MP

Biochemistry Exam Review Session

Exam Scope & Administrative Notes

  • Coverage begins at Chapter 5 material on β-turns/β-hairpins ("Beta Herb returns" in audio) and continues forward.
  • Included topics
    • Remaining Ch. 5: tertiary structure, protein-folding discussions.
    • Entire Chapter 7: dissociation & association constants, hemoglobin cooperative binding, allostery, Bohr effect, Hill plots, etc.
    • Vast majority of Chapter 8: reversible enzyme inhibition (competitive, un-/non-competitive, mixed).
      • Excluded: irreversible inhibition section (e.g., penicillin mechanism).
  • Lecture slide order may differ from recorded videos because instructor reorganized slides for narrative flow.
  • Recommendation: use white-board examples + recorded videos for mechanism walkthroughs; re-watch as needed rather than expecting live re-teaching.

Metal-Dependent Enzymes, Vitamin C & Scurvy

  • Many hydroxylases (e.g., prolyl-4-hydroxylase, lysyl-hydroxylase) require Fe²⁺/Fe³⁺ cycling during catalysis.
    • Common failure modes
      • Premature release of partially processed substrate ⇢ iron trapped in wrong oxidation state.
      • Side-reactions with non-native substrates ⇢ same oxidation-state trap.
      • Net result: enzyme exits catalytic cycle inactive.
  • Vitamin C (ascorbate)
    • Water-soluble antioxidant that reduces the mis-oxidized metal center, “resetting” the enzyme.
    • Humans cannot synthesize vitamin C → must obtain exogenously (diet).
  • Inadequate vitamin C supply ⇒ failure to reactivate enzymes ⇒ deficient post-translational modification of collagen.
    • Consequences (clinical scurvy)
      • Weak collagen triple helices & fibrils.
      • Loss of blood-vessel integrity → bleeding gums, bruising, hemorrhage.
      • Weak joints/ligaments, impaired wound healing.
      • Smooth-muscle & connective-tissue degradation.
  • Populations at risk: monotonous diets, lack of fresh fruits/vegetables, extreme food limitation.

Catalytic Triad: Serine Protease Mechanism (Broad Strokes)

  • Residues & roles
    • Ser195 (nucleophile): performs covalent attack on peptide carbonyl.
    • His57 (general acid/base): toggles protonation to activate Ser & stabilize tetrahedral intermediates.
    • Asp102: forms strong H-bond to His, electrostatically stabilizing His⁺ and aligning triad; its role is constant through entire cycle.
  • Key mechanistic phases (no arrow-pushing required for exam)
    1. Acylation – Ser O⁻ attacks carbonyl → tetrahedral oxyanion (stabilized by oxyanion hole) → scission releases C-terminal fragment; enzyme becomes acyl-enzyme.
    2. De-acylation – Water (activated by His) attacks acyl enzyme → second tetrahedral → releases N-terminal fragment, regenerating free enzyme.
  • Understand conceptual logic, catalytic roles, transition-state stabilization; memorize not the full drawing but story.

Enzyme Kinetics & Reversible Inhibition

Michaelis–Menten Refresher

  • Initial velocity v0=\frac{V{max}[S]}{K_M+[S]}.
  • Maximum velocity when all enzyme is ES: V{max}=k2[E]_{total}.

Competitive Inhibition (I binds E only)

  • I increases the apparent KM (binding looks weaker) because KM^{app}=\alpha K_M where \alpha>1.
  • V_{max} unchanged: at infinite [S] substrate out-competes I so all enzyme can reach ES.
  • Graphical cues
    • Michaelis–Menten: inhibited curve reaches same asymptote but requires higher [S].
    • Lineweaver–Burk: lines intersect on y-axis (1/V_{max} constant), different slopes.

Uncompetitive Inhibition (I binds ES only)

  • Forms ESI; cannot be overcome by high [S].
  • Both KM^{app} and V{max}^{app} decrease; parallel lines in Lineweaver–Burk.

