---

Enzymes - Complete MBBS Notes

---

1. OVERVIEW & DEFINITION

• Catalytic power: 10^6 to 10^14 times faster than uncatalyzed reactions
• Enzymes do not alter the equilibrium of a reaction - they only speed up attainment of equilibrium
• Enzymes do not change the free energy (ΔG) of reactants or products
• Turnover number (kcat): number of substrate molecules converted to product per enzyme per second; typically 10^2 to 10^4 s^-1

---

2. NOMENCLATURE

Recommended (Common) Name

• Suffix "-ase" attached to the substrate (e.g., urease, glucosidase) or the type of reaction (e.g., lactate dehydrogenase, adenylyl cyclase)
• Some retain trivial names with no descriptive value: trypsin, pepsin, chymotrypsin

Systematic Name (IUB/IUBMB Classification)

---

3. PROPERTIES OF ENZYMES

A. Active Site

• Special pocket or cleft formed by folding of the protein
• Contains amino acid residues whose side chains participate in substrate binding and catalysis
• Usually located in a cleft or crevice that excludes water from the reaction center
• Makes up only a small fraction of the total enzyme volume

B. Models of Substrate Binding

• Active site is a rigid, preformed structure perfectly complementary to the substrate
• Substrate fits exactly like a key into a lock

• Active site is flexible and dynamic
• Substrate binding causes a conformational change in the enzyme that repositions amino acid side chains
• This repositioning: improves binding, promotes the reaction, excludes water, and may activate adjacent subunits
• Example: Glucokinase - glucose binding closes the cleft, improves ATP-binding site, excludes water

C. Mechanism of Enzyme Action - How Enzymes Lower Activation Energy

1. Proximity and orientation effects - bringing substrates together in the correct geometry
2. Acid-base catalysis - amino acid side chains donate/accept protons (His is most common due to pKa ~6)
3. Covalent catalysis - formation of a transient covalent enzyme-substrate intermediate (e.g., serine proteases)
4. Metal ion catalysis - metal ions act as electrophiles, stabilize negative charges, or transfer electrons
5. Electrostatic effects - charged active site residues stabilize transition state

D. Holoenzyme, Apoenzyme, Cofactors, Coenzymes

E. Isoenzymes (Isozymes)

• Multiple forms of an enzyme that catalyze the same reaction but differ in:
  • Amino acid sequence (encoded by different genes)
  • Physical/biochemical properties (electrophoretic mobility, Km, pH optimum, heat stability)
  • Tissue distribution
• Clinical importance of LDH isoforms:

• CK (Creatine Kinase) isoforms:
  • CK-BB (CK-1): Brain
  • CK-MB (CK-2): Heart - marker for myocardial infarction
  • CK-MM (CK-3): Skeletal muscle

---

4. ENZYME KINETICS

A. Michaelis-Menten Kinetics

1. [S] >> [E] (substrate far exceeds enzyme concentration)
2. Steady-state assumption: rate of formation of ES = rate of breakdown of ES; [ES] remains constant
3. Initial velocity (v0) measured: product concentration negligible, reverse reaction ignored

v₀ = (Vmax × [S]) / (Km + [S])

• v₀ = initial reaction velocity
• Vmax = maximum velocity = kcat × [E]total
• Km = Michaelis constant = (k-1 + k2) / k1
• [S] = substrate concentration

B. Understanding Km

• Km = [S] when v₀ = ½ Vmax (definition)
• Km is a property of the enzyme-substrate pair; does NOT vary with enzyme concentration
• Km approximates the dissociation constant of the ES complex (when k2 << k-1)

C. Relationship Between Velocity and Enzyme Concentration

• Vmax is directly proportional to enzyme concentration
• Doubling [E] doubles Vmax (and all velocities at given [S])

D. Lineweaver-Burk (Double Reciprocal) Plot

1/v₀ = (Km/Vmax)(1/[S]) + 1/Vmax

• Y-intercept = 1/Vmax
• X-intercept = -1/Km
• Slope = Km/Vmax

E. Factors Affecting Enzyme Activity

---

5. ENZYME INHIBITION

A. Irreversible Inhibition

• Bind enzyme via covalent bonds - permanent inactivation
• Examples:
  • Lead (Pb2+): forms covalent bonds with -SH of cysteine; inhibits ferrochelatase (heme synthesis)
  • Organophosphate compounds (e.g., parathion, sarin nerve gas, DIPF): irreversibly inhibit acetylcholinesterase by phosphorylating its serine residue
  • Aspirin: irreversibly inhibits cyclooxygenase (COX-1 and COX-2) by acetylation
  • Penicillin: irreversibly inhibits transpeptidase (bacterial cell wall synthesis)

B. Reversible Inhibition

1. Competitive Inhibition

• Inhibitor structurally resembles substrate and competes for the same active site
• Vmax: UNCHANGED (can be overcome by high [S])
• Km: INCREASED (apparent Km increases)
• Lineweaver-Burk plot: Lines intersect at Y-axis (same 1/Vmax, different X-intercepts)

