MBB 222 – Weeks 7-10 Core Concepts

Eighty question-and-answer flashcards summarizing key biochemical principles from the Week 7–10 lecture transcript, covering thermodynamics, enzymology, nucleic acid topology, protein–ligand interactions, replication machinery, and hemoglobin regulation.

1. What is the equation for the standard free energy of protein folding at pH 7 and 25 °C?

ΔG°’fold = ΔH°’fold – TΔS°’_fold

2. For myoglobin folding, what are the numeric values of ΔH°’fold and –TΔS°’fold?

ΔH°’fold ≈ –195 kJ mol⁻¹; –TΔS°’fold ≈ +171 kJ mol⁻¹.

3. Why does burying apolar groups during folding increase water entropy?

Because ordered water molecules around apolar surfaces are released, increasing the entropy of the solvent.

4. In the biochemistry ‘prime’ convention, what does the prime (′) indicate in ΔG°’?

That reactant activities are referenced to pH 7 and standard biochemical conditions (1 M activity for solutes, 55.5 M water activity = 1).

5. Why must concentrations be converted to activities before using ΔG°’ = –RT ln K?

Because the logarithm requires dimensionless quantities; activities are unit-less ratios of concentration to 1 M.

6. For ACE2–SARS-CoV-2 spike binding, what is ΔG°’_bind?

≈ –46 kJ mol⁻¹ at pH 7, 25 °C.

7. What energetic component dominates protein–protein binding vs. hydrophobic folding?

Binding often has a small –TΔS term (little hydrophobic effect) but favorable ΔH from polar interactions; folding has large unfavorable –TΔS from chain ordering.

8. Give the relationship between standard free energy and equilibrium constant.

ΔG°’ = –RT ln K_eq.

9. What is the biochemical definition of a fuel molecule?

A compound whose hydrolysis or cleavage releases a large negative ΔG°’, storing potential energy for work.

10. How much standard free energy is released by ATP phosphoanhydride hydrolysis?

About –30 kJ mol⁻¹ (to ADP + Pi).

11. State the first law of thermodynamics in biochemical terms.

Energy can change forms (chemical, mechanical, heat) but cannot be created or destroyed.

12. What is energy coupling in metabolism?

Using an exergonic reaction (e.g., ATP hydrolysis) to drive an endergonic reaction via an enzyme that links the two.

13. In glutamine synthetase, what are ΔG°’ values for acylation, ATP hydrolysis, and the net reaction?

+14 kJ mol⁻¹, –30 kJ mol⁻¹, and –16 kJ mol⁻¹ respectively.

14. Write the equation relating actual free energy change to Q and K.

ΔG’ = ΔG°’ + RT ln Q = RT ln(Q/K).

15. When Q < K for a reaction, what is the sign of ΔG’ and which direction is favored?

ΔG’ < 0; the forward reaction proceeds.

16. Why doesn’t cellular ATP spontaneously run to equilibrium?

Cells maintain [ATP] ≫ [ADP][Pi]/K by continual synthesis, so Q ≪ K and ΔG’ remains highly negative.

17. What two factors combine to give the net driving force ΔG’?

Intrinsic structural factor (ΔG°’ or K) and concentration factor (Q).

18. Define a nucleophile in biochemical reactions.

An atom with a lone pair that donates electrons to form a new bond with an electrophilic center.

19. Which atom is the electrophile in an acyl transfer reaction?

The central carbonyl carbon (or phosphoryl phosphorus) that is partially positive.

20. In peptide bond hydrolysis, identify the attacking and leaving groups.

Attacking nucleophile = water hydroxyl O; leaving group = amide nitrogen of the C-terminal fragment.

21. What kind of linkage do aminoacyl-tRNAs contain?

A carbonyl ester linkage between 3′ OH of tRNA and the amino acid carboxyl.

22. Which functional group transfer is catalyzed by myosin?

Transfer of a phosphoryl group from ATP γ-phosphate to water (ATP hydrolysis).

23. Why is hemoglobin’s O₂-binding curve sigmoidal?

Because of cooperative allosteric switching between T (low affinity) and R (high affinity) states among its subunits.

