Download Document

Amino Acids and Peptides II
Andy Howard
Introductory Biochemistry
Fall 2009, IIT
Acids, bases, amino acids
We’ll look at the acid-base properties
of amino acid main chains and side
chains
 We’ll examine other properties of
amino acids, and begin to see how
oligomers and polymers of amino
acids work

09/03/09
Biochemistry: Amino Acids
p. 2 of 47
Plans

Chirality
 Abbreviations
 Acid/base
chemistry
 Side-chain
reactivity
09/03/09

Peptides and
proteins
 Side-chain
reactivity in
context
 Disulfides
Biochemistry: Amino Acids
p. 3 of 47
Chirality
Remember:
any carbon with four non-identical
substituents will be chiral
 Every amino acid except glycine is
chiral at its alpha carbon
 Two amino acids (ile and thr) have a
second chiral carbon: C

09/03/09
Biochemistry: Amino Acids
p. 4 of 47
Ribosomally encoded amino
acids are L-amino acids
All have the same handedness
at the alpha carbon
 The opposite handedness
gives you a D-amino acid

09/03/09
Biochemistry: Amino Acids
p. 5 of 47
Do D-amino acids ever occur in
organisms?

Yes:
 There are D-amino acids in many organisms
 Bacteria incorporate them into structures of
their cell walls
 Makes those structures resistant to standard
proteolytic enzymes, which only attack amino
acids with L specificity
09/03/09
Biochemistry: Amino Acids
p. 6 of 47
The CORN mnemonic
for L-amino acids
Imagine you’re
looking from the
alpha hydrogen
to the alpha
carbon
 The substituents
are, clockwise:
C=O, R, N:

09/03/09
Biochemistry: Amino Acids
p. 7 of 47
Abbreviations for the amino
acids

3-letter and one-letter codes exist
– All the 3-letter codes are logical
– Most of the 1-letter codes are too

6 unused letters, obviously
– U used for selenocysteine
– O used for pyrrollysine
H
– B,J,Z are used for ambiguous cases: H
B is asp/asn, J is ile/leu, Z is glu/gln
H
– X for “totally unknown”

H
Se
H
H
C
O
C
C
N+
H
O-
http://www.chem.qmul.ac.uk/iupac/AminoAcid/A2021.html
09/03/09
Biochemistry: Amino Acids
p. 8 of 47
Acid-base properties
-amino acids take part in a variety of
chemical reactivities, but the one we’ll
start with is acid-base reactivity
 The main-chain carboxylate and amine
groups can undergo changes in
protonation
 Some side chains can as well

09/03/09
Biochemistry: Amino Acids
p. 9 of 47
Letters A-F: acid-base properties
Amino
Acid
Sidechain
CH3
3-lett
abbr.
ala
1- pKa,
let COOA 2.4
*
asx
B
cysteine
CH2SH
cys
C
1.9
10.7
aspartate
CH2COO- asp
D
2.0
9.9
glutamate
(CH2)2COO-
E
2.1
9.5
phenylalanine
09/03/09
CH2-phe phe
F
2.2
9.3
alanine
glu
Biochemistry: Amino Acids
pKa,
NH3+
9.9
p. 10 of 47
Letters G-L
Amino
Acid
Sidechain
H
3-lett
abbr.
gly
1- pKa,
let COOG 2.4
pKa,
NH3+
9.8
histidine
-CH2imidazole
his
H
1.8
9.3
isoleucine
CH(Me)Et ile
I
2.3
9.8
Ile/leu
*
lex?
J
2.3
9.7-9.8
lysine
(CH2)4NH3+
lys
K
2.2
9.1
leucine
CH2CHMe2
leu
L
2.3
9.7
glycine
09/03/09
Biochemistry: Amino Acids
p. 11 of 47
Letters M-S
methionine
(CH2)2-S-Me
met
M 2.1
9.3
asparagine
CH2-CONH2
asn
N 2.1
8.7
pyrrollysine
proline
see above
pyl
O 2.2
9.1
(CH2)3CH (cyc)
pro
P 2.0
10.6
glutamine
(CH2)2CONH2
gln
Q 2.2
9.1
arginine
(CH2)3guanidinium
arg
R 1.8
9.0
serine
CH2OH
ser
S 2.2
9.2
09/03/09
Biochemistry: Amino Acids
p. 12 of 47
Letters T-Z
threonine
CH(Me)OH
thr
T
2.1
9.1
selenocysteine
CH2SeH
Sec
U
1.9
10.7
valine
CH(Me)2
val
V
2.3
9.7
tryptophan
CH2-indole
trp
W
2.5
9.4
Xaa
X
2.2
9.2
unknown
tyrosine
CH2-Phe-OH
tyr
Y
glu/gln
(CH2)2-COX
glx
Z
09/03/09
Biochemistry: Amino Acids
p. 13 of 47
Remembering the abbreviations









