Amino Acids and Peptides Andy Howard Introductory Biochemistry, Fall 2008 IIT

08/28/08 Biochemistry: Amino Acidsp. 2 of 89 Here’s the rest of the thermodynamics lecture… We didn’t quite finish that, so here’s the part that we didn’t get to. I’ll also offer some help in getting the homework done. Then we’ll move on to today’s topic, which is amino acids and peptides.

08/28/08 Biochemistry: Amino Acidsp. 3 of 89 Free energy as a source of work Change in free energy indicates that the reaction could be used to perform useful work If  G o < 0, we can do work If  G o > 0, we need to do work to make the reaction occur

08/28/08 Biochemistry: Amino Acidsp. 4 of 89 What kind of work? Movement (flagella, muscles) Chemical work: –Transport molecules against concentration gradients –Transport ions against potential gradients To drive otherwise endergonic reactions –by direct coupling of reactions –by depletion of products

08/28/08 Biochemistry: Amino Acidsp. 5 of 89 Coupled reactions Often a single enzyme catalyzes two reactions, shoving them together: reaction 1: A  B  G o 1 0 Coupled reaction: A + C  B + D:  G o C =  G o 1 +  G o 2 If  G o C < 0, then reaction 1 is driving reaction 2!

08/28/08 Biochemistry: Amino Acidsp. 6 of 89 How else can we win? Concentration of product may play a role As we’ll discuss in a moment, the actual free energy depends on  G o and on concentration of products and reactants So if the first reaction withdraws product of reaction B away, that drives the equilibrium of reaction 2 to the right

08/28/08 Biochemistry: Amino Acidsp. 7 of 89 Quantitation Concentration affects  G  G is the actual determiner of spontaneity:  G < 0 means the reaction will proceed from left to right  G > 0 means the reaction will proceed from right to left

08/28/08 Biochemistry: Amino Acidsp. 8 of 89 How does  G relate to  G o ’? We’ll look at this in more detail around mid-semester; But here’s the equation:  G =  G o ’ + RT lnK eq  G =  G o ’ + RT ln[products]/[reactants] For a simple reaction A  B:  G =  G o ’ + RT ln[B]/[A] For A + C  B + D:  G =  G o ’ + RT ln([B][D])/([A][C])

08/28/08 Biochemistry: Amino Acidsp. 9 of 89 What does that mean? It means that if the concentration of products is high and the concentration of reactants is low, equilibrium will be shifted leftward; If the concentration of products is low and the concentration of reactants is high, equilibrium will be shifted rightward

08/28/08 Biochemistry: Amino Acidsp. 10 of 89 Adenosine Triphosphate ATP readily available in cells Derived from catabolic reactions Contains two high-energy phosphate bonds that can be hydrolyzed to release energy: O O - || | (AMP)-O~P-O~P-O - | || O - O

08/28/08 Biochemistry: Amino Acidsp. 11 of 89 Hydrolysis of ATP Hydrolysis at the rightmost high-energy bond: ATP + H 2 O  ADP + P i  G o = -33kJ/mol Hydrolysis of middle bond: ATP + H 2 O  AMP + PP i  G o = -33kJ/mol BUT PP i  2 P i,  G o = -33 kJ/mol So, appropriately coupled, we get twice as much!

08/28/08 Biochemistry: Amino Acidsp. 12 of 89 ATP as energy currency Any time we wish to drive a reaction that has  G o < +30 kJ/mol, we can couple it to ATP hydrolysis and come out ahead If the reaction we want has  G o < +60 kJ/mol, we can couple it to ATP  AMP and come out ahead So ATP is a convenient source of energy — an energy currency for the cell

08/28/08 Biochemistry: Amino Acidsp. 13 of 89 Coin analogy Think of store of ATP as a roll of quarters Vendors don’t give change Use one quarter for some reactions, two for others Inefficient for buying $0.35 items

08/28/08 Biochemistry: Amino Acidsp. 14 of 89 Other high-energy compounds Creatine phosphate: ~ $0.40 Phosphoenolpyruvate: ~ $0.35 So for some reactions, they’re more efficient than ATP

08/28/08 Biochemistry: Amino Acidsp. 15 of 89 Dependence on Concentration Actual  G of a reaction is related to the concentrations / activities of products and reactants:  G =  G o + RT ln [products]/[reactants] If all products and reactants are at 1M, then the second term drops away; that’s why we describe  G o as the standard free energy

08/28/08 Biochemistry: Amino Acidsp. 16 of 89 Is that realistic? No, but it doesn’t matter; as long as we can define the concentrations, we can correct for them Often we can rig it so [products]/[reactants] = 1 even if all the concentrations are small Typically [ATP]/[ADP] > 1 so ATP coupling helps even more than 33 kJ/mol!

