---

ENZYMES - Biochemistry Notes for BDS First Year

---

1. OVERVIEW

Key point for BDS: Enzymes are critical in oral biochemistry - salivary amylase (ptyalin) begins starch digestion; proteases and nucleases function in the oral cavity; alkaline phosphatase is a marker in bone metabolism relevant to dental/alveolar bone.

---

2. NOMENCLATURE

A. Recommended (Common) Name

• The suffix "-ase" is attached to the substrate name: e.g., glucosidase, urease, lipase
• Some include a description of action: e.g., lactate dehydrogenase (LDH), adenylyl cyclase
• Some retain trivial names: trypsin, pepsin (no clue to reaction from name alone)

B. Systematic Name (IUB Classification)

Memory trick: "Old Tigers Hunt Large Impala Lions" = Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases

---

3. PROPERTIES OF ENZYMES

A. Active Site

• Enzymes contain a special pocket or cleft called the active site, formed by protein folding
• Active site amino acid residues participate in substrate binding and catalysis
• The substrate binds the enzyme forming an enzyme-substrate (ES) complex
• This binding causes a conformational change - the induced fit model - converting ES to enzyme-product (EP) complex, which then dissociates to free enzyme + product

B. Efficiency

• Enzyme-catalyzed reactions proceed 10³ to 10⁸ times faster than uncatalyzed reactions
• Turnover number (kcat): Number of substrate molecules converted to product per enzyme molecule per second = typically 10² to 10⁴ s⁻¹

C. Specificity

• Enzymes are highly specific - they interact with one or very few substrates
• They catalyze only one type of chemical reaction
• The set of enzymes within a cell determines which reactions occur in that cell

D. Holoenzymes, Apoenzymes, Cofactors, and Coenzymes

Important: Coenzymes are commonly derived from vitamins. NAD⁺ contains niacin (Vit B3); FAD contains riboflavin (Vit B2); CoA contains pantothenic acid (Vit B5).

E. Location within the Cell

• Enzymes are compartmentalized in specific organelles for efficient pathway organization:
  • Cytosol: glycolysis, fatty acid synthesis, pentose phosphate pathway
  • Mitochondria: TCA cycle, electron transport chain, β-oxidation
  • Nucleus: DNA replication/repair enzymes
  • Lysosomes: hydrolytic enzymes (hydrolases) - important in lysosomal storage diseases

F. Ribozymes

• Some RNA molecules can catalyze reactions (e.g., affecting phosphodiester and peptide bonds)
• These are called ribozymes - much less common than protein enzymes

---

4. MECHANISM OF ENZYME ACTION

A. Energy Changes During Reaction

• All chemical reactions have an energy barrier between reactants and products
• This barrier is the activation energy (Ea): the energy difference between reactants and the transition state (T)* - a high-energy, short-lived intermediate
• Enzymes lower the Ea by providing an alternate reaction pathway, allowing more molecules to react at physiologic conditions
• Enzymes do NOT change the free energy (ΔG) of reactants or products, nor the equilibrium of the reaction - they only accelerate the rate at which equilibrium is reached

B. Active Site Chemistry - How Enzymes Catalyze Reactions

1. Transition-state stabilization: The active site acts as a flexible template that binds the substrate and initiates its conversion to the transition state. Enzymes bind the transition state more tightly than the substrate, lowering Ea.
2. Acid-base catalysis: Amino acid residues (e.g., histidine) at the active site donate or accept protons to facilitate catalysis.
3. Covalent catalysis: Nucleophilic residues (e.g., serine, cysteine) form transient covalent bonds with the substrate, providing a reactive intermediate.
4. Metal ion catalysis: Metal ions (e.g., Zn²⁺ in carbonic anhydrase) stabilize negative charges, orient substrates, or participate directly in redox reactions.

---

5. FACTORS AFFECTING ENZYME ACTIVITY

A. Substrate Concentration [S]

• As [S] increases, reaction velocity (v₀) increases until a maximum is reached
• Michaelis-Menten enzymes: Plot of v₀ vs. [S] gives a hyperbolic curve
• Allosteric enzymes: Show a sigmoidal (S-shaped) curve (similar to O₂-hemoglobin binding)

B. Temperature

• Velocity increases with temperature (more molecules have sufficient energy to reach transition state)
• Above the optimum temperature (~37-40°C for human enzymes), velocity decreases due to denaturation
• Thermophilic bacteria found in hot springs have enzyme optima around 70°C

C. pH

• Optimum pH varies per enzyme - reflects the pH at which it normally functions:
  • Pepsin (stomach): optimum pH ~2
  • Trypsin (intestine): optimum pH ~8
  • Alkaline phosphatase (bone, liver): optimum pH ~9-10
  • Salivary amylase (oral cavity): optimum pH ~6.7-7.0
• pH affects: (1) ionization state of active-site residues, (2) substrate ionization, (3) enzyme structure (extreme pH causes denaturation)

