

+

++

Coulombic

fission

+++++

++ ++++

+++++++

189

372

MALDI ionization (matrix-assisted laser desorption): analyte isco-crystallized with an organic molecule that has anintense UV absorption. A laser that is tuned to the absorption of the matrix, is “pulsed” at the MALDImatrix and energy is indirectly transferred to the analyte.

++

+

+

+

+

to the mass analyzer

Laser pulse

++

+

+

+

+

+

+

2002 Nobel Prize in ChemistryJohn Fenn (ESI)Koichi Tanaka (MALDI)

373

Peptide sequencing by tandem mass spectrometry

to thedetector

Q1ElectrosprayIon Source Q3Collision Cell (Q2)Nanospray

Capillary

Select peptide to be analyzed

fragment thepeptide

Analyze thepeptide fragments

1000 1500 2000 2500 3000 m/z

1116

.67

1287

.73

1375

.76

1424

.85 1505

.77

1665

.89

1811

.85

2005

.07

2476

.21

2550

.52

2719

.48

1849

.12

1574

.20

1247

.70

Peptides fragment ina predictable manner

H2N CH C

R1

OHN CH C

R2

OHN CH C

R3

OHN CH C

R4

OH

O

b1

y1

b2

y2 y3charge to

C-terminus

charge to N-terminus

b3

Select m/z 1228.7 for Q2

190

374

average exact - HN-CHR-COGlycine G 75.07 75.03 57Alanine A 89.10 89.05 71Serine S 105.09 105.04 87Proline P 115.13 115.05 97Valine V 117.15 117.08 99Threonine T 119.12 119.06 101Cysteine C 121.16 121.02 103Isoleucine I 131.18 131.09 113Leucine L 131.18 131.09 113Asparagine N 132.12 132.05 114Aspartic Acid D 133.11 133.04 115Glutamine Q 146.15 146.07 128Lysine K 146.19 146.11 128Glutamic Acid E 147.13 147.13 129Methionine M 149.21 149.05 131Histidine H 155.16 155.02 137Phenylalanine F 165.19 165.19 147Arginine R 174.20 174.11 156Tyrosine Y 181.19 181.07 163Tryptophan W 204.23 204.09 186

Amino Acids Sorted by Mass

H2N CH C

R1

OHN CH C

R2

OHN CH C

R3

OHN CH C

R4

OH

O

b1

y1

b2

y2 y3

b3

375

112.2Leu/Ile

173.1Arg

96.9Pro

101.1Thr

113.1Leu/Ile

115.3Asp

57.1Gly

146.8Phe

146.9Phe

71.2Ala

57.1Gly

71.1Ala

128.0Lys/Gln128.2

Lys/Gln

57.2Gly

57.0Gly

70.9Ala147.1

Phe

147.0Phe115.1

Asp

57.0Gly

57.1Gly

57.1Gly

71.0Ala

57.0Gly

H2N-Ile-

-Arg-CO2H

-Pro-

-Phe-

-Ile-

-Gly-

-Gly-

-Gly-

-Phe--Ala-

-Gln--Ala-

-Gly--Ala-

-Gly-

-Gln-

-Ala-

-Gly-

-Phe-

-Gly-

-Gly-

-Phe--Asp-

-Asp--Thr-

191

376

Analysis of the proteome: separate proteins of a cell by two-dimensional electrophoresis

_ _ _ _ + + + +

separateby pI

separateby mass(PAGE)

377

5.1 5.95.7 6.3 9.6

16.9

50

107

42

128

pI

MW

192

378

26.10: Peptide Synthesis

H2N CO2H

Ala

H2N CO2H

Val

+- H2O

H2N

HN

O

CO2H

Ala - Val(A - V)

H2N

HN

O

CO2H

Val - Ala(V - A)

- H2O

The need for protecting groups

Orthogonal protecting group strategy: the carboxylateprotecting group must be stable to the reaction conditions for the removal of the α-aminoprotecting group (and vice versa)

NH

Ala

H2N

Val

+

- H2O

NH

HN

O

Ala - Val(A - V)