A, C, G, H, I, L, M, P, S, T, V easy
F: phenylalanine sounds like an F
R: talk like a pirate
D,E similar and they’re adjacent
N: contains a nitrogen
W: say tryptophan with a lisp
Y: second letter is a Y
Q: almost follows N, and gln is like asn
You’re on your own for K,O,J,B,Z,U,X
09/03/09
Biochemistry: Amino Acids
p. 14 of 47
Do you need to memorize these
structures?
Yes, for the 20 major ones
(not B, J, O, U, X, Z)
 The only other complex structures I’ll
ask you to memorize are:

–
–
–
–
09/03/09
DNA, RNA bases
Ribose, glucose
Cholesterol, stearate, palmitate
A few others I won’t enumerate right now.
Biochemistry: Amino Acids
p. 15 of 47
How hard is it to
memorize the structures?
Very easy: G, A, S, C, V
 Relatively easy: F, Y, D, E, N, Q
 Harder: I, K, L, M, P, T
 Hardest: H, R, W
 Again, I’m not asking you to memorize
the one-letter codes, but they do make
life a lot easier.

09/03/09
Biochemistry: Amino Acids
p. 16 of 47
An iClicker question
What amino acids are in ELVIS?
 (a) asp - lys - val - ile - ser
 (b) asn - lys - val - ile - ser
 (c) glu - leu - val - ile - ser
 (d) glu - lys - val - ile - ser
 (e) Thank you very much.
(25 seconds)
09/03/09
Biochemistry: Amino Acids
p. 17 of 47
… and another

How many of the twenty plentiful,
ribosomally encoded amino acids have
exactly one chiral center?
– (a) zero
– (b) one
– (c) seventeen
– (d) eighteen
– (e) twenty
09/03/09
Biochemistry: Amino Acids
p. 18 of 47
Main-chain acid-base chemistry





Deprotonating the amine group:
H3N+-CHR-COO- + OH- 
H2N-CHR-COO- + H2O
Protonating the carboxylate:
H3N+-CHR-COO- + H+ 
H3N+-CHR-COOH
Equilibrium far to the left at neutral pH
First equation has Ka=1 around pH 9
Second equation has Ka=1 around pH 2
09/03/09
Biochemistry: Amino Acids
p. 19 of 47
Why does pKa depend on the
side chain?

Opportunities for hydrogen bonding or
other ionic interactions stabilize some
charges more than others
 More variability in the amino terminus,
i.e. the pKa of the carboxylate group
doesn’t depend as much on R as the
pKa of the amine group
09/03/09
Biochemistry: Amino Acids
p. 20 of 47
When do these pKa values
apply?

The values given in the table are for the
free amino acids
 The main-chain pKa values aren’t relevant
for internal amino acids in proteins
 The side-chain pKa values vary a lot
depending on molecular environment:
a 9.4 here doesn’t mean a 9.4 in a protein!
09/03/09
Biochemistry: Amino Acids
p. 21 of 47
How do we relate pKa to
percentage ionization?
Derivable from Henderson-Hasselbalch
equation
 If pH = pKa, half-ionized
 One unit below:

– 90% at more positive charge state,
– 10% at less + charge state

One unit above: 10% / 90%
09/03/09
Biochemistry: Amino Acids
p. 22 of 47
Don’t fall into the trap!

