Download 177 Chapter 26: Biomolecules: Amino Acids, Peptides, and Proteins

Chapter 26: Biomolecules: Amino Acids, Peptides,
and Proteins
monomer unit: α-amino acids
biopolymer:
peptide (< 50 amino acids)
protein (> 50 amino acids)
H NH2
R
R = sidechain
CO2H
!- Amino Acid
O
H
N
R1
R2
N
H
O
H
N
O
R3
R4
N
H
O
H
N
O
R5
R6
N
H
O
H
N
O
R7
N
H
Peptide or protein
Amino acids are linked together through amide bonds (peptide bonds)
348
26.1
Structures of Amino Acids
Amino acids exist as a zwitterion: a dipolar ion having
both a formal positive and formal negative charge
(overall charge neutral); internal salts
R
H2N
CO2H
H
+ R
_
H3N
CO2
H
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
349
177
H NH2
R
CO2H
+
N
H H
R = sidechain
_
CO2
proline
secondary α-amino acid
primary α-amino acid
20 common amino acids
19 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 L
19 are of the S stereochemistry, one (cysteine) is R
H
CHO
OH
CH2OH
HO
D-glyceraldehyde
CHO
H
CH2OH
L-glyceraldehyde
CO2H
H
CH3
H2N
L-alanine
H2N
CO2H
H
CH2SH
L-cysteine
350
Neutral amino acids
COO–
COO–
H2N
NH3
O
(S)-(+)-Alanine (Ala, A)
HS
NH3
NH3
(S)-(–)-Asparagine (Asn, N)
O
COO–
(R)-(–)-Cysteine (Cys, C)
COO–
H2N
NH3
(S)-(+)-Glutamine (Gln, Q)
pKa ~ 8.2
COO–
COO–
COO–
NH3
NH3
Glycine (Gly, G)
(2S,3S)-(+)-Isoleucine (Ile, I)
(S)-(–)-Leucine (Leu, L)
COO–
NH3
H
(S)-(–)-Phenylalanine (Phe, F)
NH3
(S)-(–)-Tryptophan (Trp, W)
H
(S)-(–)-Proline (Pro, P)
COO–
N
H
COO–
N
COO–
HO
COO–
S
NH3
NH3
(S)-(–)-Tyrosine (Tyr, Y)
HO
COO–
NH3
NH3
(S)-(–)-Methionine (Met, M)
OH
COO–
NH3
(S)-(–)-Serine (Ser, S)
(2S,3R)-(–)-Threonine (Thr, T)
pKa ~ 13
pKa ~ 13
COO–
NH3
(S)-(+)-Valine (Val, V)
pKa ~ 10.1
351
178
Acidic amino acids
-O
O
COO–
O
COO–
-O
NH3
NH3
(S)-(+)-Aspartic Acid (Asp, D)
(S)-(+)-Glutamic Acid (Glu, E)
pKa ~ 3.6
pKa ~ 4.2
Basic amino acids
COO–
H3N
NH3
N
H
H
COO–
H2N
NH3
N
H
(S)-(+)-Lysine (Lys, K)
(S)-(–)-Histidine (His, H)
pKa ~ 10.5
pKa ~ 6.0
N
H
COO–
N
H
NH3
(S)-(+)-Arginine (Arg, R)
pKa ~ 12.5
352
Humans can produce (biosynthesize) ten of the twenty amino
acids; the remaining ten must be obtained by diet and
are called essential amino acids: tryptophan, lysine,
methionine, phenylalanine, threonine, valine, leucine
and 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)
R
H2N
CO2H
H
+ R
_
H3N
CO2
H
HO
pKa2
R
H2N
_
H3O+
pKa1
+ R
H3N
CO2H
H
low pH
_
CO2
H
high pH
353
179
pKax + pKay
2
pI =
+ CH3
H3N
CO2H
H
+ CH3
H3N
CO2
H
pKa1
(2.3)
low pH
H2N
pKa2
(9.7)
CO2H
CO2
CH2
CH2
CH2
CO2H
H
pKa1
(1.9)
H3N
CO2
H
pKa3
(3.6)
pI =
H3N
CO2
H
CO2
CH2
pKa2
(9.6)
H2N
pI =
2
H
pI = 2.7
high pH
NH3
NH3
NH3
NH2
(CH2)4
(CH2)4
(CH2)4
H
pKa1 + pKa3
CO2
(CH2)4
CO2H
2
pI = 6.0
low pH
H3N
pKa1 + pKa2
high pH
CO2H
H3N
CH3
CO2
H
pKa1
(2.2)
H3N
CO2
H
pKa2
(9.0)
H2N
CO2
H
pKa3
(10.5)
H2N
CO2
pI =
pKa2 + pKa3
2
H
pI = 9.7
high pH
low pH
354
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 their
mobility through a solid support. The separation is based
on charge (pI) or molecular mass.
