1. Andy Howard Introductory Biochemistry Fall 2013, IIT
Amino Acids
Andy Howard
Introductory Biochemistry Fall 2013, IIT

2. Amino acids and what they do
We’ll show you all 20 ribosomally encoded amino acids and discuss their reactivities
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Amino Acids and Proteins

3. Amino Acids and Proteins
Plans
Amino acid properties
Amino Acid Structures
Chirality
Acid-base behavior
Abbreviations
Side-chain reactivity
Peptide bonds
Peptides
Proteins
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Amino Acids and Proteins

4. 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, even though their resting concentrations are low
We’ll build from small to large
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Thermo II; Amino Acids I

5. 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-] = M2 (+/-) fact
So pH + pOH = 14 derived formula
Neutral pH: [H+] = [OH-] = 10-7M: pH = pOH = 7.
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Thermo II; Amino Acids I

6. So what’s the equilibrium constant for this reaction?
Note that the equation is H2O  H+ + OH-
Therefore keq = [H+][OH-] / [H2O]
But we just said that [H+] = [OH-] = 10-7M
We also know that [H2O] = 55.5M (= (1000 g / L )/(18 g/mole))
So keq = (10-7M)2/55.5M = 1.8 * 10-16M
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Thermo II; Amino Acids I

7. Henderson-Hasselbalch Equation
If ionizable solutes are present, their ionization will depend on pH
Assume a weak acid HA  H+ + A- such that the ionization equilibrium constant is Ka = [A-][H+] / [HA]
Define pKa  -log10Ka
Then pH = pKa + log10([A-]/[HA])
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Thermo II; Amino Acids I

8. The Derivation is Trivial!
Ho hum: Ka = [A-][H+]/[HA]
pKa = -log10([A-][H+]/[HA]) = -log10([A-]/[HA]) - log10([H+]) = -log10([A-]/[HA]) + pH
Therefore pH = pKa + log10([A-]/[HA])
Often written pH = pKa + log([base]/[acid])
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Thermo II; Amino Acids I

9. Thermo II; Amino Acids I
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
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Thermo II; Amino Acids I

10. Reading recommendations
If the material on ionization of weak acids isn’t pure review for you, I strongly encourage you to the relevant section of Garrett & Grisham
We won’t go over this material in detail in class because it should be review, but you do need to know it!
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Thermo II; Amino Acids I

11. pKa values for weak acids
Acid Base pKa Acid Base pKa Form Form Form Form
Formic acid Formate 3.8 Acetic acid Acetate 4.8
Lactic acid Lactate 3.9 H3PO4 H2PO
H2PO4- HPO HPO4-2 PO
H2CO3 HCO NH4+ NH3 9.2
CH3NH3+ CH3NH Alanine+ Alanine0 2.4
Alanine0 Alanine- 9.9
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Thermo II; Amino Acids I

12. 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-
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Thermo II; Amino Acids I

13. Thermo II; Amino Acids I
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
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Thermo II; Amino Acids I

14. Thermo II; Amino Acids I
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
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Thermo II; Amino Acids I

15. 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)
Note that we sometimes care about -amino acids that aren’t ribosomal—like ornithine
ornithine
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Thermo II; Amino Acids I

16. Let’s learn the ribosomal 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
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Amino Acids and Proteins

17. Amino Acids and Proteins
But first: a reminder
We often characterize a carbon atom by specifying how many hydrogens are attached to it
–CH3 is methyl
–CH2– is methylene
–CH– is methine |
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Amino Acids and Proteins

18. Amino Acids and Proteins
Quick iClicker quiz…
1. The standard form of the free-energy equation is
(a) H = G - TS
(b) G = H - TS
(c) PV = nRT
(d) none of the above.
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Amino Acids and Proteins

19. iClicker quiz question 2
2. A reaction in which reactants are spontaneously being converted to products is
(a) unlikely
(b) endergonic
(c) exergonic
(d) endothermal
(e) exothermal
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Amino Acids and Proteins

20. Amino Acids and Proteins
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
Tends to abolish -helicity because of that hindered rotation
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Amino Acids and Proteins

21. The simplest amino acids
Glycine
Alanine
These are moderately nonpolar
methyl
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Amino Acids and Proteins

22. Branched-chain aliphatic aas
Valine
Isoleucine
Seriously nonpolar
isopropyl
Leucine
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Amino Acids and Proteins

23. Hydroxylated, polar amino acids
Serine
Threonine
hydroxyl
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Amino Acids and Proteins

