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Amino Acids and Peptides II

Amino Acids and Peptides II. Andy Howard Introductory Biochemistry Fall 2009, IIT. Acids, bases, amino acids. We’ll look at the acid-base properties of amino acid main chains and side chains

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Amino Acids and Peptides II

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  1. Amino Acids and Peptides II Andy Howard Introductory BiochemistryFall 2009, IIT

  2. Acids, bases, amino acids • We’ll look at the acid-base properties of amino acid main chains and side chains • We’ll examine other properties of amino acids, and begin to see how oligomers and polymers of amino acids work Biochemistry: Amino Acids

  3. Chirality Abbreviations Acid/base chemistry Side-chain reactivity Peptides and proteins Side-chain reactivity in context Disulfides Plans Biochemistry: Amino Acids

  4. 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

  5. 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 Biochemistry: Amino Acids

  6. Do D-amino acids ever occur in organisms? • Yes: • 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

  7. 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

  8. 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

  9. 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

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

  11. Letters G-L Biochemistry: Amino Acids

  12. Letters M-S Biochemistry: Amino Acids

  13. Letters T-Z Biochemistry: Amino Acids

  14. 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

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

  16. 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

  17. An iClicker 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

  18. … 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

  19. 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

  20. 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

  21. 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

  22. 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: 10% / 90% Biochemistry: Amino Acids

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

  24. 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

  25. Acid-base reactivity on 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% charged • Lys, arg: side-chain nitrogen: • Lys sidechain –NH3+ pKa = 10.5 • Arg sidechain =NH2+ pKa = 12.5 Biochemistry: Amino Acids

  26. Acid-base reactivity in histidine • It’s easy to protonate and deprotonate the imidazole group Biochemistry: Amino Acids

  27. Cysteine: a special case • The sulfur is surprisingly ionizable • Within proteins it often remains unionized even at higher pH Biochemistry: Amino Acids

  28. Ionizing 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: Biochemistry: Amino Acids

  29. Resonance-stabilized ion Biochemistry: Amino Acids

  30. 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) Biochemistry: Amino Acids

  31. 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 Biochemistry: Amino Acids

  32. Peptides and proteins • Peptides are oligomers of amino acids • Proteins are polymers • Dividing line is a little vague:~ 50-80 aa. • All are created, both formally and in practice, by stepwise polymerization • Water eliminated at each step Biochemistry: Amino Acids

  33. Growth of oligo- or polypeptide Biochemistry: Amino Acids

  34. 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 Biochemistry: Amino Acids

  35. Double-bond character of peptide Biochemistry: Amino Acids

  36. 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 Biochemistry: Amino Acids

  37. Rotations and flexibility • Planarity implies  = 180º, 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 ,  Biochemistry: Amino Acids

  38. Ramachandran angles G.N. Ramachandran Biochemistry: Amino Acids

  39. 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 Biochemistry: Amino Acids

  40. How far from 180º can w 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. Biochemistry: Amino Acids

  41. Ramachandran 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! Biochemistry: Amino Acids

  42. 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 + Pi + (n+1)-length peptide Biochemistry: Amino Acids

  43. 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 occur • Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step. Biochemistry: Amino Acids

  44. Reactivity 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 Biochemistry: Amino Acids

  45. Another iClicker question What’s the net charge on ELVIS at pH 7? • (a) 0 • (b) +1 • (c) -1 • (d) +2 • (e) -2 Biochemistry: Amino Acids

  46. Disulfides In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains Biochemistry: Amino Acids

  47. 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 Biochemistry: Amino Acids

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