_
+
+
_
_
_ _
_
+ +
+
+
355
180
26.3
Synthesis of Amino Acids
From Chapter 24: HVZ reaction followed by SN2 reaction
with NH3 or ammonia equivalents
R-CH2-CO2H
Br2, PBr3
O
Br
R C CO2H
NaN3
N3
NH3
O
NH2
H2, Pd/C
KOH, H2O
R C CO2H
R C CO2H
H
H
K
N
H
O
N
R C CO2H
H
H2, Pd/C
-orNaB(CN)H3
Reductive amination
NH3
O
O
NH2
R C CO2H
R C CO2H
356
Amidomalonate Synthesis
O
O
HN CO2Et
C
RCH2
CO2Et
EtO
Na
RCH2X
HN CO2Et
C
RCH2
CO2Et
H2N H
C
RCH2
CO2H
H3O
- CO2
26.4 Enantioselective Synthesis of Amino Acids
The syntheses in the previous section give racemic products!!
O
Br
Br2, PBr3
RCH
R-CH2-CO2H
Br
Br
R C CO2H
H
Br
Br
Br Br
H
H
R
R
CO2H
R
50:50
mixture of
enantiomers
O
Br
Br Br
H
R
S
CO2H
357
Br
181
Resolution: separation of enantiomers
H
RCH2
NH2
C
racemic
CO2H
H
N
N
H
(-)-sparteine
(chiral base)
H
RCH2
C
H
H
NH2
H
H
CO2
H2N
N
N
+
N
RCH2
N
H
C
CO2
H
H
Diastereomeric salts
(separate)
H3O
H
RCH2
H3O
H3N
NH3
C
RCH2
CO2
H
C
CO2
358
Resolutions are inherently inefficient
Enantioselective Synthesis of Amino Acids (please read)
H
R
H H
H
S
H
H
NHAc
CO2H
CO2H
R
NHAc
H H
H
R
R
H
CO2H
NHAc
H
Chiral hydrogenation catalysts can differentiate the faces of the
C=C double bond leading a products that is highly
enriched in one enantiomer.
CO2Me
HN
Ph
O
O
O
CO2Me
Rh(I) L*, H2
HN
Ph
O
O
O
CO2
H3O+
NH3
HO
OH
L-DOPA
359
182
26.5
Peptides and Proteins
Proteins and peptides are polymers made up of amino acid units
(residues) that are linked together through the formation
of an amide bonds (peptide bonds) from the amino group
of one residue and the carboxylate of a second residue
HO
+
H2N
CO2H
H2N
CO2H
HO
N-terminus
H
N
By convention, peptide sequences
are written left to right from the
N-terminus to the C-terminus
C-terminus
CO2H
O
Ser - Ala
(S - A)
O
H
N
R2
N
H
R1
C-terminus
OH
Ala - Ser
(A - S)
- H2O
H2N
CO2H
O
N-terminus
Serine
Alanine
H
N
- H2O
H2N
O
H
N
O
R4
N
H
R3
O
H
N
O
R5
R6
N
H
O
H
N
O
backbone
N
H
R7
360
26.6 Covalent Bonding in Peptides
I. The amide bond
O
H
N
N
H
R1
_
R2
H
N
H
N
O
O
+
N
H
R1
R2
C=N double bond character
due to this resonance structure
H
N
O
restricts rotations
resistant to hydrolysis
amide bond
II.
