Amino Acids and Peptides Andy Howard Introductory Biochemistry Fall 2010, IIT

Amino Acids and PeptidesAmino Acids and Peptides

Andy Howard

Introductory BiochemistryFall 2010, IIT

08/31/2010 Biochemistry: Amino Acids

p. 2 of 74

Acids, bases, amino acidsAcids, bases, amino acids

We begin looking at specific categories of small molecules by examining acid-base equilibrium, both in general and in amino acids

These simple molecules are inherently important, and they help illustrate some general principles


p. 3 of 74

PlansPlans

Review Acid-base

equilibrium Amino acid

structures Chirality Acid/base chemistry

Side-chain reactivity Peptides and

proteins Side-chain reactivity

in context Disulfides


p. 4 of 74

Review questionsReview questions

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)


p. 5 of 74

Review questions, problem 2Review questions, 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


p. 6 of 74

Review questions, problem 3Review questions, 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


p. 7 of 74

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-] = 10-7M:

pH = pOH = 7.


p. 8 of 74

So what’s the equilibrium So what’s the equilibrium constant for this reaction?constant for this reaction?

Note that the equation isH2O H+ + OH-

Therefore keq = [H+][OH-] / [H2O]But we just said that

[H+] = [OH-] = 10-7MWe also know that [H2O] = 55.5M

(= (1000 g / L )/(18 g/mole))So keq = (10-7M)2/55.5M = 1.8 * 10-16M


p. 9 of 74

Alternative approachAlternative approach

Assume the protonated species is H3O+ rather than H+

Then the reaction is2 H2O H3O+ + OH-

keq = [H3O+][OH-] / ([H2O]2)At pH=7, [H3O+] = [OH-] = 10-7MDilute solution: [H2O] = 55.5M, so

keq = 10-14 M2/ [(55.5)2 M2] = 3.24*10-18


p. 10 of 74

Henderson-Hasselbalch Henderson-Hasselbalch EquationEquation

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])


p. 11 of 74

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])


p. 12 of 74

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


p. 13 of 74

Reading recommendationsReading recommendations

If the material on ionization of weak acids isn’t pure review for you, I strongly encourage you to read the relevant sections in 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!


p. 14 of 74

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-


p. 15 of 74

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


p. 16 of 74

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


p. 17 of 74

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)

Note that we sometimes care about-amino acids that aren’t ribosomal—like ornithine ornithine


p. 18 of 74

Let’s learn the Let’s learn the ribosomal amino acids.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 sequentiallyYou do need to memorize these, both

actively and passively


p. 19 of 74

But first: a reminderBut 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

|


p. 20 of 74

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

Tends to abolish helicity because of that hindered rotation


p. 21 of 74

The simplest amino acidsThe simplest amino acids

Glycine

AlanineCN+HHHHCCOO-HHH

CN+HHHHCOO-H

methyl

These are moderately nonpolar


p. 22 of 74

CN+HHHHCCOO-CCHHHHCHHHHH

CN+HHHHCCOO-CHCHHHHCHHHH

Branched-chain aliphatic aasBranched-chain aliphatic aas

Valine

Isoleucine

Leucine

CN+HHHHCCOO-CCHHHHHHH

isopropylSeriously nonpolar


p. 23 of 74

Hydroxylated, polar amino acidsHydroxylated, polar amino acids

Serine Threonine

CN+HHHHCCOO-OHHH

CN+HHHHCCOO-OCHHHHH

hydroxyl


p. 24 of 74

Amino acids with carboxylate Amino acids with carboxylate side chainsside chains

Aspartate Glutamate

CN+HHHHCCOO-CHHO-O

CN+HHHHCCOO-HHCHHCO-O

carboxylate

methylene


p. 25 of 74

Amino Acids with amide side Amino Acids with amide side chainschains

asparagine glutamineCN+HHHHCCOO-HHCHHCNOHH

CN+HHHHCCOO-HHCNOHH

Note: these are uncharged! Don’t fall into the trap!

amide


p. 26 of 74

Sulfur-containing amino acidsSulfur-containing amino acids

Cysteine Methionine

CN+HHHHCCOO-HHSH

CN+HHHHCCOO-HHSCCHHHHH

sulfhydryl

Two differences:(1) extra methylene(2) methylated S


p. 27 of 74

Positively charged side chainsPositively charged side chains

Lysine Arginine

CN+HHHHCCOO-HHCCCHHHHHHN+HHH CN+HHHHCCOO-HHCNCHHHHHCNN+HHHH

Guani-dinium


p. 28 of 74

Aromatic Amino AcidsAromatic Amino Acids

Phenylalanine TyrosineCN+HHHHCCOO-HHCCCCCCHHHHH

CN+HHHHCCOO-HHCCCCCCHHHHOH

phenyl


p. 29 of 74

Histidine: a special caseHistidine: a special case

Histidine

imidazole


p. 30 of 74

Tryptophan: the biggest of allTryptophan: the biggest of all

TryptophanCN+HHHHCCOO-HHCCCNCCHHCCHHHH

indole


p. 31 of 74

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


p. 32 of 74

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


p. 33 of 74

The CORN mnemonicThe CORN mnemonicfor L-amino acidsfor L-amino acids

Imagine you’re looking from the alpha hydrogen to the alpha carbon

The substituents are, clockwise:C=O, R, N:


