Enzymes, Cofactors & Coenzymes – Comprehensive Exam Notes

Cofactors: Definition & Overall Significance

• Cofactor (general): Any non-protein substance that must associate with an apoenzyme to permit catalysis.
  • Enable/augment catalytic power, structural stability, substrate orientation, or group transfer.
  • May participate directly in the chemical reaction (e.g., donate/accept electrons, atoms, or functional groups).
• Two grand categories
  • Organic
  • Tightly bound → Prosthetic group (permanent component of the enzyme complex).
  • Loosely bound → Coenzyme (binds–releases each catalytic cycle).
  • Inorganic (metal ions)
  • Tightly bound → Metalloenzymes (metal is integral part of the protein; often present in the active site).
  • Loosely bound → Metal-activated enzymes / Ion activators (metal associates transiently).
• Practical implications
  • Dietary deficiency of a vitamin or metal → loss of specific enzyme activities (clinical enzymopathies).
  • Many diagnostic assays exploit metal/coenzyme dependence to modulate or measure enzyme rate.

Metal Cofactors – Key Examples

• Zinc (Zn²⁺)
  • Required for: Carbonic anhydrase, Alkaline phosphatase, DNA & RNA polymerases, Alcohol dehydrogenase.
• Iron (Fe²⁺ / Fe³⁺)
  • Present in: Catalase, Peroxidase, Cytochrome oxidase, Non-heme iron oxygenases.
• Copper (Cu²⁺)
  • Found in: Cytochrome oxidase, Superoxide dismutase (Cu/Zn-SOD).
• Magnesium (Mg²⁺)
  • Stabilises ATP/ADP complexes; essential for: Glucose-6-phosphatase, Enolase, Creatine phosphokinase (CPK), Kinases, DNA polymerases.

Terminology Recap

• Metal-activated enzyme: Enzyme whose metal ion is loosely bound; removal usually reversible by dialysis followed by re-addition of metal.
• Metalloenzyme: Enzyme with metal tightly or covalently integrated; removal causes loss of structure/function.
• Prosthetic group: The tightly bound cofactor itself when associated with a metalloenzyme.

Organic Cofactors: Coenzymes

• General properties
  • Non-protein, organic, low-molecular-weight, heat-stable, dialyzable.
  • Act mainly as group-transfer agents (electrons, acyl, amino, methyl, phosphoryl, one-carbon units, etc.).
  • Majority are derived from vitamin B-complex; lack of vitamin → specific metabolic blocks.
• Classification
  • Vitamin-derived coenzymes (B-complex)
  • Thiamine (B₁) → Thiamine pyrophosphate (TPP)
  • Riboflavin (B₂) → FMN, FAD
  • Niacin (B₃) → NAD⁺, NADP⁺
  • Pantothenic acid (B₅) → Coenzyme-A
  • Pyridoxine (B₆) → Pyridoxal-5′-phosphate (PLP)
  • Biotin (B₇) → Biocytin (biotin-lysine)
  • Folic acid (B₉) → Tetrahydrofolate (THF)
  • Cobalamin (B₁₂) → Methylcobalamin (and 5′-deoxyadenosyl-cobalamin)
  • Non-vitamin coenzymes
  • Coenzyme-Q (Ubiquinone/ubiquinol): Lipophilic electron carrier of respiratory chain.
  • Lipoic acid: Acyl-group & redox carrier in pyruvate & α-ketoglutarate dehydrogenase complexes.
  • S-Adenosyl-methionine (SAM): Universal methyl-group donor.

Relationship Between Enzyme & Coenzyme

• Apoenzyme: Inactive protein portion lacking its cofactor.
• Holoenzyme: Catalytically competent complex of apoenzyme + required cofactor(s).
• Interconversion: \text{Apoenzyme} + \text{Cofactor} \rightleftharpoons \text{Holoenzyme}

Enzymes: Fundamental Concepts

• Definition: Biological catalysts (usually proteins; exceptions – ribozymes) that increase reaction rate without altering equilibrium.
• Basic catalytic cycle
  E + S \rightleftharpoons ES \rightarrow E + P
  where E = enzyme, S = substrate, P = product.

Characteristics of Enzyme Action

1. Cannot initiate a reaction but accelerate its rate tremendously (up to 10^{17}-fold).
2. Do not change the overall Gibbs free-energy change \Delta G; hence do not alter the extent or equilibrium position.
3. Exhibit high reaction specificity (substrate, stereochemical, positional).
4. Are regenerated unchanged after reaction.
5. Effective in extremely small amounts.
6. Activity is finely regulated (allosteric control, covalent modification, synthesis/degradation).
7. Influenced by temperature, pH, ionic strength, concentrations of enzyme & substrate, and presence of inhibitors/activators.