Disulfide bonds: the thiol groups of cysteine can be
oxidized to form disulfides (Cys-S-S-Cys)
1/2 O2
NH2
2
R6
N
H
R1
N
H
O
O
HS
O
H
N
R2
H
N
N
H
O
R8
O
H
N
O
R9
R10
N
H
R9
H
N
N
H
O
O
R5
N
H
H
N
O
O
H
N
R11
N
H
O
O
H
N
O
R12
R13
N
H
H
N
O
S
1/2 O2
R4
CO2H
S
NH2
N
H
SH
H
N
O
S
HO2C
SH
HO2C
NH2
H2O
R1
N
H
O
S
O
H
N
R2
N
H
O
H
N
O
R4
R5
N
H
H
N
O
361
183
Epidermal Growth Factor (EGF):
53 amino acid, 3 disulfide linkages
Cys33-Cys42
Cys6-Cys20
Cys14-Cys31
1986 Nobel Prize in Medicine:
Stanley Cohen
Rita Levi-Montalcini
362
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
-orprotein
[H]
reduce any
disulfide
bonds
liquid
chromatography
Different amino
acids have different
chromatographic
mobilities (retention
times)
Enzymatic
digestion
-orH3O + , Δ
NH3
R
derivatize w/
ninhydrin
CO2
individual
amino acids
Detected w/
UV-vis
1972 Nobel Prize in Chemistry
William Stein
363
Stanford Moore
184
Reaction of primary amines with ninhydrin
O
O
NH3
R
O
- CO2
-H2O
+
O
N
O
N
R
O H
CO2
O
O
O
R
O
O
H2O
NH2
- RCHO
Intense
purple
color
O
O
O
N
O
O
O
O
Amino Acid
Analysis
Chromatogram
364
26.8
Ph
S
C
N
Ph N
Peptide Sequencing: The Edman Degradation
chemical method for the sequential cleavage and
identification of the amino acids of a peptide, one at a time
starting from the N-terminus.
Reagent: Ph-N=C=S, phenylisothiocyanate (PITC)
R1
+
H
N
H2N
S
pH 9.0
Ph
CO2
O
N
H
R1
N
H
H
N
H+
CO2
O
H+
H+
S
HN
H
N
CO2
Ph
S
N
O
HN
OH
R1
R1
H+
N-phenylthiohydantoin:
separated by liquid chromatography
(based of the R group) and detected
by UV-vis
Ph
N
S
+
H2N
CO2
-1 peptide with a new
N-terminal amino acid
(repeat degradation cycle)
O
HN
R1
365
185
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
O
R1
N
H
H3N
H
N
O
R3
N
H
O
H
N
CO2
O
O
trypsin
R1
N
H
H3N
H2O
H
N
R3
O
O
+
H
N
H3N
NH3
NH3
CO2
O
O
366
• chymotrypsin: cleaves at the C-terminal side of aromatic residues
Phe, Tyr, Trp
R1
O
N
H
H3N
H
N
O
O
R3
N
H
H
N
O
O
CO2
chymotrypsin
H2O
H3N
R1
N
H
H
N
O
O
R3
O
+
H
N
H3N
CO2
O
H2N–Val–Phe–Leu–Met–Tyr–Pro–Gly–Trp–Cys–Glu–Asp–Ile–Lys–Ser–Arg–His-CO2 H
trypsin
chymotrypsin
H2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-CO2H
H2N-His-CO2H
H2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-CO2 H
H2N-Ser-Arg-CO2 H
H2N-Val-Phe-CO2H
H2N-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2H
H2N-Val-Phe-Leu-Met-Tyr-CO2H
H2N-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2H
H2N-Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-CO2H
H2N-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His-CO2H
367
186
If given the sequence of the enzyme digest fragements, allign
the sequences of the fragments to get the sequence of
the full peptide
Trypsin:
Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg
Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys
Ser-Arg
His
Chymotrypsin:
Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His
Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His
Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp
Cys-Glu-Asp-Ile-Lys-Ser-Arg-His
Val-Phe-Leu-Met-Tyr
Val-Phe
Val-Phe-Leu-Met-Tyr-Pro-Gly-Trp-Cys-Glu-Asp-Ile-Lys-Ser-Arg-His
368
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)
Cys33-Cys42
Cys6-Cys20
26.9
Cys14-Cys31
Peptide Sequencing: C-Terminal Residue
Determination (please read)
369
187
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
(genome)
mRNA
Protein
(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?
