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Amino Acids and PeptidesAmino Acids and Peptides
Andy Howard
Biochemistry, Fall 2007IIT
Let’s begin, chemically!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
PlansPlans
iClicker stuff Acid-base
equilibrium Amino acid
structures Chirality Acid/base
chemistry
Side-chain reactivity
Peptides and proteins
Side-chain reactivity in context
Disulfides
Let’s get your iClickers recorded!Let’s get your iClickers recorded!
Follow the instructions here:Your student ID will appear in the
scrolling list below.They’re sorted in student ID
numbers, beginning with 102-06-464 and ending with 104-43-853
If we miss you here, email me your iClicker number today
iClicker quiz!iClicker quiz!
1. The correct form of the free energy equation is generally given as:– (a) H = G - TS– (b) PV = nRT– (c) G = H - TS– (d) S = H - G– (e) none of the above
(20 seconds for this one)
iClicker quiz, problem 2iClicker quiz, problem 2
2. Suppose a reaction is at equilibrium with H = -6 kJ mol-1 andS = -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 3iClicker quiz, problem 3
3. Suppose the reaction AB is endergonic with Go = 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 That’s the end of this part of your iClicker quiz!of your iClicker quiz!
Note that the scores don’t make much difference to your final grade, but being present does matter somewhat
Scores will be posted on Blackboard soon
Two more questions later in the lecture
Acid-Base EquilibriumAcid-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 EquationHenderson-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])
The Derivation is Trivial!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 writtenpH = pKa + log([base]/[acid])
How do we use this?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 recommendationsReading 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 acidsSo: 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 zwitterionsThese 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 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 groupsIdentities 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.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: prolineSpecial 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 acidsThe simplest amino acids
Glycine
AlanineCN+HHHHCCOO-HHH
CN+HHHHCOO-H
CN+HHHHCCOO-CCHHHHCHHHHH
CN+HHHHCCOO-CHCHHHHCHHHH
Branched-chain aliphatic aasBranched-chain aliphatic aas
Valine
Isoleucine
Leucine
CN+HHHHCCOO-CCHHHHHHH
Hydroxylated, polar amino acidsHydroxylated, polar amino acids
Serine Threonine
CN+HHHHCCOO-OHHH
CN+HHHHCCOO-OCHHHHH
Amino acids with carboxylate Amino acids with carboxylate side chainsside chains Aspartate Glutamate
CN+HHHHCCOO-CHHO-O
CN+HHHHCCOO-HHCHHCO-O
Amino Acids with amide side Amino Acids with amide side chainschains asparagine glutamine
CN+HHHHCCOO-HHCHHCNOHH
CN+HHHHCCOO-HHCNOHH
Note: these are uncharged!
Sulfur-containing amino acidsSulfur-containing amino acids
Cysteine Methionine
CN+HHHHCCOO-HHSH
CN+HHHHCCOO-HHSCCHHHHH
Positively charged side chainsPositively charged side chains
Lysine ArginineCN+HHHHCCOO-HHCCCHHHHHHN+HHH CN+HHHHCCOO-HHCNCHHHHHCNN+HHHH
Aromatic Amino AcidsAromatic Amino Acids
Phenylalanine TyrosineCN+HHHHCCOO-HHCCCCCCHHHHH
CN+HHHHCCOO-HHCCCCCCHHHHOH
Histidine: a special caseHistidine: a special case
Histidine
Tryptophan: the biggest of allTryptophan: the biggest of all
TryptophanCN+HHHHCCOO-HHCCCNCCHHCCHHHH
ChiralityChirality
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
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
Ribosomally encoded amino Ribosomally encoded amino acids are L-amino acidsacids are L-amino acids
The CORN mnemonicThe CORN mnemonicfor L-amino acidsfor L-amino acidsImagine you’re
looking from the alpha hydrogen to the alpha carbon
The substituents are, clockwise:C=O, R, N:
Abbreviations for the amino Abbreviations for the amino acidsacids 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– B,J,Z are used for ambiguous cases:
B is asp/asn, J is ile/leu, Z is glu/gln– X for “totally unknown”
CN+HHHHCOO-CHHSeH
Letters A-F: acid-base propertiesLetters A-F: acid-base propertiesAminoAcid
Side-chain
3-lettabbr.
