BIOCHEMISTRY REVIEW Session I

1. BIOCHEMISTRY REVIEWSession I Bryan Mitton mittonb@ucmail.uc.edu

2. Biochemistry is almost over!

3. Today’s Review • 1) Amino Acids and Proteins • 2) DNA and RNA • 3) Glycolysis, Krebs Cycle, and ETC. • Plus a 5 minute break between each section.

4. Guaranteed Q. Amino Acids • You need to know the basic structure of each AA, but not the pKa’s. • A few AA facts: • Hydrophobicity is a function of the positional entropy of water. (Virtually always on test.) • The only imino amino acid = _______? • Be able to calculate the isoelectric of any amino acid. • Try Histidine: pKa1 = 1.82 pKa2 = 6.0 pKa3 = 9.17

5. Isoelectric point • The “isoelectric point” of an AA or protein is the pH at which there is NO NET CHARGE. A B C D pKa1 pKa2 pKa3 1.82 6.0 9.17

6. Isoelectric point 1.82 6.0 9.17 Uncharged form. So average the pKa values around it. (9.17 + 6)/2 = 7.6

7. Definitions • Primary Structure • Linear order of Amino Acids in a chain. • Secondary Structure • Comprised of beta pleated sheets, beta turns, alpha helices. • Tertiary Structure • How the secondary structures arrange themselves with respect to each other. • Quaternary Structure • Subunit-subunit interactions. • What are the major physical forces that hold each “structure” together?

8. Forces • Primary Structure: Covalent • Secondary Structure: Hydrogen Bonding • Tertiary Structure: Hydrophobic Forces, Hydrogen Bonding, Salt Bridges, Van der Waals Forces, and Disulfide Bonds. • The strongest covalent bonds are disulfide bonds. • The strongest non-covalent bonds are salt bridges. • The force that contributes the most to tertiary structure is HYDROPHOBIC forces. • Hydrophobic residues put in core of protein and dictate stability. • Quaternary Structure: Same as tertiary. • Q: What AA is very likely to be found at beta turns? • A: Proline, as its imino structure allows for a tight turn.

9. Practice Q’s. • In the following peptide bond sketch, which atoms are coplanar? • In an alpha helix, how many AA residues are there per turn? How “long” is one turn (the “pitch”)? • What are prion diseases a result of?

10. Which atoms in a peptide bond are coplanar? The C and N are both sp2 hybridized and so adopt a trigonal planar arrangement.

11. In an alpha helix, how many AA residues are there per turn? Answer: 3.6 Amino acids, for a length of 5.4 Angstroms. The carbonyl of the 1st residue hydrogen bonds with the amino group of the fourth.

12. What causes prion diseases? • Prion diseases result from accumulation of protein misfolding. • The misfolded molecule is dubbed “PrPSc”. • The misfolding of a “PrPc” molecule initiates a cascade of further misfolding… • PrPSc induces other properly folded to misfold. This polymerizes, causing cell damage + disease.

13. Proteins • 3 Proteins you need to know about: • Hemoglobin (myoglobin too) • Collagen • Elastin

14. Hemoglobin (Hb) • Myoglobin = 1 hemoglobin chain (almost). • Myoglobin and each hemoglobin chain contains a “heme” group. • Heme sits in an apolar pocket in the middle of each chain of hemoglobin/myoglobin. • Heme is metabolized to bilirubin, the buildup of which causes of jaundice. • Heme = 1 iron atom plus a porphyrin ring. • Porphyrin ring = 4 pyrrole groups + 4 methyl, 2 vinyl, 2 propionates stuck onto it. • Porphyrin ring coordinates with 4 Fe+2 orbitals via nitrogen atoms.

15. Heme Group Blue = Nitrogen Black = Carbon Red = Iron

16. Oxygen binding • The 5th coordination position of Fe+2 is with a histidine. • HIS 93 = F8. This is the Proximal Histidine. • Oxygen will be at the 6th spot.