370
Mass spectrometry is a gas phase technique. Peptides (and
proteins) are charged, polar, high molecular weight
molecules (ions). How can peptides and proteins be
coaxed into the gas phase?
Electrospray ionization (ESI): analyte is introduced into the mass
spectrometer as an aerosol.
liquid
chromatography
or capillary
electrophoresis
(separate the
analytes)
+
+++
+++
++
++
++
++
+ ++
++++++
++
++++++
++++
+++
+
++
+
+
+
+++
++++++
++++++
++
++++++++++
++
++
++
++
+
+
++
+++++ +
+
+
+
+
+
+
+
+
to the mass
analyzer
+
Coulombic
Coulombic
fission
fission
-
+
371
188
MALDI ionization (matrix-assisted laser desorption): analyte is
co-crystallized with an organic molecule that has an
intense UV absorption. A laser that is tuned to the
absorption of the matrix, is “pulsed” at the MALDI
matrix and energy is indirectly transferred to the
analyte.
2002 Nobel Prize in Chemistry
John Fenn (ESI)
Koichi Tanaka (MALDI)
to the mass
analyzer
Laser
pulse
+
+
+
+
+
+
+
+
372
Peptide sequencing by tandem mass spectrometry
Nanospray
Capillary
Electrospray
Ion Source
Q1
Q3
fragment the
peptide
Analyze the
peptide fragments
1116.67
1247.70
Select peptide
to be analyzed
Peptides fragment in
a predictable manner
1500
2000
2500
O
H
H
N CH C N
R2
y1
3000 m/z
b3
b2
b1
O
H2N CH C
R1
2719.48
2550.52
2476.21
2005.07
charge to
N-terminus
1665.89
1811.85
1849.12
1424.85 1375.76
1574.20 1505.77
Select m/z 1228.7 for Q2
1287.73
1000
to the
detector
Collision Cell (Q2)
y2
O
O
H
CH C N CH C OH
R3
R4
y3
charge to
C-terminus
373
189
Amino Acids Sorted by Mass
O
H
H
N CH C N
R2
y1
Glycine
Alanine
Serine
Proline
Valine
Threonine
Cysteine
Isoleucine
Leucine
Asparagine
Aspartic Acid
Glutamine
Lysine
Glutamic Acid
Methionine
Histidine
Phenylalanine
Arginine
Tyrosine
Tryptophan
G
A
S
P
V
T
C
I
L
N
D
Q
K
E
M
H
F
R
Y
W
b3
b2
b1
O
H2N CH C
R1
O
O
H
CH C N CH C OH
R3
R4
y2
average
75.07
89.10
105.09
115.13
117.15
119.12
121.16
131.18
131.18
132.12
133.11
146.15
146.19
147.13
149.21
155.16
165.19
174.20
181.19
204.23
y3
exact
75.03
89.05
105.04
115.05
117.08
119.06
121.02
131.09
131.09
132.05
133.04
146.07
146.11
147.13
149.05
155.02
165.19
174.11
181.07
204.09
- HN-CHR-CO
57
71
87
97
99
101
103
113
113
114
115
128
128
129
131
137
147
156
