BIOS3010-week 27 specific activity turnover number

Recap of Enzyme Function and Kinetics

  • Enzyme-Substrate Interaction

    • Enzymes bind to substrates to form an enzyme-substrate complex at the active site.

    • This complex proceeds to form products while the enzyme is recycled for further reactions.

  • Kinetic Parameters

    • Rate Constants:

      • k1: Association rate constant of enzyme-substrate complex.

      • k-1: Dissociation rate constant of enzyme-substrate complex.

      • kcat: Slow step rate constant leading to product formation.

    • Vmax: Maximum velocity of the enzyme-catalyzed reaction, dependent on enzyme concentration; when the enzyme is in the enzyme-substrate complex, no free enzyme is available.

      • Vmax increases with higher enzyme concentrations (e.g., doubling enzyme concentration results in double Vmax).

    • Km: Affinity of the enzyme for the substrate; independent of enzyme concentration.

Comparing Enzymes

  • Using Parameters for Comparison

    • Vmax cannot be used for direct enzyme comparisons due to its dependence on enzyme concentration.

    • Km is useful for comparing enzyme affinities as it is independent of concentration.

    • Need for normalized parameters.

  • New Parameters Introduced

    • Specific Activity:

      • Calculated as catalytic activity (mole of substrate converted per second) divided by mass of enzyme (or protein).

      • Unit: mole of substrate / s / g of enzyme.

    • Turnover Number (kcat):

      • Defines the efficiency of an enzyme in converting substrate into product, expressed as the number of substrate molecules converted per enzyme molecule per second.

      • Unit: s^-1.

Specific Activity Calculation

  • Example Calculation

    • Given: Vmax = 60 µM/min, reaction volume = 3 mL, enzyme prep = 10 µL of 1 µg/mL.

    • Convert Vmax to seconds: 60 µM/min equals 1 × 10^-6 moles/L/s.

    • Catalytic Activity Calculation:

      • Vmax × volume = (1 × 10^-6 moles/L/s) × (3 × 10^-3 L) = 3 × 10^-9 moles/s.

    • **Mass of Enzyme Calculation:

      • 10 µL (1 µg/mL) = 10 × 10^-6 L × 1 × 10^-6 g/mL = 10 × 10^-12 g.

    • Specific Activity Calculation:

      • Specific Activity = Catalytic Activity (3 × 10^-9 moles/s) / Mass of Enzyme (10 × 10^-12 g) = 300 mole/s/g.

Impact of Purity on Specific Activity

  • Purity of Enzyme Preparations

    • Higher purity leads to increased specific activity.

    • Specific activity increases as the contaminants decrease, thus yielding a greater measurement per dietary mass.

  • Threshold of Purity:

    • At 100% purity, specific activity will plateau and represent maximum efficiency of the enzyme.

Turnover Number (kcat)

  • Understanding kcat:

    • The number of substrate molecules converted per enzyme molecule per second, highlighting enzyme efficiency.

    • Typically, efficient enzymes range between 100 and 1,000,000 substrate conversions per second.

  • Independence from Concentration:

    • Turnover number is independent of enzyme concentration, making it suitable for comparing enzyme efficiencies across various preparations.

Key Takeaways

  • Specific activity and turnover number are essential for comparing enzyme efficiency.

  • These metrics are independent of the concentration of the enzyme, allowing for standardized comparisons.

  • Understanding these principles is critical for assessing enzyme functions and capabilities.