Ionization of leucine:
pH
%+ve
2.3 3.3
50 10
8.7
0
9.7
0
10.7
0
% neutral 10
50
90
90
50
10
%-ve
0
0
0
10
50
90
Main
species
NH3+CHRCOOH
NH3+
CHRCOO-
NH3+
CHRCOO-
09/03/09
1.3
90
Biochemistry: Amino Acids
NH2CHRCOO-
p. 23 of 47
Side-chain reactivity

Not all the chemical reactivity of amino
acids involves the main-chain amino and
carboxyl groups
 Side chains can participate in reactions:
– Acid-base reactions
– Other reactions

09/03/09
In proteins and peptides,
the side-chain reactivity is more important
because the main chain is locked up!
Biochemistry: Amino Acids
p. 24 of 47
Acid-base reactivity
on side chains

Asp, glu: side-chain COO-:
– Asp sidechain pKa = 3.9
– Glu sidechain pKa = 4.1
– That means that at pH = 5.1, a glutamate
will be ~90.9% charged

Lys, arg: side-chain nitrogen:
– Lys sidechain –NH3+ pKa = 10.5
– Arg sidechain =NH2+ pKa = 12.5
09/03/09
Biochemistry: Amino Acids
p. 25 of 47
Acid-base reactivity in histidine

It’s easy to protonate and
deprotonate the imidazole group
09/03/09
Biochemistry: Amino Acids
p. 26 of 47
Cysteine: a special case
The sulfur is surprisingly ionizable
 Within proteins it often remains
unionized even at higher pH

H
H+
S-
S
H
H
pKa = 8.4
H
H
O
C
C
N+
H
H
H+
C
09/03/09
C
O
C
C
N+
H
H
H
H
H
O-
H
Biochemistry: Amino Acids
O-
p. 27 of 47
Ionizing hydroxyls
X–O–H  X–O- + H+
 Tyrosine is easy, ser and thr hard:
– Tyr pKa = 10.5
– Ser, Thr pKa = ~13
 Difference due to resonance
stabilization of phenolate ion:

09/03/09
Biochemistry: Amino Acids
p. 28 of 47
Resonance-stabilized ion
09/03/09
Biochemistry: Amino Acids
p. 29 of 47
Other side-chain reactions
Little activity in hydrophobic amino acids
other than van der Waals
 Sulfurs (especially in cysteines) can be
oxidized to sulfates, sulfites, …
 Nitrogens in his can covalently bond to
various ligands
 Hydroxyls can form ethers, esters
 Salt bridges (e.g. lys - asp)

09/03/09
Biochemistry: Amino Acids
p. 30 of 47
Phosphorylation
ATP donates terminal phosphate to
side-chain hydroxyl of ser, thr, tyr
 ATP + Ser-OH  ADP + Ser-O-(P)
 Often involved in activating or
inactivating enzymes
 Under careful control of enzymes called
kinases and phosphatases

09/03/09
Biochemistry: Amino Acids
p. 31 of 47
Peptides and proteins
Peptides are oligomers of amino acids
 Proteins are polymers
 Dividing line is a little vague:
~ 50-80 aa.
 All are created, both formally and in
practice, by stepwise polymerization
 Water eliminated at each step

09/03/09
Biochemistry: Amino Acids
p. 32 of 47
Growth of oligo- or polypeptide
R1
H
H
O
C
C
N+
H
+
O-
O
C
C
N+
H
H
H
H
R2
H
O-
R1
O
H
C
O
C
H
H
N
H
09/03/09
H
C
N+
H
H2O
C
R2
O-
Biochemistry: Amino Acids
p. 33 of 47
The peptide bond
The amide bond between two
successive amino acids is known
as a peptide bond
 The C-N bond between the first
amino acid’s carbonyl carbon and
the second amino acid’s amine
nitrogen has some double bond
character

09/03/09
Biochemistry: Amino Acids
p. 34 of 47
Double-bond character of
peptide
H
N
C
N+
H
O
H
R1
H
C
C
C
O
R2
H
H
H
R1
H
C
N+
N+
H
O
H
C
C
C
O-
R2
H
H
09/03/09
Biochemistry: Amino Acids
p. 35 of 47
The result: planarity!