24. Define P₅₀ for a gas-binding protein.

The partial pressure of gas that yields 50 % saturation of binding sites.

25. What is the Bohr effect?

Lower pH (higher [H⁺]) stabilizes the T state of hemoglobin, promoting O₂ release.

26. How does 2,3-bisphosphoglycerate affect hemoglobin?

Binds the central cavity, stabilizes T state, decreases O₂ affinity, aiding release in tissues.

27. Why does fetal hemoglobin bind O₂ more tightly than adult Hb?

Its γ subunits bind BPG and CO₂ less effectively, favoring the R state.

28. What mutation causes sickle-cell hemoglobin?

β-chain Glu6→Val, creating a hydrophobic patch that promotes fiber formation in T state.

29. Describe transition-state stabilization by enzymes.

Enzymes bind the transition state more tightly than ground state, lowering ΔG‡ and accelerating the reaction.

30. State the Michaelis-Menten equation.

V₀ = Vmax [S]/([S] + Km).

31. Define k_cat.

Turnover number: maximum number of substrate molecules converted to product per enzyme molecule per unit time under saturating [S].

32. What does K_m represent mechanistically?

The substrate concentration at which V₀ = ½ Vmax; approximates (koff + kcat)/kon.

33. Give the equation relating V_max to enzyme concentration.

Vmax = kcat [E]_total.

34. What kinetic parameter compares catalytic efficiency for different substrates?

kcat/Km (specificity constant).

35. In rate constants, what are the units of kon and koff?

kon: M⁻¹ s⁻¹ (second-order); koff: s⁻¹ (first-order).

36. What are the two phases of chymotrypsin catalysis?

(A) Acylation: peptide acyl group transferred to Ser; (B) De-acylation: acyl group hydrolyzed by water, regenerating enzyme.

37. Name the residues of the catalytic triad in serine proteases.

Aspartate, histidine, and serine.

38. How does histidine assist serine during nucleophilic attack?

Acts as a base, accepting Ser-OH proton to generate a more reactive Ser-O⁻ alkoxide.

39. What is the role of the oxyanion hole?

Stabilizes the negative charge on the tetrahedral oxyanion intermediate via backbone NH hydrogen bonds.

40. Which amino acid side chains position chymotrypsin’s specificity pocket?

Hydrophobic residues forming a deep pocket accommodating bulky apolar side chains (Phe, Trp, Tyr, Met).

41. What is covalent catalysis?

Temporary formation of a covalent bond between enzyme and substrate to lower activation energy.

42. How does diisopropylfluorophosphate (DIFP) inhibit serine proteases?

Irreversibly forms a phospho-triester bond with the reactive serine, preventing de-acylation.

43. Which DNA groove usually participates in sequence-specific protein recognition?

The major groove.

44. Why are histones rich in Lys and Arg residues?

Their cationic side chains neutralize DNA’s negative phosphate backbone, enabling non-specific binding.

45. What structural unit is formed by DNA wrapped around a histone octamer?

A nucleosome core particle.

46. Define twist (Tw) in DNA topology.

Number of helical turns of the two strands around each other inside the duplex (≈1 per 10.5 bp).

47. Define writhe (Wr).

Number of supercoil turns formed by the DNA duplex axis coiling upon itself.

48. Write the equation linking Lk, Tw, and Wr.

Lk = Tw + Wr (linking number equals twist plus writhe).

49. What is negative supercoiling and why is it useful?

Wr < 0; stores energy that facilitates unwinding for processes like replication and transcription.

50. Name the bacterial enzyme that removes positive supercoils ahead of the replication fork.

DNA gyrase (a type II topoisomerase).

51. Which protein initiates E. coli chromosome replication by unwinding oriC?

DnaA replication-initiator protein.

52. In what direction does helicase move along the strand it binds?

5′ → 3′ along the single strand to which it is bound.

53. What keeps exposed single-stranded DNA from re-annealing during replication?

Single-Strand Binding protein (SSB).

54. What enzyme synthesizes short RNA primers in bacteria?

Primase (DnaG).