17. The DistalHis is near where the oxygen binds. This is E7, or His 64. • The distal is present to DECREASEFe+2 AFFINITY FOR CARBON MONOXIDE. • Q: What happens if Fe+2 turns into Fe+3? • A: It binds to water, becoming “methemoglobin”.

18. Nomenclature • Adult hemoglobin is normally an a2b2 tetramer. This is called “HbA.” • Also, 2% of total blood Hb is a2d2. This is “HbA2”. • Fetally, here is the progression: • z2e2 a2g2a2b2 plus a2d2 • A question about this was on my board exam: • Which one is “fetal hemoglobin”? A: alpha 2 gamma 2.

19. Fetal vs. Adult Hb • Important difference between gamma and beta chains: • BPGbinds in a pocket that forms in the middle of all four chains when Hb is in the “taut” form. • Recall: TAUT = low affinity for oxygen, so usually NO oxygen bound. RELAXED = high affinity for oxygen, so usually oxygen is bound to Hb. More on this later. • When bound, BPG lowers the affinity of Hb for oxygen because it stabilizes the taut form of Hb. • Gamma chain – a Serine is replaced with a Histidine, so BPG doesn’t bind to fetal Hb very well. • Thus, fetal Hb has HIGHER affinity for oxygen, because BPG doesn’t bind to it and Hb remains in a relaxed conformation.

20. BPG Importance • Q: When happens to plasma BPG concentration at high altitude, and why? • A: Its concentration increases in the blood, so that hemoglobin spends more time in the taut conformation and lets go of oxygen more easily. • Remember: It lowers oxygen affinity by stabilizing the taut conformation of Hb.

21. Cooperativity • Cooperativity – Once the first oxygen is bound to Hb, it is easier for the other 3 to bind. P50 = 27 torr

22. Cooperativity • Compare the curves for myoglobin and hemoglobin. • In the absence of BPG, Hb oxygen affinity curve looks like that for myoglobin.

23. Hill coefficient = 2.8 for Hb. Any Hill coefficient >1 implies functional cooperativity among the molecule’s subunits. • What is the Hill coefficient for myoglobin?

24. Hb Oxygenation • When taut form gets oxygenated, Fe is pulled forward in heme group. • This pulls on His 92 (the proximal Histidine, or F8) and this ends up breaking a hydrogen bond between Val 98 and Tyr 145. • Breaking Val 98 – Tyr 145 bond has two effects: • 1) An H-bond between His 146 – Asp 94 is broken. • 2) An H-bond between His 146 and a Lysine on the alpha chain is broken. • As for the His 146 – Asp 94 bond….

25. Bohr Effect • Breaking the His 146 – Lysine bond is how the beta chain tells the alpha chain it has picked up an oxygen. • The His 146 – Asp 94 bond is responsible for the Bohr effect.

26. So, uh, what was the Bohr Effect again? • Hemoglobin’s oxygen affinity is dependent upon the local pH and carbon dioxide level. • It works like this: • If this bond is intact, Hb adopts Taut form. • If this bond is broken, Hb adopts the Relaxed form.

27. Thus, when oxygenated Hb enters a low pH zone, the His 146 gets protonated. It then forms the bond with Asp 94. • When this bond is made, Hb will switch to the Taut form, and let go of oxygen. • So protonation of His 146 is the key.

28. Q: List the 3 major variables/molecules that affect the affinity of Hb for oxygen.

29. #1 factor: pH • #2 factor: pCO2 • #3 factor: BPG

30. Which graph is the one with LOWER oxygen affinity? If we RAISE the following, WHICH WAY will the curve shift? BPG pH pCO2

31. Hb • Molecule can carry carbon dioxide, oxygen, and protons. • 10% of all carbon dioxide in blood bound to 1st amino acid of Hb, Valine. • Other 90% is carried as bicarbonate. The proton formed is part of Bohr effect. • What enzyme catalyzes this reaction?