• Statins (atorvastatin, pravastatin) → competitive inhibitors of HMG-CoA reductase (cholesterol synthesis)
• Methotrexate → competitive inhibitor of dihydrofolate reductase (DHFR)
• Sulfonamides → competitive inhibitors of dihydropteroate synthase in bacteria
• Allopurinol → competitive inhibitor of xanthine oxidase (gout)
• Metformin → inhibits Complex I (NADH dehydrogenase) in mitochondria

2. Noncompetitive (Pure) Inhibition

• Inhibitor binds at a site other than the active site (allosteric site), on either free enzyme or ES complex
• Vmax: DECREASED (cannot be overcome by high [S])
• Km: UNCHANGED (affinity for substrate unchanged)
• Lineweaver-Burk plot: Lines intersect at X-axis (same -1/Km, different Y-intercepts)

3. Uncompetitive Inhibition

• Inhibitor binds only to the ES complex (not free enzyme)
• Vmax: DECREASED
• Km: DECREASED (apparent)
• Lineweaver-Burk plot: Parallel lines (same slope)

Summary Table - Types of Inhibition

---

6. ENZYME REGULATION

A. Allosteric Regulation

• Allosteric enzymes have regulatory sites separate from the active site
• Effectors (modulators) bind non-covalently at allosteric sites → conformational change
• Positive allosteric effectors = activators → increase activity
• Negative allosteric effectors = inhibitors → decrease activity
• Typically seen in regulatory enzymes (often the first committed/rate-limiting step)
• Feedback (end-product) inhibition: final product of a pathway inhibits the first enzyme in the pathway

• Allosteric enzymes often show sigmoidal v₀ vs [S] curves due to cooperativity
• Multiple subunits - binding of substrate to one subunit increases affinity of other subunits (positive cooperativity)
• Example: Hemoglobin (not an enzyme, but classic cooperativity model), ATCase, phosphofructokinase-1

B. Covalent Modification (Post-translational)

• Phosphorylation/Dephosphorylation is most important:
  • Protein kinases (use ATP as phosphate donor) → add -PO₄ to Ser, Thr, or Tyr residues
  • Protein phosphatases → remove -PO₄
  • Effect depends on the specific enzyme:
    • Activates: Glycogen phosphorylase, hormone-sensitive lipase
    • Inhibits: Glycogen synthase, pyruvate kinase (liver)
• Other covalent modifications: acetylation, methylation, ubiquitination, adenylation, ADP-ribosylation

C. Zymogen Activation (Proteolytic Cleavage)

• Zymogens = inactive enzyme precursors
• Activated by irreversible proteolytic cleavage (one-time event, cannot be reversed)
• Examples:

D. Enzyme Synthesis and Degradation (Slow Regulation)

• Induction: increased synthesis of enzyme (hours to days); e.g., insulin induces glycolytic enzymes
• Repression: decreased synthesis
• Enzyme degradation: ubiquitin-proteasome pathway
• Much slower than allosteric or covalent modification

---

7. ENZYMES AS DIAGNOSTIC MARKERS (Clinical Applications)

---

8. ENZYMES IN GENETIC/METABOLIC DISEASES

---

9. SPECIAL ENZYME TOPICS

A. Multienzyme Complexes

• Multiple enzymes organized together to increase efficiency (substrate channeling)
• Examples:
  • Pyruvate dehydrogenase complex (PDC): E1 (pyruvate decarboxylase-TPP), E2 (dihydrolipoyl transacetylase-lipoic acid, CoA), E3 (dihydrolipoyl dehydrogenase-FAD, NAD+)
  • Fatty acid synthase (FAS): multifunctional enzyme
  • α-Ketoglutarate dehydrogenase complex: structurally similar to PDC

B. Rate-Limiting Enzyme

• The slowest step in a metabolic pathway determines the overall rate
• This step is most commonly regulated (allosteric + covalent modification)
• Often at the first committed step of a pathway
• Examples: PFK-1 (glycolysis), HMG-CoA reductase (cholesterol synthesis), carbamoyl phosphate synthetase (urea cycle)

C. Transition State Theory

• Reactants must pass through a high-energy transition state
• Enzymes work by stabilizing the transition state (lowering its energy), not the reactants or products
• This is why transition state analogs are potent competitive inhibitors (bind more tightly than substrate)
• Examples of drugs as transition state analogs: HIV protease inhibitors (ritonavir, saquinavir)

D. Serine Proteases

• Have serine, histidine, aspartate in the catalytic triad (charge relay system)
• Examples: trypsin, chymotrypsin, elastase, thrombin, plasmin, kallikrein
• Mechanism: serine acts as nucleophile; histidine acts as proton shuttle; aspartate stabilizes histidine

---

10. HIGH-YIELD EXAM POINTS

---

• Lippincott's Illustrated Reviews: Biochemistry, 8th Edition, Chapter 5 (pp. 167-200)
• Basic Medical Biochemistry: A Clinical Approach, 6th Edition, Chapter 8
• Harper's Illustrated Biochemistry, 32nd Edition, Chapters on enzyme kinetics and regulation