Enzyme Kinetics Lecture 2 Notes

Enzyme Kinetics Lecture 2 Notes

Learning Outcomes

  • Lecture 1

    • LO 1: Understand the role of enzymes as biological catalysts.

    • LO 2: Explain the factors affecting enzyme activity.

  • Lecture 2

    • LO 3: Describe the basic principles of enzyme kinetics.

    • LO 4: Explain the different types of enzyme inhibition.

    • LO 5: Understand the basic mechanism of action of allosteric enzymes.

Enzyme Kinetics Overview (LO 3-5)

  • Revision of Kinetics

    • Zero Order Kinetics

    • First Order Kinetics

    • Michaelis-Menten Theory:

    • Understanding of parameters Km and Vmax.

    • Lineweaver-Burk Plot:

    • A graphical representation to determine Vmax and Km.

    • Enzyme Inhibition:

    • Effects on Vmax and Km.

    • Types of Inhibitors:

    • Irreversible

    • Reversible Competitive

    • Reversible Non-competitive

    • Allosteric Enzymes

Kinetics Types

  • Zero Order Kinetics:

    • Saturation kinetics with a constant rate of reaction.

    • Linear change in enzyme concentration irrespective of substrate concentration.

  • First Order Kinetics:

    • Rate dependent on the concentration of the substrate.

    • Half-Life (t1/2):

    • Time required to decrease or increase the substrate concentration by 50% (e.g., t1/2 = 1 hour).

Steps in Determining Enzyme Kinetics

  1. Calculate the Molar Extinction Coefficient:

    • Prepare a standard curve of absorbance against known concentrations of substrate/product.

  2. Enzyme Kinetic Study:

    • Plot change of absorbance over time for the substrate/product at different concentrations.

  3. Calculate the Rate of Reaction:

    • Using the Michaelis-Menten model.

  4. Calculate Vmax and Km:

    • Using the Lineweaver-Burk Plot.

Molar Extinction Coefficient and Beer’s Law

  • Beer’s Law explains the relationship between absorbance (A) and concentration (c):

    • A=extεLCA = ext{ε} L C

    • A: Absorbance

    • ε: Molar absorptivity constant (depends on the comb. of solute/solvent/λ)

    • L: Length of path traveled by light

    • C: Concentration of solution (mol/L)

  • Relation of absorbance with concentration is linear, allowing for calculations of concentrations from absorbance measurements.

Key Concepts in Enzyme Kinetics

  • Enzyme interacts with substrate at the active site to form the enzyme-substrate complex (ES), which decomposes to form product (P) and regenerate free enzyme (E).

  • The rate of reaction (V) is the number of moles of product formed per time, about extμmolL−1s−1ext{μmol L}^{-1} s^{-1}.

  • Initial Rate (V0) is determined by substrate concentration ([S]):

    • At low [S], V is linearly proportional to [S].

    • At high [S], V is independent of [S] as enzymes become saturated.

Michaelis-Menten Equation

  • The Michaelis-Menten equation is given by:

    • V0=racVmaximes[S]Km+[S]V_0 = rac{V_{max} imes [S]}{K_m + [S]}

    • V0: Initial velocity of the reaction

    • Vmax: Maximal rate of reaction

    • [S]: Concentration of substrate

    • Km: Michaelis-Menten constant; substrate concentration at which velocity is half Vmax.

  • Km Interpretation:

    • A low Km indicates high affinity between the enzyme and substrate, while a high Km indicates lower affinity.

Lineweaver-Burk Plot

  • This plot helps to linearize the Michaelis-Menten equation for easier extrapolation:

    • Plot rac1[S]rac{1}{[S]} vs. rac1V0rac{1}{V_0}.

    • X-intercept: −rac1Km- rac{1}{K_m}

    • Y-intercept: rac1Vmaxrac{1}{V_{max}}

  • This approach allows for visual comparison of kinetic parameters under different conditions (with or without inhibitors).

Types of Enzyme Inhibition

  • Inhibitors: Substances that bind to enzymes and interfere with their activity. Types include:

    • Irreversible Inhibition:

    • Inhibitor binds irreversibly, usually creating covalent bonds.

    • Permanent reduction in enzyme activity.

    • Reversible Inhibition:

    • Can be competitive, non-competitive, or uncompetitive.

    • Competitive Inhibition:

    • Inhibitor resembles substrate, competes for active site.

    • Raises Km while Vmax remains unchanged.

    • Non-competitive Inhibition:

    • Inhibitor binds to an allosteric site, affecting enzyme activity without affecting substrate binding.

    • Lowers Vmax without changing Km.

    • Uncompetitive Inhibition:

    • Inhibitor binds only to ES complex.

    • Lowers both Km and Vmax in equal proportions.

Allosteric Enzymes

  • Enzymes with allosteric sites that modulate activity:

    • Allosteric behavior results in a sigmoidal curve of the reaction rate as a function of [S].

    • Positive modulation enhances activity, while negative modulation inhibits activity.

  • Feedback Inhibition: A mechanism where end products inhibit the action of the regulatory enzyme in a pathway.

  • Different conformational changes occur due to ligand binding at allosteric sites.

Summary of Enzyme Kinetics and Inhibition Principles

  • The Michaelis-Menten model describes the kinetics of simple enzymes, while allosteric enzymes display cooperative behavior and are often regulated by feedback inhibition.

  • Key parameters: Km and Vmax characterize enzyme activity and substrate affinity.

  • Different types of inhibition influence enzyme kinetics through various mechanisms affecting either substrate binding or catalysis.

Learning Check Example

  • For kinetic experiments performed with increasing concentrations of irreversible inhibitor, which outcome can be expected?

    • (A) Km would increase and Vmax would decrease.

    • (B) Km would decrease and Vmax would increase.

    • (C) Km and Vmax would both remain the same.

    • (D) Km would remain the same and Vmax would decrease.

    • (E) None of the above.