Physicochemical Properties

• Protein in nature; generally colloidal & water-soluble.
• Heat labile: denatured at high temperature.
• Exhibit a characteristic isoelectric pH (pI) where solubility is minimal.
• Mostly non-dialyzable due to macromolecular size, except small coenzymes.

Kinetic Parameters & Active-Site Terminology

• Active site: 3-D cleft/pocket formed by specific amino-acid side chains; complements substrate in geometry & chemical properties.
• Substrate: Molecule upon which enzyme acts.
• ES complex: Transient association preceding catalysis; stabilised by non-covalent interactions.
• Michaelis constant (Km): Substrate concentration required to reach half-maximal velocity ( V_{max}/2 ); indicator of enzyme’s affinity for substrate.

Enzyme “Sidekicks” Cartoon Analogy

• Cofactors (metal ions) → “I’m helping!” provide charge stabilisation or redox capability.
• Coenzymes (vitamins) → “I’m a hero!” shuttle functional groups/electrons between multiple enzymes, integrating metabolic networks.

Enzyme Inhibition (Key Concept Mentioned)

• Reversible:
  • Competitive (bind active site; overcome by ↑[S]).
  • Non-competitive (bind allosteric site; ↓Vmax without affecting Km).
• Irreversible: Covalent modification or suicide inhibition; permanently inactivates enzyme.
• Allosteric regulation: Effector binds site distinct from active site; alters conformation & activity (positive or negative).

IUBMB Classification – The Six Major Classes (Mnemonic: OTHLIL)

1. Oxidoreductases
   • Catalyse oxidation-reduction (electron/hydrogen/oxygen transfer).
   • Examples: Oxidase, Peroxidase, Dehydrogenase, Oxygenase, Catalase.
   • Generic reaction: AH2 + B \rightarrow A + BH2
2. Transferases
   • Transfer functional group (excl. electrons) between molecules.
   • Example: Aminotransferases (ALT/AST), Kinases, Methyltransferases.
   • Reaction: A\text{-}X + B \rightarrow A + B\text{-}X
3. Hydrolases
   • Cleave bonds by adding water.
   • Digestive enzymes: Pepsin, Amylase, Lipase, Maltase, Sucrase, Lactase.
   • Reaction: A\text{-}B + H_2O \rightarrow AH + BOH
4. Lyases
   • Remove groups leaving double bonds or add groups to double bonds; do not use water/oxidation.
   • Examples: Decarboxylase, Aldolase, Carbonic anhydrase, Glycogen synthase.
   • Carbonic anhydrase example: H2CO3 \rightarrow H2O + CO2
5. Isomerases
   • Catalyse intramolecular rearrangements to yield isomers.
   • Examples: Phosphoglucose isomerase, Epimerase, Mutase.
   • Glucose-6-P ↔ Fructose-6-P conversion.
6. Ligases (Synthetases)
   • Couple two molecules using energy from ATP hydrolysis.
   • Examples: DNA ligase, Pyruvate carboxylase, Glutamine synthetase.
   • Generic: A + B + ATP \rightarrow AB + ADP + P_i

Extended Examples & Reaction Equations

• ALT (Alanine transaminase)
  \text{Glutamate} + \text{Pyruvate} \xrightarrow{ALT} \text{Alanine} + \alpha\text{-ketoglutarate}
• DNA Ligase
  • Seals nick in DNA backbone by forming phosphodiester bond; critical in replication & repair.
• Lactate dehydrogenase
  \text{Pyruvate} + NADH + H^+ \xrightarrow{LDH} \text{Lactate} + NAD^+

Integrative & Clinical Connections

• Serum ALT/AST levels → hepatic cell damage.
• Creatine phosphokinase (CPK) isoforms serve as myocardial infarction markers; Mg²⁺ is obligatory cofactor.
• Superoxide dismutase (Cu/Zn-SOD) mutations linked to amyotrophic lateral sclerosis (ALS).
• Carbonic anhydrase (Zn²⁺) inhibitors (acetazolamide) treat glaucoma & altitude sickness.

Summary Checklist for Exam Review

• Distinguish cofactor vs. coenzyme vs. prosthetic group vs. metalloenzyme.
• Memorise vitamin → coenzyme table (TPP, FAD, NAD, CoA, PLP, Biocytin, THF, Methyl-B₁₂).
• Recall metal requirements (Zn, Fe, Cu, Mg) and representative enzymes.
• Understand catalytic mechanism sequence E + S \rightleftharpoons ES \rightarrow EP \rightarrow E + P and influence of Km, Vmax.
• Know six IUBMB classes, their reaction type, and textbook examples.
• Be able to classify inhibitors (competitive vs. non-competitive vs. irreversible).
• Recognise factors affecting enzyme activity (temperature optima, pH optima, [substrate], [enzyme]).
• Apply concepts to clinical scenarios (enzyme assays, deficiency diseases, drug targets).