Enzyme kinetics

Enzyme Kinetics Workshop

Introduction

  • Instructor: Rebecca Stover

  • Experience: Over a dozen years of teaching for Kaplan

  • Focus: Enzymes in biology and biochemistry, emphasizing the mathematical aspects of enzyme kinetics.

Understanding Enzymes and Catalysts

  • Enzyme vs Catalyst:
      - A catalyst is a substance that speeds up a chemical reaction without being consumed in the process.
      - Enzymes are biological catalysts, specifically defined in the context of biological reactions.

Activation Energy and Reaction Coordinate

  • **Reaction Coordinate Graph:
      - Reactants on the left, products on the right.
      - Green line: High transition energy barrier (no catalyst).
      - Red line: Lower transition energy barrier (with catalyst).
      - *Role of Catalysts:*
          - Lower the activation energy needed for reactions.
          - Facilitates both forward and reverse reactions.
          - Example: Glycolysis and gluconeogenesis share enzymes to convert glucose in both directions.

Delta G and Equilibrium Constant

  • The overall change in Gibbs free energy (G) remains unchanged by catalysts.

  • The equilibrium constant (004K_eq) is unaffected, indicating the favorability of the reaction remains the same.

Characteristics of Catalysts

  • **Key Points about Catalysts/Enzymes:
      - They *do not alter thermodynamics*.
      - They *speed up both forward and reverse reactions*.
      - They must *interact* with substrates to catalyze reactions.
      - Enzymes must regenerate after the reaction, implying they do chemically interact with substrates.

Example Question on Catalysts

  • **Select Incorrect Statement about Catalysts:
      - (A) Doesn't change thermodynamics (True)
      - (B) Speeds up both reactions (True)
      - (C) Gives molecular weight info (True but misleading context)
      - (D) Doesn't interact with substrates (False, must interact)

  • Correct Answer: (C)

Enzyme Models

  • Lock and Key Model:
      - Outdated, suggests enzymes and substrates fit perfectly without changes.

  • Induced Fit Model:
      - Enzyme and substrate change shape upon interaction, fitting each other.
      - Enzymes match the transition state of substrates, indicating dynamic interactions.

Regulation of Enzymes

  • Active Site: Where substrate binds to the enzyme.

  • Allosteric Site: Separate site for regulating activity, can activate or inhibit enzyme activity.

  • Inhibitors: Molecules that reduce enzyme activity by binding to the allosteric site, changing the enzyme shape so it no longer fits the substrate.

Cofactors and Coenzymes

  • Cofactors: Typically inorganic substances (e.g., magnesium, zinc).

  • Coenzymes: Organic molecules, often derived from vitamins.

  • Example: Magnesium as a cofactor helps in the formation of ATP from ADP and inorganic phosphate by shielding negative charges.

Classifications of Enzymes

  • Mnemonic: LIL HOT
      - Ligases: Join molecules, require energy (e.g., ATP).
      - Isomerases: Rearrange isomers (e.g., aconitase).
      - Lyases: Break apart compounds without water.
      - Hydrolases: Break apart compounds with water (e.g., peptidases).
      - Oxidoreductases: Catalyze redox reactions (e.g., dehydrogenases).
      - Transferases: Transfer functional groups (e.g., kinases).

Enzyme Activity Considerations

  • Enzymes generally do not work alone; they require cofactors or coenzymes for optimum activity.

  • Optimal conditions for activity include appropriate pH and temperature.

Example Question on Enzyme Characteristics

  • Identify statements regarding enzyme activity. Use elimination strategies based on known enzyme principles.

Enzyme Kinetics

  • Enzyme Velocity: Refers to the reaction rate of an enzyme.

  • Michaelis-Menten Model:
      - Enzymes have maximum velocity (004V_max) under saturation.
      - 004K_m: Substrate concentration at half of V_max (indicates affinity).
      - V_max corresponds to maximum enzyme capacity when all active sites are saturated.

  • Graphing Enzyme Activity:
      - Plotting substrate concentration on the x-axis versus reaction velocity on the y-axis yields a characteristic hyperbolic curve.
      - As substrate concentration increases, the reaction rate increases until V_max is reached, indicating saturation of enzymatic activity.
      - At half of V_max, the substrate concentration is equal to K_m.

Relationship between K_m & Enzyme Affinity

  • Inverse Relationship:
      - A higher K_m indicates lower affinity between enzyme and substrate, while a lower K_m indicates higher affinity.

Enzyme Inhibition and Drug Design Example (Acyclovir)

  • Acyclovir is a synthetic purine nucleotide analog that mimics natural purines in DNA polymerase.

  • Binding affinity of acyclovir can lead to increased effectiveness against viruses due to differing affinities with host enzymes.

  • K_m Inference:
      - Acyclovir binds with higher affinity to viral enzymes, resulting in a lower K_m value.

Lineweaver-Burk Plot

  • A double reciprocal plot:
      - Allows more accurate determination of K_m and V_max by converting the Michaelis-Menten equation into a straight line, y = mx + b, where:
        - y-axis: 1/V
        - x-axis: 1/[S]
        - Slope: K_m/V_max
        - y-intercept: 1/V_max
        - x-intercept: -1/K_m

  • Comparison of different enzymes can be made easily by looking at the intercepts.

  • Movement of the intercepts provides insight into how the inhibitor or activation affects enzyme activity.

Conclusion

  • Summary of important enzyme concepts: Structure, function, kinetics, classifications, Michaelis constant K_m, and V_max.

  • Invitation to join a follow-up workshop on enzyme inhibition.

  • Encouragement and wishing success on exams (e.g., MCAT).