Amino Acids and Peptides

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

2. 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. Biochemistry: Amino Acids

3. Free energy as a source of work • Change in free energy indicates that the reaction could be used to perform useful work • If Go < 0, we can do work • If Go > 0, we need to do work to make the reaction occur Biochemistry: Amino Acids

4. 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 Biochemistry: Amino Acids

5. Coupled reactions • Often a single enzyme catalyzes two reactions, shoving them together:reaction 1: A  B Go1 < 0 reaction 2: C D Go2 > 0 • Coupled reaction:A + C  B + D: GoC = Go1 + Go2 • If GoC < 0,then reaction 1 is driving reaction 2! Biochemistry: Amino Acids

6. 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 Go 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 Biochemistry: Amino Acids

7. Quantitation • Concentration affects DG • DG is the actual determiner of spontaneity: • DG < 0 means the reaction will proceed from left to right • DG > 0 means the reaction will proceed from right to left Biochemistry: Amino Acids

8. How does DG relate to DGo’? • We’ll look at this in more detail around mid-semester; • But here’s the equation:DG = DGo’ + RT lnKeqDG = DGo’ + RT ln[products]/[reactants] • For a simple reaction A  B:DG = DGo’ + RT ln[B]/[A] • For A + C  B + D:DG = DGo’ + RT ln([B][D])/([A][C]) Biochemistry: Amino Acids

9. 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 Biochemistry: Amino Acids

10. 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 Biochemistry: Amino Acids

11. Hydrolysis of ATP • Hydrolysis at the rightmost high-energy bond:ATP + H2O  ADP + PiGo = -33kJ/mol • Hydrolysis of middle bond:ATP + H2O  AMP + PPiGo = -33kJ/mol • BUT PPi  2 Pi, Go = -33 kJ/mol • So, appropriately coupled,we get twice as much! Biochemistry: Amino Acids

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

13. Coin analogy • Think of store of ATPas a roll of quarters • Vendors don’t give change • Use one quarter for some reactions, two for others • Inefficient for buying $0.35 items Biochemistry: Amino Acids

14. Other high-energy compounds • Creatine phosphate: ~ $0.40 • Phosphoenolpyruvate: ~ $0.35 • So for some reactions, they’re more efficient than ATP Biochemistry: Amino Acids

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

16. 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] = 1even if all the concentrations are small • Typically [ATP]/[ADP] > 1 so ATP coupling helps even more than 33 kJ/mol! Biochemistry: Amino Acids

17. 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 even if Go > 0 Biochemistry: Amino Acids

18. 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! Biochemistry: Amino Acids

19. A bit more detail • Suppose we couple two equations:A + B  C + D, DGo’ = xC + F  B + G, DGo’ = y • The result is:A + B + C + F  B + C + D + GorA + F  D + G, DGo’ = x + y • … since B and C appear on both sides Biochemistry: Amino Acids

20. What do we mean by hydrolysis? • It simply means a reaction with water • Typically involves cleaving a bond: • U + H2O  V + Wis described as hydrolysis of Uto yield V and W Biochemistry: Amino Acids

21. 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 Biochemistry: Amino Acids

22. 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 Biochemistry: Amino Acids

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

24. iClicker quiz! • 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

25. iClicker quiz, 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

26. iClicker quiz, 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

27. 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 Biochemistry: Amino Acids

28. 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-] = 10-7:pH = pOH = 7. Biochemistry: Amino Acids

29. 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

30. 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

31. 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

32. 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

33. 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! Biochemistry: Amino Acids

34. 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

35. 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

36. 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

37. 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) Biochemistry: Amino Acids

38. 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 Biochemistry: Amino Acids

39. 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

40. The simplest amino acids • Glycine • Alanine methyl Biochemistry: Amino Acids

41. Valine Isoleucine Leucine Branched-chain aliphatic aas isopropyl Biochemistry: Amino Acids

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

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

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

45. Cysteine Methionine Sulfur-containing amino acids sulfhydryl Biochemistry: Amino Acids

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

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

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

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

50. 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