Enzyme Kinetics and Inhibition Reviewer

Enzyme Kinetics and Inhibition Overview

Enzyme Activity and Rate Equations

  • Reaction Progress:

    • Substrate concentration decreases over time.
    • Product concentration increases over time.
    • Example: Triose Phosphate Isomerase
    • Converts glyceraldehyde-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP).
    • The concentration of substrate and product changes at identical rates.

  • Velocity of Reaction:

    • Defined as the rate of substrate depletion or product formation.
    • Directly proportional to enzyme concentration: More enzyme = more product.
    • Ford Factory Analogy:
    • More machines working leads to increased production output.

  • Effect of Substrate Concentration on Velocity:

    • The graph exhibits a hyperbolic shape.
    • At low substrate concentrations, velocity increases rapidly.
    • At high substrate concentrations, enzymes become saturated, reaching Vmax (Maximum Velocity).
    • At Vmax, all enzyme molecules are occupied with substrate.
Rate Constants and Reaction Orders

  • First-order Reaction:

    • Involves a single reactant: A → B.
    • Velocity (V) = k[A].

  • Second-order Reaction:

    • Involves two reactants: A + B → C.
    • Velocity (V) = k[A][B].
    • Example: Enzyme-substrate binding.
Michaelis-Menten Equation

  • Developers: Leonor Michaelis & Maud Menten (1913).

  • Reaction Scheme:

    • E + S ⇌ ES → E + P
    • Rate Constants:
    • k1 = Formation of ES.
    • k-1 = Dissociation of ES.
    • k2 = Conversion of ES to product (irreversible).

  • Key Assumptions:

    • Single substrate and product.
    • k2 is irreversible.
    • Steady-State Assumption:
    • Concentration of ES remains constant over time.
    • Formation rate of ES = Depletion rate of ES.
Key Parameters of the Michaelis-Menten Equation
  • Vmax: Maximum reaction velocity (all enzymes bound to substrate).
  • Km (Michaelis Constant):
    • The substrate concentration at which reaction velocity is half of Vmax.
    • Low Km = High affinity (enzyme binds substrate tightly).
Graph Interpretation
  • Hyperbolic Curve:
    • Plotted velocity (v) vs. substrate concentration ([S]).
    • Key values:
    • Vmax: Maximum achievable velocity.
    • Km: Substrate concentration at 1/2 Vmax.
Significance of Km and Vmax
  • Km Interpretation:
    • Low Km → High affinity (substrate binds tightly).
    • High Km → Low affinity (substrate binds weakly).
  • Vmax Interpretation:
    • Indicates catalytic efficiency when all enzymes are saturated.
    • Higher Vmax correlates to more product formed per unit time.
Experimental Measurements of Enzyme Kinetics
  • Determining Km and Vmax:
    • Through plotting reaction velocity vs. substrate concentration.
    • Lineweaver-Burk plot (double reciprocal plot) provides Km and Vmax values.
Enzyme Kinetics and Inhibition
  • Kinetics Variants:
    • Some enzymes, such as allosteric enzymes, exhibit sigmoidal kinetics rather than hyperbolic.
Types of Kinetic Behaviors
  1. Michaelis-Menten Model:
    • Plot of initial velocity (v0) vs. substrate concentration ([S]) illustrates Michaelis-Menten kinetics.
  2. Vmax and Turnover Number (kcat):
    • Vmax is the maximum reaction rate at enzyme saturation.
    • kcat represents substrate molecules converted to product per enzyme unit time.
    • Example values:
      • Carbonic Anhydrase: kcat = 1 million s⁻¹
      • Lysozyme: kcat = 0.5 s⁻¹
  3. Km as a Measure of Affinity:
    • A collection of rate constants indicates ES complex dynamics.
    • Lower Km = Higher substrate affinity.
    • Example concentrations:
      • Carbonic Anhydrase: Km = 8000 µM
      • Lysozyme: Km = 6 µM
  4. Catalytic Efficiency (kcat/Km):
    • High efficiency from high kcat and low Km.
  5. Lineweaver-Burk Equation:
    • Rearrangement of Michaelis-Menten for clearer Km and Vmax determination.
    • Advantages: More accurate than the hyperbolic plot.
    • Disadvantages: Uneven data points affect accuracy at low [S].
  6. Non-Michaelis-Menten Kinetics:
    • Examples: Multi-substrate reactions and allosteric regulation.
Enzyme Inhibition Types
  1. Irreversible Inhibition:
    • Permanently inactivates the enzyme through irreversible covalent modifications.
    • Example: Kyrion Inactivation (serine residue modified).
  2. Suicide Substrates:
    • Bind like normal substrates but stop the reaction irreversibly once they enter the active site.
    • Example: Thymidylate Synthase Inhibition with fluoridated dUMP.
  3. Reversible Inhibition:
    • Binds to the enzyme in a reversible manner, affecting kinetics (Kcat, Km).
    • Competitive Inhibition: Inhibitor resembles substrate and competes for the active site.
    • Example: Malonate inhibits succinate dehydrogenase.
      • Key Characteristics: Vmax remains unchanged, Km increases.
Effects of Competitive Inhibition on Kinetics
  • Vmax remains unchanged; enzyme can still function if sufficiently substrate is present.
  • Km increases, leading to decreased affinity due to competition.
  • Graphical representation: Hyperbolic plot shifts right on the Km axis, Lineweaver-Burk plot shows varying intercepts.
  • Inhibition Factor (α):
    • Defined as α = 1 + [I]/Ki; impacts on inhibition concentration and affinity.
  • Drug Development Insight: Lower Ki values indicate more potent inhibitors, and transition state analogs are generally more effective as competitive inhibitors.
Conclusion
  • The understanding of enzyme kinetics is crucial for regulation and inhibition in biochemical processes. Mastery of the concepts of Vmax, Km, and inhibition types is essential for deeper biochemical applications.