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Amino Acids and Peptides

Amino Acids and Peptides. Andy Howard Introductory Biochemistry Fall 2010, IIT. Acids, bases, amino acids. We begin looking at specific categories of small molecules by examining acid-base equilibrium, both in general and in amino acids

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Amino Acids and Peptides

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  1. Amino Acids and Peptides Andy Howard Introductory BiochemistryFall 2010, IIT

  2. Acids, 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 Biochemistry: Amino Acids

  3. Review Acid-base equilibrium Amino acid structures Chirality Acid/base chemistry Side-chainreactivity Peptides and proteins Side-chain reactivity in context Disulfides Plans Biochemistry: Amino Acids

  4. Review questions • 1. The correct form of the free energy equation is generally given as: • (a) DH = DG - TDS • (b) PV = nRT • (c) DG = DH - TDS • (d) DS = DH - DG • (e) none of the above • (20 seconds for this one) Biochemistry: Amino Acids

  5. Review questions, problem 2 • 2. Suppose a reaction is at equilibrium with DH = -6 kJ mol-1 andDS = -0.02 kJ mol-1K-1.Calculate the temperature. • (a) 250K • (b) 280K • (c) 300K • (d) 310K • (e) 340K • 45 seconds for this one Biochemistry: Amino Acids

  6. Review questions, problem 3 • 3. Suppose the reaction AB is endergonic with DGo = 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 Biochemistry: Amino Acids

  7. Acid-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. Biochemistry: Amino Acids

  8. So what’s the equilibrium constant for this reaction? • Note that the equation isH2O  H+ + OH- • Therefore keq = [H+][OH-] / [H2O] • But we just said that [H+] = [OH-] = 10-7M • We also know that [H2O] = 55.5M(= (1000 g / L )/(18 g/mole)) • So keq = (10-7M)2/55.5M = 1.8 * 10-16M Biochemistry: Amino Acids

  9. Alternative 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-] = 10-7M • Dilute solution: [H2O] = 55.5M, sokeq = 10-14 M2/ [(55.5)2 M2] = 3.24*10-18 Biochemistry: Amino Acids

  10. 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 Ka = [A-][H+] / [HA] • Define pKa -log10Ka • Then pH = pKa + log10([A-]/[HA]) Biochemistry: Amino Acids

  11. 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]) Biochemistry: Amino Acids

  12. 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 - pKathen 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 Biochemistry: Amino Acids

  13. 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 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! Biochemistry: Amino Acids

  14. So: 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- Biochemistry: Amino Acids

  15. 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 Biochemistry: Amino Acids

  16. 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 Biochemistry: Amino Acids

  17. 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=-CH2-indole) • Note that we sometimes care about-amino acids that aren’t ribosomal—like ornithine ornithine Biochemistry: Amino Acids

  18. Let’s learn the 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 sequentially • You do need to memorize these, both actively and passively Biochemistry: Amino Acids

  19. But 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 | Biochemistry: Amino Acids

  20. 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 Biochemistry: Amino Acids

  21. The simplest amino acids • Glycine • Alanine These are moderately nonpolar methyl Biochemistry: Amino Acids

  22. Valine Isoleucine Leucine Branched-chain aliphatic aas Seriously nonpolar isopropyl Biochemistry: Amino Acids

  23. Serine Threonine Hydroxylated, polar amino acids hydroxyl Biochemistry: Amino Acids

  24. Aspartate Glutamate Amino acids with carboxylate side chains carboxylate methylene Biochemistry: Amino Acids

  25. asparagine glutamine Amino Acids with amide side chains amide Note: these are uncharged! Don’t fall into the trap! Biochemistry: Amino Acids

  26. Cysteine Methionine Sulfur-containing amino acids sulfhydryl Two differences:(1) extra methylene(2) methylated S Biochemistry: Amino Acids

  27. Lysine Arginine Positively charged side chains Guani-dinium Biochemistry: Amino Acids

  28. Phenylalanine Tyrosine Aromatic Amino Acids phenyl Biochemistry: Amino Acids

  29. Histidine: a special case • Histidine imidazole Biochemistry: Amino Acids

  30. Tryptophan: the biggest of all • Tryptophan indole Biochemistry: Amino Acids

  31. 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 Biochemistry: Amino Acids

  32. Ribosomally encoded amino acids are L-amino acids • 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 Biochemistry: Amino Acids

  33. The CORN mnemonicfor L-amino acids • Imagine you’re looking from the alpha hydrogen to the alpha carbon • The substituents are, clockwise:C=O, R, N: Biochemistry: Amino Acids

  34. 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” • http://www.chem.qmul.ac.uk/iupac/AminoAcid/A2021.html Biochemistry: Amino Acids

  35. Acid-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 Biochemistry: Amino Acids

  36. Letters A-F: acid-base properties Biochemistry: Amino Acids

  37. Letters G-L Biochemistry: Amino Acids

  38. Letters M-S Biochemistry: Amino Acids

  39. Letters T-Z Biochemistry: Amino Acids

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

  41. 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, glucose, glyceraldehyde • Cholesterol, stearate, palmitate • A few others I won’t enumerate right now. Biochemistry: Amino Acids

  42. 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. Biochemistry: Amino Acids

  43. Another 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) Biochemistry: Amino Acids

  44. … and 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 Biochemistry: Amino Acids

  45. Main-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 Biochemistry: Amino Acids

  46. Why does pKa 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 pKa of the carboxylate group doesn’t depend as much on R as the pKa of the amine group Biochemistry: Amino Acids

  47. When do these pKa values 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! Biochemistry: Amino Acids

  48. How do we relate pKa to percentage ionization? • Derivable from Henderson-Hasselbalch equation • If pH = pKa, half-ionized • One unit below: • 90% at more positive charge state, • 10% at less + charge state • One unit above: 9% / 91% Biochemistry: Amino Acids

  49. Don’t fall into the trap! • Ionization of leucine: Biochemistry: Amino Acids

  50. 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! Biochemistry: Amino Acids

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