08/28/08 Biochemistry: Amino Acidsp. 17 of 89 How does this matter? Often coupled reactions involve withdrawal of a product from availability If that happens, [product]/[reactant] shrinks, the second term becomes negative, and  G 0

08/28/08 Biochemistry: Amino Acidsp. 18 of 89 How to solve energy problems involving coupled equations General principles: –If two equations are added, their energetics add –An item that appears on the left and right side of the combined equation can be cancelled This is how you solve the homework problem!

08/28/08 Biochemistry: Amino Acidsp. 19 of 89 A bit more detail Suppose we couple two equations: A + B  C + D,  G o ’ = x C + F  B + G,  G o ’ = y The result is: A + B + C + F  B + C + D + G or A + F  D + G,  G o ’ = x + y … since B and C appear on both sides

08/28/08 Biochemistry: Amino Acidsp. 20 of 89 What do we mean by hydrolysis? It simply means a reaction with water Typically involves cleaving a bond: U + H 2 O  V + W is described as hydrolysis of U to yield V and W

08/28/08 Biochemistry: Amino Acidsp. 21 of 89 Phew. We’re done with thermodynamics… for now! We’ll come back to this after from time to time in the semester We’ll cover kinetics in some detail when we discuss enzyme dynamics and mechanisms

08/28/08 Biochemistry: Amino Acidsp. 22 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 23 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 24 of 89 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)

08/28/08 Biochemistry: Amino Acidsp. 25 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 26 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 27 of 89 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 Two more questions later in the lecture

08/28/08 Biochemistry: Amino Acidsp. 28 of 89 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.

08/28/08 Biochemistry: Amino Acidsp. 29 of 89 So what’s the equilibrium constant for this reaction? Note that the equation is H 2 O  H + + OH - Therefore k eq = [H + ][OH - ] / [H 2 O] But we just said that [H + ] = [OH - ] = M We also know that [H 2 O] = 55.5M (= (1000 g / L )/(18 g/mole)) So k eq = (10 -7 M) 2 /55.5M = 1.8 * M

08/28/08 Biochemistry: Amino Acidsp. 30 of 89 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])

08/28/08 Biochemistry: Amino Acidsp. 31 of 89 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])

08/28/08 Biochemistry: Amino Acidsp. 32 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 33 of 89 Reading recommendations If the material on ionization of weak acids isn’t pure review for you, I strongly encourage you to read the relevant sections of chapter 2 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!

08/28/08 Biochemistry: Amino Acidsp. 34 of 89 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 -

08/28/08 Biochemistry: Amino Acidsp. 35 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 36 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 37 of 89 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)

08/28/08 Biochemistry: Amino Acidsp. 38 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 39 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 40 of 89 The simplest amino acids Glycine Alanine methyl

08/28/08 Biochemistry: Amino Acidsp. 41 of 89 Branched-chain aliphatic aas Valine Isoleucine Leucine isopropyl

08/28/08 Biochemistry: Amino Acidsp. 42 of 89 Hydroxylated, polar amino acids Serine Threonine hydroxyl

08/28/08 Biochemistry: Amino Acidsp. 43 of 89 Amino acids with carboxylate side chains Aspartate Glutamate carboxylate methylene

08/28/08 Biochemistry: Amino Acidsp. 44 of 89 Amino Acids with amide side chains asparagine glutamine Note: these are uncharged! amide

08/28/08 Biochemistry: Amino Acidsp. 45 of 89 Sulfur-containing amino acids Cysteine Methionine sulfhydryl

08/28/08 Biochemistry: Amino Acidsp. 46 of 89 Positively charged side chains Lysine Arginine Guani- dinium

08/28/08 Biochemistry: Amino Acidsp. 47 of 89 Aromatic Amino Acids Phenylalanine Tyrosine phenyl