55. Why is a sliding clamp required for DNA polymerase III?

To confer high processivity, preventing the polymerase core from dissociating during long synthesis.

56. What protein complex loads the sliding clamp onto DNA?

Clamp loader (γ complex) using ATP hydrolysis.

57. Distinguish leading and lagging strand synthesis.

Leading: continuous 5′→3′ synthesis toward the fork; Lagging: discontinuous synthesis away from the fork as Okazaki fragments.

58. Approximately how long are bacterial Okazaki fragments?

About 1–2 kilobases.

59. Which enzyme removes RNA primers and fills resulting gaps?

DNA polymerase I, via 5′→3′ exonuclease and polymerase activities.

60. How are adjacent Okazaki fragments finally joined?

DNA ligase forms a phosphodiester bond using ATP (or NAD⁺) energy.

61. What subunits tether the two DNA pol III cores to the helicase?

Tau (τ) subunits of the clamp loader complex.

62. What is processivity in DNA polymerase terminology?

The number of nucleotides added per binding event before the enzyme dissociates.

63. Explain the ‘proofreading’ function of many DNA polymerases.

A 3′→5′ exonuclease removes mis-incorporated nucleotides, allowing the polymerase to retry.

64. How does the active site of DNA polymerase discriminate against mismatches?

Incorrect base pairs distort geometry, preventing proper fit and reduced catalytic rate; they dissociate before incorporation.

65. Which DNA polymerase in E. coli possesses 5′→3′ exonuclease activity for primer removal?

DNA polymerase I.

66. What is the name of the large fragment of DNAP I lacking the 5′→3′ exonuclease domain?

Klenow fragment.

67. Define homotropic vs. heterotropic modulation in hemoglobin.

Homotropic: modulation by the ligand itself (O₂); heterotropic: modulation by other molecules (H⁺, CO₂, BPG).

68. What is Le Châtelier’s principle applied to biochemical reactions?

If Q differs from K, the reaction proceeds in the direction that drives Q toward K until equilibrium (ΔG’ = 0).

69. Give the standard free energy change of hydrolysis for an amide compared to an anhydride.

Amide hydrolysis ΔG°’ ≈ –23 kJ mol⁻¹ (less exergonic) vs. anhydride ≈ –30 to –32 kJ mol⁻¹ (more exergonic).

70. What is the chemical reason phosphoanhydrides store high potential energy?

Electrostatic repulsion and strain between adjacent phosphoryl groups plus resonance stabilization of products.

71. In nucleophilic substitution, what determines a good leaving group?

Its ability to stabilize the extra electrons after bond cleavage (i.e., lower pKa, better anion stability).

72. Why does protein folding generally have a negative ΔG°’?

Favorable enthalpy from optimized interactions outweighs the unfavorable conformational entropy loss.

73. What does the specificity pocket of trypsin prefer?

Positively charged side chains (Arg, Lys) due to an Asp residue at the pocket bottom.

74. How does aspartate in the catalytic triad aid histidine?

Stabilizes the positive charge developing on His imidazole, increasing its basicity toward serine or water.

75. Why do enzymes that bind transition states also bind competitive transition-state analog inhibitors tightly?

Such analogs mimic the geometry and charge distribution of the TS, exploiting the enzyme’s highest-affinity interactions.

76. Describe the concerted (MWC) model of hemoglobin cooperativity.

Tetramer exists in equilibrium between all-T and all-R states; ligand binding shifts equilibrium toward R, enhancing affinity of remaining sites.

77. What is the effect of CO₂ carbamylation on hemoglobin?

Forms carbamate at N-termini, stabilizes T state, promotes O₂ release, and carries CO₂ to lungs.

78. Identify two physiological roles of serine proteases.

Digestion (trypsin, chymotrypsin) and blood clot dissolution (plasmin).

79. How does Single-Strand Binding protein facilitate replication beyond protection?

Prevents secondary structures in ssDNA and recruits other replication proteins.

80. What is the significance of negative supercoiling for replication origin melting?

Stored torsional energy lowers the energy required to denature A:T-rich regions, aiding initiator proteins in opening the duplex.