Transcript aminoacids

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
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 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

Glycine
H
H
O
C
C
N+
H
H
H

Alanine
OH
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
H
H
C
H
C
C
H
N+
H
H
H
O
C
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
C
N+
H
H
O-
H
O
C
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
H
H
C
N+
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
C
pKa = 8.4
H
H
O
C
C
N+
H
H
H+
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