24. Amino acids with carboxylate side chains
Aspartate
Glutamate
carboxylate
methylene
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Amino Acids and Proteins

25. Amino Acids with amide side chains
asparagine
glutamine
amide
Note: these are uncharged! Don’t fall into the trap!
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Amino Acids and Proteins

26. Sulfur-containing amino acids
Cysteine
Methionine
sulfhydryl
Two differences: (1) extra methylene (2) methylated S
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Amino Acids and Proteins

27. Positively charged side chains
Lysine
Guani- dinium
Arginine
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Amino Acids and Proteins

28. Amino Acids and Proteins
Aromatic Amino Acids
Phenylalanine
Tyrosine
phenyl
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Amino Acids and Proteins

29. Histidine: a special case
imidazole
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Amino Acids and Proteins

30. Tryptophan: the biggest of all
indole
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Amino Acids and Proteins

31. Amino Acids and Proteins
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
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Amino Acids and Proteins

32. 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
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
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Amino Acids and Proteins

33. 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:
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Amino Acids and Proteins

34. 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 pyrrolysine
B,J,Z are used for ambiguous cases: B is asp/asn, J is ile/leu, Z is glu/gln
X for “totally unknown”
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Amino Acids and Proteins

35. Amino Acids and Proteins
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
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Amino Acids and Proteins

36. Letters A-F: acid-base properties
Amino Acid
Side-chain
3-lett abbr.
1-let
pKa, COO-
pKa, NH3+
alanine
CH3
ala A
2.4
9.9 *
asx B
cysteine
CH2SH
cys C
1.9
10.7
aspartate
CH2COO-
asp D
2.0
glutamate
(CH2)2COO-
glu E
2.1
9.5
phenyl-alanine
CH2-phe
phe F
2.2
9.3
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Amino Acids and Proteins

37. Amino Acids and Proteins
Letters G-L
Amino Acid
Side-chain
3-lett abbr.
1-let
pKa, COO-
pKa, NH3+
glycine H
gly G
2.4
9.8
histidine
-CH2-imidazole
his
1.8
9.3
isoleucine
CH(Me)Et
ile I
2.3
Ile/leu *
lex? J
lysine
(CH2)4NH3+
lys K
2.2
9.1
leucine
CH2CHMe2
leu L
9.7
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Amino Acids and Proteins

38. Amino Acids and Proteins
Letters M-S
pyrrolysine
methionine
(CH2)2-S-Me
met M
2.1
9.3
asparagine
CH2-CONH2
asn N
8.7
pyrrol- lysine
see above
pyl O
2.2
9.1
proline
(CH2)3CH (cyc)
pro P
2.0
10.6
glutamine
(CH2)2CONH2
gln Q
arginine
(CH2)3-guanidinium
arg R
1.8
9.0
serine
CH2OH
ser S
9.2
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Amino Acids and Proteins

39. Amino Acids and Proteins
Letters T-Z
threonine
CH(Me)OH
thr T
2.1
9.1
seleno- cysteine
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
unknown
Xaa X
tyrosine
CH2-Phe-OH
tyr Y
2.2
9.2
glu/gln
(CH2)2-COX
glx Z
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Amino Acids and Proteins

40. 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
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Amino Acids and Proteins

41. 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, glucose, glyceraldehyde
Cholesterol, stearate, palmitate
A few others I won’t enumerate right now.
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Amino Acids and Proteins

42. 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.
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Amino Acids and Proteins

43. Amino Acids and Proteins
iClicker question #3
3. 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)
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Amino Acids and Proteins

44. Amino Acids and Proteins
… and another
4. 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
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Amino Acids and Proteins

45. 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
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Amino Acids and Proteins

46. 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
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Amino Acids and Proteins

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!
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Amino Acids and Proteins

48. How do we relate pKa to percentage ionization?
Derivable from Henderson-Hasselbalch equation
If pH = pKa, half-ionized
One unit below:
91% at more positive charge state,
9% at less + charge state
One unit above: 9% / 91%
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Amino Acids and Proteins

49. Don’t fall into the trap!
Ionization of leucine: pH
1.3
2.3
3.3
8.7
9.7
10.7
%+ve 91 50 9
% neutral
%-ve
Main species
NH3+-CHR-COOH
NH3+CHR-COO-
NH3+ CHR-COO-
NH2-CHR-COO-
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Amino Acids and Proteins

50. 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!
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Amino Acids and Proteins