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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)