---

6. ENZYME KINETICS (Michaelis-Menten)

A. The Michaelis-Menten Equation

• v₀ = initial reaction velocity
• Vmax = maximum velocity (when all enzyme active sites are saturated with substrate)
• [S] = substrate concentration
• Km (Michaelis constant) = [S] at which v₀ = ½ Vmax

B. Significance of Km

C. Lineweaver-Burk Plot (Double-Reciprocal Plot)

• A straight-line plot of 1/v₀ (y-axis) vs 1/[S] (x-axis)
• y-intercept = 1/Vmax
• x-intercept = -1/Km
• Useful for determining kinetic constants and identifying type of inhibition

---

7. ENZYME INHIBITION

A. Irreversible Inhibition

• Bind via covalent bonds to the enzyme
• Cannot be reversed by dilution
• Example: Lead (Pb²⁺) forms covalent bonds with cysteine (-SH) groups, inhibiting ferrochelatase (heme synthesis) - clinically seen in lead poisoning
• Organophosphates (e.g., nerve agents, some pesticides) irreversibly inhibit acetylcholinesterase

B. Reversible Inhibition

BDS relevance: Methotrexate (used in oral cancer/lichen planus) is a competitive inhibitor of dihydrofolate reductase, blocking folate metabolism in rapidly dividing cells. Many antibiotics act as enzyme inhibitors.

---

8. ENZYME REGULATION

A. Allosteric Regulation

• Allosteric enzymes do NOT follow Michaelis-Menten kinetics; regulated by effectors binding at sites other than the active site
• Always multi-subunit proteins
• Positive effectors: increase enzyme activity
• Negative effectors: decrease enzyme activity
• Show a sigmoidal v₀ vs. [S] curve (cooperative binding)

• Homotropic effectors: Substrate itself acts as effector (cooperative binding, e.g., hemoglobin + O₂)
• Heterotropic effectors: A different molecule acts as effector (e.g., feedback inhibition)

B. Covalent Modification

• Enzymes can be activated or inactivated by phosphorylation (addition of phosphate group by kinases)
• Phosphorylation can turn enzymes on or off depending on the enzyme
• Example: Glycogen phosphorylase is activated by phosphorylation; glycogen synthase is inactivated by phosphorylation

C. Zymogens (Inactive Precursors)

• Some enzymes are synthesized as inactive forms called zymogens (or proenzymes)
• Activated by proteolytic cleavage when and where needed
• Examples:
  • Pepsinogen → Pepsin (in stomach, activated by HCl and pepsin itself)
  • Trypsinogen → Trypsin (in small intestine, activated by enteropeptidase)
  • Prothrombin → Thrombin (blood coagulation cascade)
  • Chymotrypsinogen → Chymotrypsin
• Purpose: Protects the cell from self-digestion (especially important for digestive enzymes and blood clotting enzymes)

D. Regulation of Enzyme Quantity

• Increased synthesis (induction): Cells can produce more enzyme in response to physiologic signals (e.g., liver enzymes induced by drugs)
• Decreased synthesis (repression): Cells can reduce enzyme production
• Increased degradation: Enzyme levels can be reduced by targeted protein degradation

---

9. ISOENZYMES (ISOZYMES)

• Isoenzymes are multiple forms of an enzyme that catalyze the same reaction but differ in:
  • Amino acid sequence (encoded by different genes)
  • Physical, chemical, and immunological properties
  • Tissue distribution and kinetic properties
• Clinical significance: Measurement of specific isoenzymes is used in diagnosis

BDS Relevance: Alkaline phosphatase isoenzymes are particularly relevant in dentistry - bone isoform is elevated during active bone formation (e.g., during alveolar bone remodeling, fracture healing). Acid phosphatase is found in osteoclasts.

---

10. PLASMA ENZYMES - DIAGNOSTIC SIGNIFICANCE

---

11. CLINICALLY IMPORTANT ENZYME CONCEPTS FOR BDS

Salivary Amylase (Ptyalin)

• Secreted by parotid, submandibular, and sublingual glands
• Begins starch digestion in the mouth; cleaves α-1,4 glycosidic bonds in starch
• Optimum pH ~6.7-7.0 (oral environment)
• Inactivated by stomach acid (pH 2)

Alkaline Phosphatase in Dentistry

• Produced by osteoblasts - marker of osteoblastic activity
• Elevated in: Paget's disease of bone, hyperparathyroidism, healing fractures, alveolar bone remodeling
• Useful marker for monitoring jaw bone diseases

Lysozyme (Muramidase)

• Present in saliva, tears, mucus
• Hydrolyzes β-1,4 glycosidic bonds between NAM and NAG in bacterial cell walls
• First line of defense against oral bacteria

Matrix Metalloproteinases (MMPs)

• Zinc-dependent endopeptidases
• Degrade extracellular matrix components (collagen, elastin, proteoglycans)
• Important in periodontal disease - elevated MMPs degrade periodontal ligament and alveolar bone matrix
• Doxycycline (sub-antimicrobial dose) inhibits MMPs - used in periodontal therapy

---

QUICK REVISION SUMMARY

---