08/28/08 Biochemistry: Amino Acidsp. 48 of 89 Histidine: a special case Histidine imidazole

08/28/08 Biochemistry: Amino Acidsp. 49 of 89 Tryptophan: the biggest of all Tryptophan indole

08/28/08 Biochemistry: Amino Acidsp. 50 of 89 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 

08/28/08 Biochemistry: Amino Acidsp. 51 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 52 of 89 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:

08/28/08 Biochemistry: Amino Acidsp. 53 of 89 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”

08/28/08 Biochemistry: Amino Acidsp. 54 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 55 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 56 of 89 Letters M-S methionine (CH 2 ) 2 -S-Me metM asparagine CH 2 -CONH 2 asnN pyrrol- lysine see above pylO proline (CH 2 ) 3 CH (cyc) proP glutamine (CH 2 ) 2 CONH 2 glnQ arginine (CH 2 ) 3 - guanidinium argR serineCH 2 OH serS2.29.2

08/28/08 Biochemistry: Amino Acidsp. 57 of 89 Letters T-Z threonine CH(Me)OH thrT seleno- cysteine CH 2 SeH SecU valineCH(Me) 2 valV tryptophanCH 2 -indole trpW unknown XaaX tyrosineCH 2 -Phe-OH tyrY glu/gln (CH 2 ) 2 -COX glxZ

08/28/08 Biochemistry: Amino Acidsp. 58 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 59 of 89 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 I won’t enumerate right now.

08/28/08 Biochemistry: Amino Acidsp. 60 of 89 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.

08/28/08 Biochemistry: Amino Acidsp. 61 of 89 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)

08/28/08 Biochemistry: Amino Acidsp. 62 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 63 of 89 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, i.e. the pK a of the carboxylate group doesn’t depend as much on R as the pK a of the amine group

08/28/08 Biochemistry: Amino Acidsp. 64 of 89 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%

08/28/08 Biochemistry: Amino Acidsp. 65 of 89 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 -

08/28/08 Biochemistry: Amino Acidsp. 66 of 89 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!

08/28/08 Biochemistry: Amino Acidsp. 67 of 89 Acid-base reactivity on side chains 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

08/28/08 Biochemistry: Amino Acidsp. 68 of 89 Acid-base reactivity in histidine It’s easy to protonate and deprotonate the imidazole group

08/28/08 Biochemistry: Amino Acidsp. 69 of 89 Cysteine: a special case The sulfur is surprisingly ionizable Within proteins it often remains unionized even at higher pH

08/28/08 Biochemistry: Amino Acidsp. 70 of 89 Ionizing hydroxyls X–O–H  X–O - + 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:

08/28/08 Biochemistry: Amino Acidsp. 71 of 89 Resonance-stabilized ion

08/28/08 Biochemistry: Amino Acidsp. 72 of 89 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)

08/28/08 Biochemistry: Amino Acidsp. 73 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 74 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 75 of 89 Growth of oligo- or polypeptide

08/28/08 Biochemistry: Amino Acidsp. 76 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 77 of 89 Double-bond character of peptide

08/28/08 Biochemistry: Amino Acidsp. 78 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 79 of 89 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 , 

08/28/08 Biochemistry: Amino Acidsp. 80 of 89 Ramachandran angles G.N. Ramachandran

08/28/08 Biochemistry: Amino Acidsp. 81 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 82 of 89 How far from 180º can  vary? Remember what we said about the partial double bond character of the C-N main-chain bond That imposes planarity In practice it rarely varies by more than a few degrees from 180º.

08/28/08 Biochemistry: Amino Acidsp. 83 of 89 Ramachandran plot Cf. figures in G&G If you submit a structure to the PDB with Ramachandran angles far from the yellow regions, be prepared to justify them!

08/28/08 Biochemistry: Amino Acidsp. 84 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 85 of 89 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.

08/28/08 Biochemistry: Amino Acidsp. 86 of 89 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

08/28/08 Biochemistry: Amino Acidsp. 87 of 89 What’s the net charge in ELVIS at pH 7? (a) 0 (b) +1 (c) -1 (d) +2 (e) -2

08/28/08 Biochemistry: Amino Acidsp. 88 of 89 Disulfides In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains

08/28/08 Biochemistry: Amino Acidsp. 89 of 89 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