1-let
pKa,COO-
pKa, NH3
+
alanine CH3 ala A 2.4 9.9
* asx Bcysteine CH2SH cys C 1.9 10.7aspartate CH2COO- asp D 2.0 9.9glutamate (CH2)2COO- glu E 2.1 9.5phenyl-alanine
CH2-phe phe F 2.2 9.3
Letters G-LLetters G-LAminoAcid
Side-chain
3-lettabbr.
1-let
pKa,COO-
pKa, NH3
+
glycine H gly G 2.4 9.8histidine -CH2-
imidazolehis H 1.8 9.3
isoleucine CH(Me)Et ile I 2.3 9.8
Ile/leu * lex? J 2.3 9.7-9.8lysine (CH2)4NH3
+ lys K 2.2 9.1leucine CH2CHMe2 leu L 2.3 9.7
Letters M-SLetters M-S
methionine (CH2)2-S-Me met M 2.1 9.3
asparagine CH2-CONH2 asn N 2.1 8.7
pyrrol-lysine
see above pyl O 2.2 9.1
proline (CH2)4 (cyc) pro P 2.0 10.6glutamine (CH2)2CONH2 gln Q 2.2 9.1
arginine (CH2)3-guanidinium
arg R 1.8 9.0
serine CH2OH ser S 2.2 9.2
Letters T-ZLetters 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 9.7tryptophan CH2-indole trp W 2.5 9.4unknown Xaa X
tyrosine CH2-Phe-OH tyr Y 2.2 9.2
Glu/gln (CH2)2-COX glx Z
Remembering the abbreviationsRemembering 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 Do you need to memorize these structures?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?How hard is it to memorize them?
Very easy: G, A, S, C, VRelatively easy: F, Y, D, E, N, QHarder: I, K, L, M, P, THardest: H, R, W
What amino acids are in ELVIS?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 chemistryMain-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 pWhy does pKKaa depend on the side depend on the side
chain?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 pHow do we relate pKKaa to to
percentage ionization?percentage ionization?Derivable from Henderson-
Hasselbalch equationIf 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!Don’t fall into the trap!
Ionization of leucine:
pH 1.3 2.3 3.3 8.7 9.7 10.7
%+ve 90 50 10 0 0 0
% neutral 10 50 90 90 50 10
%-ve 0 0 0 10 50 90
Main species
NH3+-
CHR-COOH
NH3+C
HR-COO-
NH3+
CHR-COO-
NH2-
CHR-COO-
Side-chain reactivitySide-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 reactivityAcid-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 histidineAcid-base reactivity in histidine
It’s easy to protonate and deprotonate the imidazole group
Cysteine: a special caseCysteine: a special case
The sulfur is surprisingly ionizableWithin proteins it often remains
unionized even at higher pHCN+HHHHCOO-CHHSHCN+HHHHCOO-CHHS-H+H+pKa = 8.4
Ionizing hydroxylsIonizing 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 ionResonance-stabilized ion
Other side-chain reactionsOther 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, estersSalt bridges (e.g. lys - asp)
PhosphorylationPhosphorylation
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 enzymesUnder careful control of enzymes
called kinases and phosphatases
Peptides and proteinsPeptides and proteins
Peptides are oligomers of amino acids
Proteins are polymersDividing line is a little vague:
~ 50-80 aa.All are created, both formally and in
practice, by stepwise polymerizationWater eliminated at each step
Growth of oligo- or polypeptideGrowth of oligo- or polypeptideCN+HHHHCOO-R1CN+HHHCOO-+H2OCN+HHHHCOR1CNCOO-HR2HR2H
The peptide bondThe 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 peptideDouble-bond character of peptideCN+HHHHCOR1NCHR2HCOCN+HHHHCO-R1N+CHR2HCO
The result: planarity!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 flexibilityRotations and flexibility
Planarity implies = 180, 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 ,
Ramachandran anglesRamachandran angles
G.N. Ramachandran
Preferred Values of Preferred Values of and 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 plotRamachandran plot
Cf. fig. 4.9 in Horton
How are oligo- and polypeptides How are oligo- and polypeptides synthesized?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?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 occurCyclization doesn’t happen at the
ribosome: it involves a separate, enzymatic step.
Reactivity in peptides & proteinsReactivity 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 ELVISWhat’s the net charge in ELVISat pH 7?at pH 7?(a) 0(b) +1(c) -1(d) +2(e) -2
DisulfidesDisulfides
In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains
CHHSHCHHSH+(1/2)O2SSHCHHCHH2O
What could this do?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