1.4 Enzyme Classification & Mechanisms

The Six Enzyme Commission (EC) Classes

Enzymes are biological catalysts that accelerate chemical reaction rates without being consumed. The International Union of Biochemistry and Molecular Biology classifies enzymes into six major classes based on the reaction mechanism catalyzed. Remember the mnemonic OTH LIL:

1. EC 1: Oxidoreductases

• Mechanism: Catalyze oxidation-reduction (redox) reactions involving the transfer of electrons, hydrides ($\text{H}^-$), or hydrogen atoms between donor and acceptor molecules.
• Common Subclasses: Dehydrogenases, Reductases, Oxidases, Peroxidases.
• Metabolic Examples:
  • Lactate Dehydrogenase (LDH): Converts pyruvate to lactate while oxidizing $\text{NADH}$ to $\text{NAD}^+$.
  • Alcohol Dehydrogenase (ADH): Converts ethanol to acetaldehyde.

2. EC 2: Transferases

• Mechanism: Catalyze the transfer of a functional group (e.g., methyl, phosphate, amino, acyl) from one donor molecule to another acceptor molecule.
• Common Subclasses: Kinases, Transaminases / Aminotransferases, Polymerases.
• Metabolic Examples:
  • Hexokinase: Transfers a phosphate group from $\text{ATP}$ to glucose, forming glucose-6-phosphate.
  • Alanine Aminotransferase (ALT): Transfers an amino group from alanine to $\alpha$-ketoglutarate.

3. EC 3: Hydrolases

• Mechanism: Catalyze the cleavage of chemical bonds by the addition of water ($\text{H}_2\text{O}$).
• Common Subclasses: Phosphatases, Proteases / Peptidases, Nucleases, Lipases.
• Metabolic Examples:
  • Glucose-6-Phosphatase: Hydrolyzes glucose-6-phosphate to produce free glucose and inorganic phosphate ($P_i$).
  • Trypsin & Chymotrypsin: Hydrolyze internal peptide bonds in proteins.

4. EC 4: Lyases

• Mechanism: Catalyze the cleavage of $\text{C}-\text{C}$, $\text{C}-\text{O}$, or $\text{C}-\text{N}$ bonds by elimination, forming a double bond or ring structure without hydrolysis or oxidation. The reverse reaction (adding a group to a double bond) is catalyzed by synthases.
• Common Subclasses: Decarboxylases, Aldolases, Dehydratases.
• Metabolic Examples:
  • Pyruvate Decarboxylase: Cleaves the carboxyl group from pyruvate to yield acetaldehyde and $\text{CO}_2$.
  • Fructose-1,6-Bisphosphate Aldolase: Cleaves F-1,6-BP into DHAP and GAP in glycolysis.

5. EC 5: Isomerases

• Mechanism: Catalyze structural or geometric rearrangements of atoms within a single molecule to interconvert constitutional or stereoisomers.
• Common Subclasses: Mutases, Epimerases, Racemases, Cis-Trans Isomerases.
• Metabolic Examples:
  • Triosephosphate Isomerase (TPI): Interconverts dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP).
  • Phosphoglycerate Mutase: Transfers a phosphate group from C-3 to C-2 within phosphoglycerate.

6. EC 6: Ligases

• Mechanism: Catalyze the joining (condensation) of two large molecules, coupled with the hydrolysis of $\text{ATP}$ or a similar high-energy nucleoside triphosphate. Also called synthetases.
• Metabolic Examples:
  • DNA Ligase: Joins Okazaki fragments by forming phosphodiester bonds.
  • Pyruvate Carboxylase: Carboxylates pyruvate to oxaloacetate using $\text{ATP}$ and $\text{CO}_2$.

---

Active Site Models & Energetics of Catalysis

Lock-and-Key vs Induced-Fit Model

• Lock-and-Key Model (Fischer): Assumes the active site of the enzyme is rigid and perfectly complementary to the substrate's ground state prior to binding. This model fails to account for transition-state stabilization or conformational flexibility.
• Induced-Fit Model (Koshland): The accepted model. Substrate binding induces a dynamic conformational change in the enzyme's active site, forming an optimal fit around the high-energy transition state ($EX^\ddagger$).