Pn OH

O

OPc

O

PnOPc

Oselectivelyremove Pn

H2N

HN

O

Ala - Val(A - V)

OPc

O

peptidecoupling

peptidecoupling(-H2O)

NH

Pn OH

O

Ph

Phe (F)

NH

HN

O

OPc

OOHN

Ph

Pn

Phe - Ala - Val(F - A - V)

Repeat peptide synthesis

379

C-terminal protecting group:benzyl ester: removes by catalytic hydrogenation

(H2, Pd/ C)

α-amino protecting group: tert-butyloxycarbamoyl (BOC): removed with strong

protic acid (F3CCO2H)

Peptide coupling reagent: dicyclohexylcarbodiimide (DCC)

C6H11 N C N C6H11

R OH

O

C6H11 N C NH

C6H11

R O

O

+

R O

O

C

N

C6H11

C6H11

NH

R'-NH2

••

R O

O

C

HNC6H11

C6H11

NH

NHR' +

R NH

O

R'NH

NH

O

C6H11 C6H11+

(DCC)

Amide DCU

"activated acid"

R O

O

C

N

C6H11

C6H11

NH

NR'

++H

H

Mechanism: Figure 26.5, page 1006

193

380

In order to practically synthesize peptides and proteins, time consuming purifications steps must be avoided until the very end of the synthesis.

Large excesses of reagents are used to drive reactions forward and accelerate the rate of reactions.

How are the excess reagents and by-products from the reaction,which will interfere with subsequent coupling steps, removed without a purification step?

NH

Ala

H2N

Val

+ NH

HN

OBOC OH

O

OBn

O

BOCOBn

O

selectivelyremove N-protecting

group

H2N

HN

O

OBn

O

NH

BOC OH

O

Ph

Phe (F)

NH

HN

O

OBn

OOHN

Ph

BOC

DCC

peptidecoupling

CF3CO2H DCC

CF3CO2H

NH

HN

O

OBn

OO

H2N

Ph

H2, Pd/CNH

HN

O

OH

OO

H2N

Ph

Phe - Ala - Val(F - A - V)

381

26.11 Automated peptide synthesis: The Merrifield Solid-Phase Techniques: Peptides and proteins up to ~ 100 residues long are synthesized on a solid, insoluble, polymer support. Purification is conveniently accomplishedafter each step by a simple wash and filtration.

The solid support (Merrifield resin): polystyrene polymer

~ 1 - 10% of the available phenyl groups are functionalized

O

R

OHN_

O

O

R

NH

CF3CO2H

O

O

R

NH2

BOC

BOC

+

polymerization

styrene

divinylbenzene(crosslinker, ~1 %)

initiator

Ph

Ph Ph Ph Ph

PhPhPh Ph

Ph

Ph

Ph

Ph

Ph

Ph

Ph

H3COCH2ClZnCl2

CH2Cl

commericallyavailable

194

382

H2N

Val

O

Opeptidecoupling

NH

BOC OH

ODCC

O

O

HN

BOC NH

Opurify:

wash & filter

remove N-protecting

group

CF3CO2H

O

O

H2NNH

O

NH

BOC OH

O

Ph

Phe (F)

DCC

O

O

NH

O

purify:wash & filter

NH

BOCHN

O

Ph

purify:wash & filter HF

remove N-protecting

group and cleavefrom solid-support

OH

O

NH

O

H2N

HN

O

Phpurify by liquidchromatograrphy

or electrophoresis

Solid-phase peptide synthesis

383

LYS GLU THR ALA ALA ALA LYS PHE GLU ARG GLN HIS MET ASP SER SER THR SER ALA ALA SER SER SER ASN TYR CYS ASN GLN MET MET LYS SER ARG ASN LEU THR LYS ASP ARG CYS LYS PRO VAL ASN THR PHE VAL HIS GLU SER LEU ALA ASP VAL GLN ALA VAL CYS SER GLN LYS ASN VAL ALA CYS LYS ASN GLY GLN THR ASN CYS TYR GLN SER TYR SER THR MET SER ILE THR ASP CYS ARG GLU THR GLY SER SER LYS TYR PRO ASN CYS ALA TYR LYS THR THR GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE ASP ALA SER VAL

His-12 AHis-119 A

His-12 BHis-119 B

pdb code: 1AFL

Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNASolid-phase synthesis of RNase A:B. Gutte & R. B. Merrifield, J. Am. Chem. Soc. 1969, 91, 501-2.

