Enzymes – Biosciences I (CH4006)

Introduction to Enzymes

• Definition & Nature

  • Enzymes are globular proteins that act as biological catalysts, i.e. they speed up biochemical reactions without being consumed.
  • General catalytic scheme: (\text{Substrate} + \text{Enzyme} \;\rightarrow\; ES \;\rightarrow\; \text{Enzyme} + \text{Product})
  • Produced under genetic control (gene transcription → translation → enzyme), therefore cellular regulation of gene expression directly regulates enzyme abundance.

• Learning Aims (module CH4006 – Biosciences I)

  • Explain enzyme structure/function and mechanism of catalysis.
  • Describe requirements for enzyme activity, factors influencing it, and strategies for regulation.
  • Clarify the role of enzymes inside biochemical/metabolic pathways, including feedback inhibition and energetic coupling.

Types of Biochemical Reactions Catalysed

• Anabolic (biosynthetic) reactions
  • Form bonds, consume energy, build complex molecules.
  • Example : protein synthesis from amino acids.
• Catabolic (degradative) reactions
  • Break bonds, release energy, generate simpler molecules.
  • Example : cellular respiration (glucose + \text{O}2 → \text{CO}2 + \text{H}_2\text{O} + energy).
• Typical reaction pairings in the cell
  • Dehydration synthesis (condensation) vs hydrolysis.
  • Both directions require enzymes to overcome kinetic barriers.

Energetics: Why Enzymes Are Needed

• Reactions possess an activation energy (E_A) barrier even when overall free-energy change \Delta G is favourable.
• Enzymes lower E_A but do not alter \Delta G:
  • Graphically: two curves (with/without enzyme). The peak for the catalysed pathway is lower while initial and final free energies remain unchanged.
• Consequence : dramatically faster rates (often \ge10^{6}-fold acceleration) at physiological temperature, pH, salinity.

Properties & Characteristics

• Catalytic power – one molecule can process thousands of substrates per second.
• Specificity – each enzyme typically recognises one (or a narrow set of) substrate(s) via precise chemical fit at the active site.
• Reversibility – catalysts speed both forward and reverse directions equally; net direction depends on \Delta G and reactant/product concentrations.
• Sensitivity to cellular conditions – protein conformation (hence activity) is affected by temperature, pH, ionic strength, etc.

Models of Enzyme–Substrate Interaction

• Lock-and-Key model
  • Historical, simplistic. Substrate shape exactly complements rigid active site; once product released, enzyme unchanged.
• Induced-Fit model (modern view)
  • Active site is flexible; initial binding triggers conformational change → tighter fit, optimally positions catalytic residues.
  • Enhances transition-state stabilisation.

How Active Sites Lower Activation Energy

• Transition-state stabilisation – binding lowers energy of the transition state relative to substrates.
• Orientation – brings two substrates together in correct spatial arrangement.
• Induced strain – strains specific bonds (makes them less stable) promoting breakage/formation.
• Micro-environment – provides optimal pH, polarity, or electrostatic landscape within active site (e.g. shielding from water).

Catalytic Cycle (stepwise)

1. Substrates diffuse into active site → form enzyme–substrate (ES) complex through weak interactions (H-bonds, electrostatics, Van der Waals).
2. Active site undergoes adjustments (induced fit).
3. Chemical reaction occurs; E_A lowered, substrates converted → products.
4. Products have lower affinity → released.
5. Enzyme active site re-opens, ready for next catalytic event (enzyme not consumed).

Nomenclature Conventions

• Enzymes named after substrate/reaction + suffix “-ase”.
  • Sucrase – hydrolyses sucrose.
  • Proteases – hydrolyse peptide bonds in proteins.
  • Lipases – hydrolyse triglycerides/lipids.

Factors Affecting Enzyme Activity

• Temperature
  • Reaction rate rises with temperature due to increased kinetic energy up to an optimum (often 35–40 °C for human enzymes); beyond that, denaturation drops activity sharply.
• pH
  • Each enzyme has an optimum pH reflecting its environment (e.g. pepsin ≈ pH 2 in stomach; trypsin ≈ pH 8 in intestine).
  • Deviations alter ionisation of active-site residues, upsetting binding/catalysis.
• Substrate concentration
  • At low [S], rate ∝ [S]; as [S] increases active sites saturate → maximum velocity V_{\text{max}} reached.
• Cofactors & Coenzymes
  • Non-protein helpers essential for some enzymes.
  • Inorganic ions (Mg²⁺, Zn²⁺, Fe²⁺).
  • Coenzymes (organic, often vitamins: NAD⁺, FAD, CoA).
  • Prosthetic groups – tightly bound cofactors (e.g. haem).

Enzyme Kinetics (Michaelis–Menten)

• Rate equation: V = \dfrac{V{\text{max}}[S]}{Km + [S]}
  • V = initial rate; V{\text{max}} = maximal rate at saturation; Km = [S] at \tfrac12 V{\text{max}} (indicator of substrate affinity: lower Km ⇒ higher affinity).
• Graphical features
  • Hyperbolic approach to asymptote V_{\text{max}} as [S] → ∞.

Regulation & Control Strategies

• Gene-level control – transcription/translation of enzyme gene turned on/off.
• Post-translational modification – phosphorylation (adds \text{PO}_4^{3−}), methylation, acetylation alter activity or localisation.
• Inhibitors
  • Irreversible – covalently attach, permanently inactivate (e.g. nerve gas on acetylcholinesterase).
  • Competitive – resemble substrate; bind active site; raise apparent K_m; overcome by high [S].
  • Non-competitive – bind allosteric site; decrease V{\text{max}} without changing Km.
• Activators – small molecules or ions that increase activity (e.g. Ca²⁺ for protein kinase C).

Enzymes in Metabolic Pathways

• Metabolic pathway = ordered sequence of enzyme-catalysed steps converting a starting molecule → product.
  • Each step’s product becomes next enzyme’s substrate.
• Enables efficiency & regulation:
  • Coupling exergonic and endergonic reactions (e.g. ATP hydrolysis powers biosynthesis).
  • Spatial organisation: enzymes often form complexes or scaffold on membranes (e.g. glycolysis enzymes loosely associated in cytosol; ETC embedded in inner mitochondrial membrane).

Example Pathway Illustration

• Starting molecule A \xrightarrow{\text{Enz 1}} B \xrightarrow{\text{Enz 2}} C \xrightarrow{\text{Enz 3}} D.
• Loss of any enzyme blocks the chain → metabolic disease or alternative routing.

Feedback Inhibition (End-Product Inhibition)

• A classic negative-feedback regulatory scheme ensuring resources aren’t wasted.
• End product binds allosteric site on an early enzyme → conformational change → pathway slows/stops.
• Dynamic: as product concentration falls, inhibitor dissociates, pathway resumes.

Specific Example – Isoleucine Biosynthesis

• Pathway: threonine → … → isoleucine (multi-step)
• Isoleucine (end product) acts as non-competitive inhibitor of enzyme 1 (threonine deaminase).
  • High [isoleucine] ⇒ binding frequency > threonine binding ⇒ pathway suppressed.
  • When demand increases (levels drop), inhibition relieved, flux restarts.

Key Takeaways & Broader Connections

• Enzymes make life’s chemistry possible under mild conditions compatible with delicate biomolecules.
• They integrate with cellular physiology via genetic regulation, post-translational modifications, and metabolic feedback loops.
• Understanding enzyme kinetics and regulation underpins biotechnology, pharmacology (designing inhibitors), and clinical diagnostics (enzyme deficiencies, inborn errors of metabolism).