This partial double bond character means the
nitrogen is sp2 hybridized
 Six atoms must lie in a single plane:
–
–
–
–
–
–
09/03/09
First amino acid’s alpha carbon
Carbonyl carbon
Carbonyl oxygen
Second amino acid’s amide nitrogen
Amide hydrogen
Second amino acid’s alpha carbon
Biochemistry: Amino Acids
p. 36 of 47
Rotations and flexibility
Planarity implies  = 180º, where  is
the rotation angle about N-C bond
 Free rotations are possible about N-C
and C-C bonds

– Define  = rotation about N-C
– Define  = rotation about C-C

We can characterize main-chain
conformations according to , 
09/03/09
Biochemistry: Amino Acids
p. 37 of 47
Ramachandran angles
G.N. Ramachandran
09/03/09
Biochemistry: Amino Acids
p. 38 of 47
Preferred Values of  and 
Steric hindrance makes some values
unlikely
 Specific values are characteristic of
particular types of secondary
structure
 Most structures with forbidden values
of  and  turn out to be errors

09/03/09
Biochemistry: Amino Acids
p. 39 of 47
How far from 180º can  vary?
Remember what we said about the
partial double bond character of the C-N
main-chain bond
 That imposes planarity
 In practice it rarely varies by more than
a few degrees from 180º.

09/03/09
Biochemistry: Amino Acids
p. 40 of 47
Ramachandran plot

Cf. figures in text
 If you submit a
structure to the
PDB with
Ramachandran
angles far from
the yellow
regions, be
prepared to justify
them!
09/03/09
Biochemistry: Amino Acids
p. 41 of 47
How are oligo- and polypeptides
synthesized?
Formation of the peptide linkages
occurs in the ribosome under careful
enzymatic control
 Polymerization is endergonic and
requires energy in the form of GTP (like
ATP, only with guanosine):
 GTP + n-length-peptide + amino acid 
GDP + Pi + (n+1)-length peptide

09/03/09
Biochemistry: Amino Acids
p. 42 of 47
What happens at the ends?
Usually there’s a free amino end and a
free carboxyl end:
 H3N+-CHR-CO-(peptide)n-NH-COO Cyclic peptides do occur
 Cyclization doesn’t happen at the
ribosome: it involves a separate,
enzymatic step.

09/03/09
Biochemistry: Amino Acids
p. 43 of 47
Reactivity in peptides & proteins

Main-chain acid-base reactivity
unavailable except on the ends
 Side-chain reactivity available but with
slightly modified pKas.
 Terminal main-chain pKavalues modified
too
 Environment of protein side chain is often
hydrophobic, unlike free amino acid side
chain
09/03/09
Biochemistry: Amino Acids
p. 44 of 47
Another iClicker question
What’s the net charge on ELVIS at
pH 7?
 (a) 0
 (b) +1
 (c) -1
 (d) +2
 (e) -2
09/03/09
Biochemistry: Amino Acids
p. 45 of 47
Disulfides
In oxidizing
environments, two
neighboring
cysteine residues
can react with an
oxidizing agent to
form a covalent
bond between the
side chains
09/03/09
H
H
S
S
H
H
C
H
+
H
C
(1/2)O 2
H2O
H
H
C
C
S
H
Biochemistry: Amino Acids
S
H
p. 46 of 47
What could this do?
Can bring portions of a protein
that are distant in amino acid
sequence into close proximity
with one another
 This can influence protein stability

09/03/09
Biochemistry: Amino Acids
p. 47 of 47