Synthetic RNase A: 78 % activity 0.4 mg was synthesized 2.9 % overall yield average yield ~ 97% per coupling step

R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry: “for his development of methodology for chemical synthesis on a solid matrix.”

195

384

26.12 Protein Classification (please read)

26.13 Protein Structureprimary (1°) structure: the amino acid sequence.secondary (2°) structure: recurring sub-structural motifs of

proteins. These include α-helices, β-sheets, turns, disulfide bonds and others. These sub-structures arelargely held together by H-bonds and other non-covalentinteractions.

tertiary (3°) structure: The overall three-dimensional structure(conformation) of a singe polypeptide chain.

quaternary (4°) structure: overall organization of non-covalentlylinked subunits of a functional protein

385

Common secondary (2°) sub-structural motifs; α-helix: collagen 3.6 amino acids per coil, 5.4 Å

3.6 AA,5.4 Å

196

386

Common secondary (2°) sub-structural motifs; β-helix:

387

Hydrophobic (on the inside) and hydrophilic (on the outside)residues of myoglobin

Pro • Ile • Lys • Tyr • Leu • Glu • Phe • Ile • Ser • Asp • Ala • Ile • Ile • His •Val • His • Ser • Lys

197

388

26.14 Enzymes: a catalyst of a biological reaction.accelerates the rate of a reaction by lowering the

activation energy (ΔG‡)

Proteases: catalyzes the hydrolysis of peptide bonds

NH

HN

NH

O

RO

HN

N

O R

RO

R

H

O protease

H2OH3N

CO2 NH

HN

H3N

O

RO

HN

N

O R

RO

R

H

O

H3NCO2O +

chymotrypsin: cleaves at the C-terminal side of aromatic residues Phe, Tyr, Trptrypsin: cleaves at the C-terminal side of basic residues Arg, Lys but not His

389

Many enzymes catalyze reactions by using the function groupson the amino acid sidechains.

Ser-195

His-57

Asp-102

Catalytic triad of α-chymotrypsin

pdb code: 5CHA

198

390

Some reactions require additional organic molecules or metalions. These are referred to as cofactors (coenzymes)

N N

SO

P-O O

O PO-

O-

O

NH2

N

+

Thiamin diphosphate (Vitamin B1)

N

2-O3POOH

O H

Pyridoxal Phosphate (Vitamin B6)

N

N

NH

N O

O

O

OH

HO

OH

O-

P

O

P O

O

O-

O

HO

N

OH

O

N

N

N

NH2

Flavin Adenine Diphosphate(Vitamin B2)

NN

N N

H 2N

O NH2

O

NH2

NH2O

O

O

Co

O

H

H2N

O

NH

O

C

PO

O

-O

O

N

N

HO H

H

NH2

H

H

OH

N

Cyanocolbalmin(Vitamin B12)

N

NN

N

O

HOO

OH

Fe(III)

Heme

NHHN

S

O

CH4CO2H

Biotin

N

HNN

N

O

H2N

HN

O

HN CO2H

CO2H

Folic acid

391

The protein part in such an enzyme is called an apoenzyme, and the combination of apoenzyme plus cofactor is called a holoenzyme

26.15 How Do Enzymes Work? Citrate SynthaseBy bringing reactants together and holding them in the optimal

orientation for the reaction to occur

26.16 Protein DenaturationThe unfolding of the three-dimensional structure of a protein to a “random coil” state. This results in the lossof its secondary, tertiary and quaternary structures andany biological activity. Proteisn can be denatured byheat, pH or chemicals (urea)