                                ENERGY PROFILE
   Free Energy (G)
        ^
        |       /\  <-- Uncatalyzed Transition State (High Ea)
        |      /  \  
        |     /    \   /\  <-- Catalyzed Transition State (Lower Ea)
        |    /      \ /  \
Reactants --+        V    \       Ea (catalyzed) < Ea (uncatalyzed)
        |                  \      delta G_rxn remains UNCHANGED!
        |                   +-- Products
        +------------------------------------------> Reaction Coordinate

Thermodynamic vs Kinetic Effects

• Activation Energy ($E_a$ or $\Delta G^\ddagger$): Enzymes lower the activation energy barrier for both the forward and reverse reactions by stabilizing the transition state.
• Equilibrium & $\Delta G$: Enzymes do NOT alter the overall free energy change of the reaction ($\Delta G$), the enthalpy change ($\Delta H$), the entropy change ($\Delta S$), or the equilibrium constant ($K_{\text{eq}}$). Enzymes only accelerate the rate ($k$) at which equilibrium is attained.

---

Catalytic Strategies & Case Study: Serine Proteases

Enzymes employ several fundamental physical-chemical mechanisms within their active sites to achieve enormous rate enhancements:

1. Acid-Base Catalysis: Active site side chains act as proton donors or acceptors (e.g., Histidine, Glutamate, Aspartate).
2. Covalent Catalysis: The active site residue forms a transient covalent bond with the substrate (e.g., nucleophilic Serine in proteases).
3. Metal Ion Catalysis: Bound metal cofactors orient substrates, shield negative charges, or participate in redox electron transfer.
4. Proximity & Orientation Effects: Active site binding aligns substrates in optimal spatial orientation for reaction.

Serine Protease Catalytic Triad & Oxyanion Hole

Serine proteases (Trypsin, Chymotrypsin, Elastase) illustrate these catalytic principles:

• Catalytic Triad: Consists of Serine-195, Histidine-57, and Aspartate-102.
  • Asp-102 forms a hydrogen bond with His-57, increasing the basicity of His-57.
  • His-57 acts as a general base, deprotonating the hydroxyl group of Ser-195.
  • Alkoxide-like Ser-195 acts as a potent nucleophile, attacking the peptide carbonyl carbon to form a tetrahedral intermediate.
• Oxyanion Hole: Backbone amide hydrogens of Ser-195 and Gly-193 form hydrogen bonds with the negatively charged oxygen atom of the tetrahedral intermediate, stabilizing the high-energy transition state.

---

Apoenzymes, Holoenzymes, Cofactors & Coenzymes

Many enzymes require non-protein helper components to achieve catalytic activity.

• Apoenzyme: The inactive protein component lacking its required cofactor/coenzyme.
• Holoenzyme: The complete, catalytically active complex consisting of the apoenzyme bound to its essential cofactor/coenzyme.
• Prosthetic Group: A cofactor or coenzyme that is covalently or extremely tightly bound to the enzyme (e.g., heme in cytochromes, biotin in pyruvate carboxylase).

  Apoenzyme (Inactive Protein) + Cofactor / Coenzyme  --->  Holoenzyme (Active Complex)

1. Inorganic Cofactors

Inorganic metal ions that bind to the active site to stabilize negative charges, orient substrates, or participate in redox catalysis.

• $\text{Mg}^{2+}$: Essential for all kinases; shields negative charges on $\text{ATP}$ polyphosphate chains.
• $\text{Zn}^{2+}$: Acts as a Lewis acid in carbonic anhydrase and carboxypeptidases.
• $\text{Fe}^{2+} / \text{Fe}^{3+}$: Electron transfer in cytochromes, hemoglobin, and catalase.
• $\text{Cu}^{2+} / \text{Cu}^+$: Redox center in cytochrome c oxidase.

2. Organic Coenzymes (Vitamin Derivatives)

Small non-protein organic molecules, mostly synthesized from dietary water-soluble B vitamins.