Download Biochemical Thermodynamics

Amino Acids and Peptides
Andy Howard
Biochemistry, Fall 2007
IIT
Let’s begin, chemically!
Amino acids are important on their
own and as building blocks
 We need to start somewhere:

– Proteins are made up of amino acids
– Free amino acids and peptides play
significant roles in cells
– We’ll build from small to large
Plans





iClicker stuff
Acid-base
equilibrium
Amino acid
structures
Chirality
Acid/base
chemistry

Side-chain
reactivity
 Peptides and
proteins
 Side-chain
reactivity in
context
 Disulfides
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
iClicker quiz!

1. The correct form of the free energy
equation is generally given as:
– (a) DH = DG - TDS
– (b) PV = nRT
– (c) DG = DH - TDS
– (d) DS = DH - DG
– (e) none of the above

(20 seconds for this one)
iClicker quiz, problem 2

2. Suppose a reaction is at equilibrium
with DH = -6 kJ mol-1 and
DS = -0.02 kJ mol-1K-1.
Calculate the temperature.
–
–
–
–
–

(a) 250K
(b) 280K
(c) 300K
(d) 310K
(e) 340K
45 seconds for this one
iClicker quiz, problem 3

3. Suppose the reaction AB is
endergonic with DGo = 37 kJ/mol. What
would be a suitable exergonic reaction to
couple this reaction to in order to drive it
to the right?
–
–
–
–

(a) hydrolysis of ATP to AMP + PPi
(b) hydrolysis of glucose-1-phosphate
(c) hydrolysis of pyrophosphate
(d) none of the above
30 seconds for this one
That’s the end of this part
of your iClicker quiz!
Note that the scores don’t make
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but being present does matter
somewhat
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soon
 Two more questions later in the
lecture

Acid-Base Equilibrium

In aqueous solution, the concentration of
hydronium and hydroxide ions is nonzero
 Define:
– pH  -log10[H+]
– pOH  -log10[OH-]
Product [H+][OH-] = 10-14 M2 (+/-)
 So pH + pOH = 14
 Neutral pH: [H+] = [OH-] = 10-7:
pH = pOH = 7.

Henderson-Hasselbalch Equation
If ionizable solutes are present, their
ionization will depend on pH
 Assume a weak acid HA  H+ + Asuch that the ionization equilibrium
constant is Ka = [A-][H+] / [HA]
 Define pKa  -log10Ka
 Then pH = pKa + log10([A-]/[HA])

The Derivation is Trivial!
Ho hum:
 pKa = -log([A-][H+]/[HA])
= -log([A-]/[HA]) - log([H+])
= -log([A-]/[HA]) + pH
 Therefore pH = pKa + log([A-]/[HA])
 Often written
pH = pKa + log([base]/[acid])

How do we use this?
Often we’re interested in calculating
[base]/[acid] for a dilute solute
 Clearly if we can calculate
log([base]/[acid]) = pH - pKa
then you can determine
[base]/[acid] = 10(pH - pKa)
 A lot of amino acid properties are
expressed in these terms
 It’s relevant to other biological acids and
bases too, like lactate and oleate

Reading recommendations
If the material on ionization of weak
acids isn’t pure review for you, I
strongly encourage you to read
sections 2.7 to 2.10 in Horton.
 We won’t go over this material in
detail in class because it should be
review, but you do need to know it!

So: let’s look at amino acids
The building blocks of
proteins are of the form
H3N+-CHR-COO-;
these are -amino acids.
 But there are others,
e.g. beta-alanine:
H3N+-CH2-CH2-COO
These are zwitterions

Over a broad range of pH:
– the amino end is protonated and is
therefore positively charged
– the carboxyl end is not protonated and is
therefore negatively charged

Therefore both ends are charged
 Free -amino acids are therefore
highly soluble, even if the side chain
is apolar
At low and high pH:

At low pH, the
carboxyl end is
protonated
At high pH, the amino
end is deprotonated
 These are molecules
with net charges

Identities of the R groups
Nineteen of the twenty ribosomally
encoded amino acids fit this form
 The only variation is in the identity of
the R group (the side chain
extending off the alpha carbon)
 Complexity ranging from glycine
(R=H) to tryptophan (R=-CH2-indole)

Let’s learn the amino acids.
We’ll walk through the list of 20, one
or two at a time
 We’ll begin with proline because it’s
weird
 Then we’ll go through them
sequentially
 You do need to memorize these, both
actively and passively

Special case: proline
Proline isn’t an amino
acid: it’s an imino acid
 Hindered rotation
around bond between
amine N and alpha
carbon is important to
its properties

The simplest amino acids

H
Glycine
H
O
C
C
N+
H
H
H

Alanine
O-
H
H
H
C
H
N+
H
H
O
C
C
H
O-
Branched-chain aliphatic aas
H
H

Valine
H
H
H
C
H

C
C
Leucine
H
H H
H
H
H
C
C
H
H
N+
H
Isoleucine
H
C
C
C
H
H
H

H
O
C
H
OH
H
H
C
H
C
H
H
O
C
N+
H
C
C
H
H
H
H
C
H
O-
N+
H
H
O
C
H
C
H
O-
Hydroxylated, polar amino acids