163
186
146.8
128.0 57.1
Phe
Lys/ Gly
128.2 Gln
71.2
57.2 Lys/
Ala
147.0
Gly Gln
71.1
115.1 Phe
57.1
Ala
70.9
71.0
Asp
Gly 146.9
Ala 57.0 Ala
Phe
147.1
Gly
Phe
57.0
57.1
Gly
Gly
57.0
Gly
115.3
Asp
57.1
Gly
374
101.1 173.1
Thr Arg
113.1 96.9
Leu/Ile Pro
112.2
Leu/Ile
H2N-Ile--Pro--Ile--Gly--Phe--Ala--Gly--Ala--Gln--Gly--Gly--Phe--Asp-Gly--Phe--Ala--Gly--Ala--Gln--Gly--Gly--Phe--Asp--Thr--Arg-CO2H
375
190
Analysis of the proteome: separate proteins of a cell by
two-dimensional electrophoresis
separate
by pI
_
_
_ _
+ +
+
+
separate
by mass
(PAGE)
376
128
107
50
MW
42
16.9
5.1
5.7
pI
5.9
6.3
9.6
377
191
26.10: Peptide Synthesis
H
N
H2N
- H2O
+
- H2O
CO2H
H2N
H2N
CO2H
O
CO2H
O
Val
Ala
Val - Ala
(V - A)
H
N
H2N
CO2H
Ala - Val
(A - V)
The need for protecting groups
Pn
OH
N
H
peptide
coupling
+
Pn
OPc
H2N
Ala - Val
(A - V)
Val
Ala
H
N
H2N
peptide
coupling
(-H2O)
O
OPc
Pn
Ph
O
Pn
Ala - Val
(A - V)
selectively
remove Pn
OPc
O
- H2O
O
O
O
H
N
N
H
N
H
O
H
N
H
N
N
H
O
Repeat
OPc
peptide
synthesis
O
Ph
OH
Phe - Ala - Val
(F - A - V)
O
Phe (F)
Orthogonal protecting group strategy: the carboxylate
protecting group must be stable to the reaction conditions
for the removal of the α-aminoprotecting group
378
(and vice versa)
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 (F3 CCO2H)
Peptide coupling reagent:
dicyclohexylcarbodiimide (DCC)
O
R
O
OH
R
+
C6H11 N C N C6H11
O
R
R'
O
+
C6H11 N C N C6H11
H
(DCC)
C6H11
O
R
N
C6H11
C6H11
"activated acid"
O
O
NH
O C
NH
HN
+ C6H11
Mechanism: Figure 26.5, page 1006
NH
O C
+N
N
R'
H
H
C6H11
R
O C
••
R'-NH2
R
C6H11
O
NH
N
H
Amide
R'
+
C6H11
N
H
N
H
C6H11
DCU
379
192
BOC
N
H
DCC
+
OH
H2N
O
O
O
H
N
BOC
peptide
coupling
N
H
O
H
N
OBn
H
N
N
H
O
O
CF3CO2H
H2N
OBn
O
Ph
N
H
Ph
CF3CO2H
O
H
N
O
H
N
H2N
selectively
remove Nprotecting
group
O
Val
Ala
BOC
OBn
DCC
OBn
Ph
O
BOC
N
H
OH
O
Phe (F)
O
H2, Pd/C
H2N
OBn
O
Ph
N
H
H
N
O
OH
O
Phe - Ala - Val
(F - A - V)
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?
380
26.11 Automated peptide synthesis: The Merrifield SolidPhase Techniques: Peptides and proteins up to ~ 100
residues long are synthesized on a solid, insoluble,
polymer support. Purification is conveniently accomplished
after each step by a simple wash and filtration.