32. Sickle Cell Anemia • The main problem in sickle cell anemia is a point mutation…. • Glutamate 6 is substituted with what residue? • [This was also on my board exam!] • When Hb is deoxygenated, this residue is exposed and causes polymerization of Hb. • The chains form and deforms the cell, giving it the sickle shape. • Having this mutation offers resistance to Plasmodium falciparum, the bug that causes malaria. • Called HbS. Two bad chains = SS, one bad one good = AS (heterozygote).

33. Remember – Glutamic Acid 6 is mutated to Valine, and causes polymerization of Hb.

34. Thalassemia • b-Thalassemia - Not enough b chains produced by cell, so a chains accumulate. • a-Thalassemia – Not enough a chains produced, and b chains accumulate. • This disease is usually caused by a problem with splicing… the mRNA isn’t spliced correctly, so it gets destroyed.

35. Bone marrow expands in skull to make more RBC, so you get this “crew-cut appearance”. Again… a thalassemia means you don’t make a chains.

36. That’s all for Hb! • On to collagen.

37. Collagen • The Amino Acid Sequence = Gly-X-Y. • Gly = 33%, Pro = 20%, 10% = Ala. 5% = lysine. • Hydroxyproline and hydroxylysine are also part of primary structure. • Prolylhydroxylase and lysylhydroxylase both require Ascorbic Acid, Vitamin C to work. • These enzymes modify the individual polypeptides before they wrap up into the triple helix. • Without Vitamin C, what disease do you get?

38. Scurvy, characterized by spontaneous bleeding from joints and hair follicles.

39. Collagen – The Vocab • The confusing nomenclature of collagen: • One collagen polypeptide has a helical structure. This is the “minor helix”. • Three polypeptides (three minor helices) wrap up to form the “triple helix”. • A triple helix gets cleaved after it is exported from the cell at both the the N and C termini. After trimming, it is called “tropocollagen”. • Tropocollagens line up to form fibrils. • Fibrils line up to form the overall structure.

40. Enzyme lysyl oxidase forms lysine cross-links in fibrils. Also requires vitamin C. • Disulfide bonds form at both N and C termini: • At C termini, the disulfides form to help line up the 3 minor helices. • At N termini, they form to stop intracellular fibrinogenesis.

41. Elastin • Weird amino acids in it: desmosine, etc. • Has “coiled-coil regions.” • No hydroxyproline or lysine. • 1/3 = ala + val. • Elastin is made of tropoelastin monomers.

42. Take a 5 minute break!

43. II – DNA and RNA

44. DNA and RNA • Some facts: • Purines = Adenine + Guanine • Pyrimidines = Thymine + Cytidine + Uracil. • Uracil found only in RNA. • In double stranded DNA, G-C base pairing is stronger than T-A base pairing. Why?

46. Chagraff’s rule problems: (T/F) • In dsDNA: • If T>35%, then G>15%. • Answer: F. • If T>35%, then A+T>70%, and G<15%. • If A=15% of the bases in one strand, G must =35% of the bases in the whole ds molecule. • Answer: F. Who knows how many A’s there are in the other strand. • If G+C = 40%, then T+G = 50% • Answer: T

47. In ssRNA, if T=24%, then A=24% • Answer: F. It is single stranded, so there is no relationship. • Lieberman = Good Teacher • Answer: F • He’s a GREAT teacher!!!

48. DNA and RNA synthesis • RNA and DNA are made in the 5’ to 3’ direction. • Be careful: The template strand is read 3’ to 5’. Strands always run antiparallel. • Drugs that stop HIV replication have some modification of the 3’ OH group, such that phosphodiester bonds cannot be made after viral incorporation of these nucleotides. So, these drugs terminate viral replication. • DNA Replication is semi-conservative. • The “parent” strands are separated from each other, and after replication each parent strand is base-paired with newly-synthesized DNA.

49. Replication • Replication begins at ori sites and proceeds bidirectionally. • Helicase first unravels the DNA. Topoisomerase relieves tension developed during the unraveling at the other end. • Leading and lagging strands form at each replication fork… • The following will demonstrate the names of the enzymes involved in prokaryotic DNA synthesis and the order in which they act.

50. Bacterial Replication