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