The solid support (Merrifield resin): polystyrene polymer
Ph
styrene
initiator
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H3COCH2Cl
ZnCl2
Ph
+
polymerization
Ph
CH2Cl
Ph
Ph
~ 1 - 10% of the
available phenyl
groups are
functionalized
Ph
Ph
divinylbenzene
(crosslinker, ~1 %)
Ph
Ph
O
H
N
_
O
R
BOC
CF3CO2H
O
O
R
NH
BOC
commerically
available
O
O
NH2
R
381
193
Solid-phase peptide synthesis
BOC
O
H2N
DCC
O
BOC
H
N
O
N
H
O
O
N
H
purify:
wash & filter
O
H2N
O
peptide
coupling
Val
purify:
wash & filter
OH
N
H
remove Nprotecting
group
O
Ph
DCC
BOC
Ph
O
BOC
N
H
CF3CO2H
OH
N
H
H
N
O
N
H
O
O
O
O
Phe (F)
purify:
wash & filter
purify by liquid
chromatograrphy
HF
Ph
H
N
H2N
remove Nor electrophoresis
protecting
group and cleave
from solid-support
O
O
N
H
OH
O
382
Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNA
Solid-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
LYS
GLN
SER
LYS
LYS
LEU
LYS
ASN
ILE
LYS
GLN
GLU
ASP
GLU
HIS
SER
SER
PRO
ALA
ASN
CYS
THR
TYR
ALA
GLY
ALA
THR
MET
SER
ARG
VAL
ASP
VAL
TYR
ASP
PRO
ASN
ASN
SER
ALA
ASP
ASN
ASN
ASN
VAL
ALA
GLN
CYS
ASN
LYS
PRO
VAL
ALA
SER
TYR
LEU
THR
GLN
CYS
SER
ARG
CYS
HIS
TYR
ALA
SER
CYS
THR
PHE
ALA
LYS
TYR
GLU
ALA
ILE
VAL
LYS
THR
ASN
LYS
VAL
VAL
ASN
SER
THR
TYR
ILE
PRO
PHE
SER
GLN
ASP
HIS
CYS
GLY
THR
GLY
LYS
VAL
VAL
GLU
ALA
MET
ARG
GLU
SER
GLN
MET
SER
THR
ALA
HIS
ARG
ALA
MET
CYS
SER
GLN
THR
SER
SER
THR
CYS
PHE
His-119 A
His-12 A
His-12 B
His-119 B
pdb code: 1AFL
R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry:
“for his development of methodology for chemical synthesis on a solid matrix.”
383
194
26.12 Protein Classification (please read)
26.13 Protein Structure
primary (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 are
largely held together by H-bonds and other non-covalent
interactions.
tertiary (3°) structure: The overall three-dimensional structure
(conformation) of a singe polypeptide chain.
quaternary (4°) structure: overall organization of non-covalently
linked subunits of a functional protein
384
Common secondary (2°) sub-structural motifs; α-helix: collagen
3.6 amino acids per coil, 5.4 Å
3.6 AA,
5.4 Å
385
195
Common secondary (2°) sub-structural motifs; β-helix:
386
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
387
196
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
O
H3N
R
N
H
O
H
N
O
R
R
N
H
O
H
N
O
R
O
protease
N
H
CO2
H3N
H2O
R
N
H
O
H
N
O
R
O
R
O
H
N
+ H N
3
O
R
N
H
CO2
chymotrypsin: cleaves at the C-terminal side of aromatic residues Phe, Tyr, Trp
trypsin: cleaves at the C-terminal side of basic residues Arg, Lys but not His
388
Many enzymes catalyze reactions by using the function groups
on the amino acid sidechains.
Catalytic triad of α-chymotrypsin
His-57
Asp-102
Ser-195
pdb code: 5CHA
389
197
Some reactions require additional organic molecules or metal
ions. These are referred to as cofactors (coenzymes)
S
N
O
N
+
-
O
P
O
O
O
O
OP
O-
H
Pyridoxal Phosphate
(Vitamin B6)
O
OH
O
O-
OH
N
N
P
O
O
N
HO
N
OH
HO
O
-
O
P
O
H
HO
N
H
O
O
H
H
OH
O
Cyanocolbalmin
(Vitamin B12)
OH
H2N
N
O
O
Flavin Adenine Diphosphate
(Vitamin B2)
N
(III)
Fe N
N
NH
N
O
N
NH
N
O-
O
N
N H2
O
N
O
O P
N
H2 N
NH2
N
N
Co
H
O
Thiamin diphosphate
(Vitamin B1)
NH 2
N
C
N
O
N
NH2
N H2
O
H 2N
OH
2-O PO
3
N
HO
N H2
O
O
Heme
N
O
HN
N
O
HN
HN
H
N
Folic acid
CO2H
O
NH
S
CH4CO2H
Biotin
CO2H
390
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 Synthase
By bringing reactants together and holding them in the optimal
orientation for the reaction to occur
26.16 Protein Denaturation
The unfolding of the three-dimensional structure of a
protein to a “random coil” state. This results in the loss
of its secondary, tertiary and quaternary structures and
any biological activity. Proteisn can be denatured by
heat, pH or chemicals (urea)
391
198