Serine
Threonine
H
H
H
C
H
H
H
O
C
H
H
N+
H
C
H
O-
H
H
O
C
O
C
N+
H
C
H
H
O
C
H
O-
Amino acids with carboxylate
side chains


Aspartate
O-
Glutamate
C
O
H
H
H
H
C
H
H
C
H
O
C
N+
H
H
C
C
H
O
O
C
N+
H
H
O-
H
O
C
C
H
O-
Amino Acids with amide side
chains


asparagine
H
glutamine
O
N
H
H
C
O
N
H
H
C
H
C
H
H
H
C
H
H
H
O
C
N+
H
O-
H
C
O
C
N+
H
C
Note: these are
uncharged!
H
H
C
H
O-
Sulfur-containing amino acids
H


Cysteine
Methionine
H
C
H
H
S
S
H
H
H
C
H
H
C
H
C
H
N+
H
H
O
C
H
O
C
C
N+
H
H
O-
H
C
H
O-
Positively charged side chains
H
H

Lysine
H
H

N+
H
H
H
Arginine N+
C
H
H
N
C
H
H
H
H
H
C
H
C
H
H
N
H
C
H
C
H
H
H
C
C
H
H
O
C
H
C
N+
H
H
H
C
N+
H
O
C
H
O-
O-
Aromatic Amino Acids
H

Phenylalanine

H
Tyrosine
C
C
H
H
O
H
H
H
C
C
C
C
C
C
C
C
H
H
C
C
H
H
H
C
H
H
H
O
C
N+
H
C
N+
H
H
O
C
H
C
C
H
H
O-
H
O-
Histidine: a special case

Histidine
Tryptophan: the biggest of all

Tryptophan
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

Ribosomally encoded amino
acids are L-amino acids

All have the same handedness at the
alpha carbon
 The opposite handedness gives you a Damino acid
– 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
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:

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
H
Se

6 unused letters, obviously
– U used for selenocysteine
H
– 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
– X for “totally unknown”
H
H
C
O
C
C
N+
H
O-
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-
glu
E
2.1
9.5
phenylalanine
CH2-phe phe
F
2.2
9.3
alanine
pKa,
NH3+
9.9
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
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)4 (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
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
unknown
tyrosine
CH2-Phe-OH
tyr
Y
Glu/gln
(CH2)2-COX
glx
Z
2.2
9.2
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
You’re on your own for K,O,Q,J,B,Z,U,X
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:
–
–
–
–
DNA, RNA bases
Ribose
Cholesterol
A few others that I can’t think of right now.
How hard is it to memorize them?
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

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.

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
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

How do we relate pKa to
percentage ionization?
Derivable from HendersonHasselbalch 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%
Don’t fall into the trap!

Ionization of leucine:
pH
%+ve
1.3
90
2.3 3.3
50 10
8.7
0
9.7
0
10.7
0
%
neutral
%-ve
10
50
90
90
50
10
0
0
0
10
50
90
Main
species
NH3+CHRCOOH
NH3+
CHRCOO-
NH3+
CHRCOO-
NH2CHRCOO-
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

In proteins and peptides,
the side-chain reactivity is more important
because the main chain is locked up!
Acid-base reactivity

Asp, glu: side-chain COO-:
– Asp sidechain pKa = 3.9
– Glu sidechain pKa = 4.1

Lys, arg: side-chain nitrogen:
– Lys sidechain NH3+ pKa = 10.5
– Arg sidechain =NH2+ pKa = 12.5
Acid-base reactivity in histidine

It’s easy to protonate and
deprotonate the imidazole group
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
C
O
C
C
N+
H
H
H
H
H
O-
H
O-
Ionizing hydroxyls
X-O-H  XO- + H+
 Tyrosine is easy, ser and thr hard:

– Tyr pKa = 10.5
– Ser, Thr pKa = ~13

Difference due to resonance
stabilization of phenolate ion:
Resonance-stabilized ion
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)

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

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

Growth of oligo- or polypeptide
R1
H
H
O
C
C
N+
H
+
O-
C
R2
H
O-
R1
O
H
C
H2O
H
C
N+
H
O
C
H
H
N
H
O
C
N+
H
H
H
H
C
R2
O-
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

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
The result: planarity!

This partial double bond character means
the nitrogen is sp2 hybridized
 Six atoms must lie in a single plane:
–
–
–
–
–
–
First amino acid’s alpha carbon
Carbonyl carbon
Carbonyl oxygen
Second amino acid’s amide nitrogen
Amide hydrogen
Second amino acid’s alpha carbon
Rotations and flexibility
Planarity implies  = 180, where  is
the rotation angle about N-C bond
 Free rotations are possible about NC and C-C bonds

– Define  = rotation about N-C
– Define  = rotation about C-C

We can characterize main-chain
conformations according to , 
Ramachandran angles
G.N. Ramachandran
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

Ramachandran plot

Cf. fig. 4.9
in Horton
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

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.

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
What’s the net charge in ELVIS
at pH 7?
(a) 0
 (b) +1
 (c) -1
 (d) +2
 (e) -2

Disulfides
In oxidizing
environments, two
H
neighboring
cysteine residues
can react with an
oxidizing agent to
form a covalent
bond between the
side chains
H
H
S
S
H
C
H
+
(1/2)O 2
H2O
H
H
C
C
S
H
S
H
H
C
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