p. 34 of 74

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”

http://www.chem.qmul.ac.uk/iupac/AminoAcid/A2021.html

CN+HHHHCOO-CHHSeH


p. 35 of 74

Acid-base propertiesAcid-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


p. 36 of 74

Letters A-F: acid-base propertiesLetters A-F: acid-base properties

AminoAcid

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


p. 37 of 74

Letters G-LLetters G-L

AminoAcid

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


p. 38 of 74

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)3CH (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


p. 39 of 74

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


p. 40 of 74

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 Q: almost follows N, and gln is like asn You’re on your own for K,O,J,B,Z,U,X


p. 41 of 74

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, glucose, glyceraldehyde

– Cholesterol, stearate, palmitate

– A few others I won’t enumerate right now.


p. 42 of 74

How hard is it to How hard is it to memorize the memorize the structuresstructures??

Very easy: G, A, S, C, VRelatively easy: F, Y, D, E, N, QHarder: I, K, L, M, P, THardest: H, R, WAgain, I’m not asking you to memorize

the one-letter codes, but they do make life a lot easier.


p. 43 of 74

Another review questionAnother review 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)


p. 44 of 74

… … and anotherand 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


p. 45 of 74

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


p. 46 of 74

Why does pWhy does pKKaa depend on the depend on the

side chain?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


p. 47 of 74

When do these pWhen do these pKKaa values values

apply?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!


p. 48 of 74

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: 9% / 91%


p. 49 of 74

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-


p. 50 of 74

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!


p. 51 of 74

Acid-base reactivity Acid-base reactivity on side chainson 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% chargedLys, arg: side-chain nitrogen:

– Lys sidechain –NH3+ pKa = 10.5

– Arg sidechain =NH2+ pKa = 12.5


p. 52 of 74

Acid-base reactivity in histidineAcid-base reactivity in histidine

It’s easy to protonate and deprotonate the imidazole group


p. 53 of 74

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


p. 54 of 74

Ionizing hydroxylsIonizing 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:


p. 55 of 74

Resonance-stabilized ionResonance-stabilized ion


p. 56 of 74

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)


p. 57 of 74

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

enzymes Under careful control of enzymes called

kinases and phosphatases This is an instance of post-translational

modification


p. 58 of 74

Amino acid frequencies and Amino acid frequencies and importance in active sitesimportance in active sites

Polar amino acids, particularly S, H, D, E, K, are at the heart of most active sites of enzymes and other globular proteins

Yet they’re relatively uncommon overall in proteins

Nonpolar amino acids (V, L, I, A) occur with higher frequencies overall


p. 59 of 74

Peptides and proteinsPeptides and proteins

Peptides are oligomers of amino acidsProteins 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


p. 60 of 74

Growth of oligo- or polypeptideGrowth of oligo- or polypeptide

CN+HHHHCOO-R1CN+HHHCOO-+H2OCN+HHHHCOR1CNCOO-HR2HR2H


p. 61 of 74

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


p. 62 of 74

Double-bond character of Double-bond character of peptidepeptide

CN+HHHHCOR1NCHR2HCOCN+HHHHCO-R1N+CHR2HCO


p. 63 of 74

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


p. 64 of 74

Rotations and flexibilityRotations and flexibility

Planarity implies = 180º, where is the torsion angle about N-C bond

Free rotations are possible about N-C and C-C bonds– Define = torsional rotation about N-C– Define = torsional rotation about C-C

We can characterize main-chain conformations according to ,


p. 65 of 74

Ramachandran anglesRamachandran angles

G.N. Ramachandran


p. 66 of 74

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


p. 67 of 74

How far from 180º can How far from 180º can vary? vary?

Remember what we said about the partial double bond character of the C-N main-chain bond

That imposes planarityIn practice it rarely varies by more than

a few degrees from 180º.


p. 68 of 74

Ramachandran plotRamachandran 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!


p. 69 of 74

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


p. 70 of 74

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.


p. 71 of 74

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


p. 72 of 74

Another iClicker questionAnother iClicker question

What’s the net charge on ELVIS at pH 7?

(a) 0(b) +1(c) -1(d) +2(e) -2


p. 73 of 74

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


p. 74 of 74

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

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