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)  H =  G - T  S –(b) PV = nRT –(c)  G =  H - T  S –(d)  S =  H -  G –(e) none of the above (20 seconds for this one)

iClicker quiz, problem 2 2. Suppose a reaction is at equilibrium with  H = -6 kJ mol -1 and  S = kJ mol -1 K -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  G o = 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 + PP i –(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 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 Equilibrium In aqueous solution, the concentration of hydronium and hydroxide ions is nonzero Define: –pH  -log 10 [H + ] –pOH  -log 10 [OH - ] Product [H + ][OH - ] = M 2 (+/-) So pH + pOH = 14 Neutral pH: [H + ] = [OH - ] = : pH = pOH = 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 K a = [A - ][H + ] / [HA] Define pK a  -log 10 K a Then pH = pK a + log 10 ([A - ]/[HA])

The Derivation is Trivial! Ho hum: pK a = -log([A-][H+]/[HA]) = -log([A-]/[HA]) - log([H+]) = -log([A-]/[HA]) + pH Therefore pH = pK a + log([A-]/[HA]) Often written pH = pK a + 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 - pK a 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 H 3 N + -CHR-COO - ; these are  -amino acids. But there are others, e.g. beta-alanine: H 3 N + -CH 2 -CH 2 -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=-CH 2 -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 Alanine

Branched-chain aliphatic aas Valine Isoleucine Leucine

Hydroxylated, polar amino acids Serine Threonine

Amino acids with carboxylate side chains Aspartate Glutamate

Amino Acids with amide side chains asparagine glutamine Note: these are uncharged!

Sulfur-containing amino acids Cysteine Methionine

Positively charged side chains Lysine Arginine

Aromatic Amino Acids Phenylalanine Tyrosine

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 

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 acids are L-amino acids

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 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”

Letters A-F: acid-base properties Amino Acid Side- chain 3-lett abbr. 1- let pK a, COO - pK a, NH 3 + alanine CH 3 alaA *asxB cysteineCH 2 SH cysC aspartateCH 2 COO - aspD glutamate (CH 2 ) 2 COO - gluE phenyl- alanine CH 2 -phepheF2.29.3

Letters G-L Amino Acid Side- chain 3-lett abbr. 1- let pK a, COO - pK a, NH 3 + glycine HglyG histidine -CH 2 - imidazole hisH isoleucineCH(Me)Et ileI Ile/leu * lex?J lysine (CH 2 ) 4 NH 3 + lysK l eucine CH 2 CHMe 2 leuL2.39.7

Letters M-S methionine (CH 2 ) 2 -S-Me metM asparagine CH 2 -CONH 2 asnN pyrrol- lysine see above pylO proline (CH 2 ) 4 (cyc) proP glutamine (CH 2 ) 2 CONH 2 glnQ arginine (CH 2 ) 3 - guanidinium argR serineCH 2 OH serS2.29.2

Letters T-Z threonine CH(Me)OH thrT seleno- cysteine CH 2 SeH SecU valineCH(Me) 2 valV tryptophan CH 2 -indole trpW unknown XaaX tyrosine CH 2 -Phe-OH tyrY Glu/gln (CH 2 ) 2 -COX glxZ

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: H 3 N + -CHR-COO - + OH -  H 2 N-CHR-COO - + H 2 O Protonating the carboxylate: H 3 N + -CHR-COO - + H +  H 3 N + -CHR-COOH Equilibrium far to the left at neutral pH First equation has K a =1 around pH 9 Second equation has K a =1 around pH 2

Why does pK a 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 pK a to percentage ionization? Derivable from Henderson- Hasselbalch equation If pH = pK a, 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 % neutral %-ve Main species NH 3 +- CHR- COOH NH 3 + CHR- COO - NH 2 - CHR- COO -

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 pK a = 3.9 –Glu sidechain pK a = 4.1 Lys, arg: side-chain nitrogen: –Lys sidechain NH 3 + pK a = 10.5 –Arg sidechain =NH 2 + pK a = 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

Ionizing hydroxyls X-O-H  XO - + H + Tyrosine is easy, ser and thr hard: –Tyr pK a = 10.5 –Ser, Thr pK a = ~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: ~ aa. All are created, both formally and in practice, by stepwise polymerization Water eliminated at each step

Growth of oligo- or polypeptide

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

The result: planarity! This partial double bond character means the nitrogen is sp 2 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 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 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 + P i + (n+1)-length peptide

What happens at the ends? Usually there’s a free amino end and a free carboxyl end: H 3 N + -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 pK a s. Terminal main-chain pK a values 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 neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains

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