Non-competitive / Mixed Inhibition (I binds E & ES)

  • Pure non-competitive: KM unchanged, V{max} lowered because ESI removes active enzyme.
  • Mixed: both parameters change; magnitude depends on individual K_i values.
  • Key concept: Any pathway that diverts enzyme into ESI limits maximal catalytic rate even at saturating [S].

Hemoglobin & Sickle-Cell Disease (E6V Mutation)

  • Mutation: Glu6 → Val in β-chain (E6V); generates hydrophobic patch.
  • Polymerization behavior
    • Occurs only in T-state (deoxy Hb).
    • Hydrophobic Val inserts into complementary hydrophobic pocket of neighboring tetramer → filamentous polymer.
  • Pathophysiology
    • Polymer growth locks tetramers in T-state ⇒ low O_2 affinity; impaired oxygen delivery ⇢ anemia.
    • Filament bundles rupture RBC membrane → hemolysis, inflammation, pain crises.
    • Essentially irreversible once fibers form.
  • Required knowledge: mutation identity, T-state specificity, consequences on oxygen transport & cell integrity.

Storage Polysaccharides & Osmotic Considerations

  • Purposes of converting glucose → glycogen (animals) / starch (plants)
    • Lower intracellular [glucose] to favor continued uptake/transport (concentration-gradient component of \Delta G for transport).
    • Reduce osmotic pressure by tying many glucose units into one particle & maximizing intra-molecular H-bonding (less need for water solvation).
  • Branching & rate of mobilization
    • Glycogen/amylopectin: α(1→4) backbones with α(1→6) branches every 8-12 residues (glycogen) or ~25 residues (amylopectin).
    • Enzymes (glycogen phosphorylase, debranching enzymes) remove glucose from non-reducing ends.
    • More branch points ⇒ more non-reducing ends ⇒ higher parallel cleavage rate ⇒ faster glucose release (critical for “fight-or-flight”).
    • Linear amylose (10 000 residues) has only one non-reducing end, hence much slower mobilization.
  • Exam-relevant connections: osmotic stress, transport energetics, structural features enabling rapid energy access in animals.

Study & Logistics Tips from Q&A

  • Re-watch mechanism videos repeatedly while narrating steps aloud.
  • For kinetics, focus on conceptual interpretations of plots & parameter changes rather than heavy math (no α, α' calculations expected).
  • Make sure you can verbally explain mechanistic roles, binding equilibria, and physiological implications.
  • Instructor responded to repetitive direct-message queries; consolidate common questions to save review time.

Key Equations & Definitions (Quick Reference)

• Michaelis–Menten: v0=\frac{V{max}[S]}{K_M+[S]}

• Max velocity: V{max}=k2[E]_{total}

• Competitive inhibition effect: KM^{app}=\alpha KM\ \ (\alpha=1+\frac{[I]}{Ki}); V{max}^{app}=V_{max}

• Uncompetitive inhibition: KM^{app}=\frac{KM}{\alpha'}, V{max}^{app}=\frac{V{max}}{\alpha'} (α' from ESI binding).

• Arrhenius notion: catalytic triad lowers \Delta G^{\ddagger} by stabilizing tetrahedral oxyanion intermediate.

Checklist of Must-Know Topics for the Exam

  • β-turn motifs & overall tertiary structure principles.
  • Protein folding energetics & chaperone roles (from Ch. 5).
  • Hemoglobin: cooperative binding, Bohr effect, Hill coefficient, sickle-cell polymerization mechanism.
  • Enzyme kinetics fundamentals; interpretation of Lineweaver–Burk, Eadie-Hofstee.
  • Reversible inhibition types & their signature kinetic changes.
  • Serine protease catalytic triad function & reaction coordinate.
  • Vitamin C biochemistry, collagen PTMs, scurvy symptoms.
  • Storage polysaccharide structure/function; glycogen vs plant starch; osmotic & energetic rationale.