Enzymes: Michealis- Menten Kinetics and Inhibition

Lecture Overview

  • Title: Molecular Biology & Biochemistry Lecture 14: Michaelis Menten Kinetics & Enzyme Regulation

  • Instructor: Dr. Emily Flack

Learning Outcomes

  • Ability to define key determinants of enzyme effectiveness:

    • Km (Michaelis constant)

    • Vmax (maximum reaction velocity)

    • Kcat (turnover number)

    • Specificity

    • Rate enhancement

  • Explain progress in enzyme-catalyzed reactions in terms of energy change.

  • Describe substrate binding and transition state formation in enzyme catalysis.

  • Explain how effectors regulate enzyme activity and distinguish types of inhibition.

  • Discuss strategies enzymes use for catalysis.

Enzyme Catalysis

  • Function of Enzymes: Catalysts that lower activation energy barriers.

    • Lower activation energy increases the number of reactants with sufficient energy, thus speeding up the reaction rate.

  • Energy Profile:

    • Free Energy (G): Indicates the energy change during the reaction.

    • Activation Energy (Ea): The energy required to initiate a reaction, reduced by enzymes.

Enzyme-Substrate Complex

  • Diagram Analysis: Shows the states in the reaction:

    • E + S → ES (Enzyme-Substrate Complex) → EP (Enzyme-Product Complex)

    • Represents reaction kinetics with constants (k1, k2, k-1).

    • Free energy changes for different stages of the reaction.

Models of Enzyme Action

1. Lock-and-Key Model

  • Early concept of enzyme-substrate specificity based on shape and charge matching.

  • Active site recognized substrate but considered a passive fit.

2. Induced Fit Model

  • Active site undergoes conformational changes upon substrate binding.

  • Strain induced by substrate stabilizes the transition state, promoting reaction progression.

Transition State Concept

  • Definition: The highest energy state within the reaction pathway.

  • Key role of enzymes is to stabilize this state, reducing energy required and increasing reaction speed.

Enzyme Efficiency Parameters

  • Km: Substrate concentration required to reach half of Vmax; varies among enzymes.

  • Vmax: Maximum turnover rate of an enzyme at saturation.

  • Kcat: Turnover number indicating substrate conversion rate per enzyme.

    • Example: Catalase's Kcat is 40 million, indicating its rapid action on H2O2.

Enzyme Specificity and Activity

  • Enzyme activity is highly reliant on environmental substrate concentration.

  • Km reflects biological availability, usually within millimolar to micromolar range.

  • Vmax can change with varying enzyme concentrations; it reveals enzyme efficiency under substrate saturation.

Regulation of Enzyme Activity

Types of Inhibition:

  1. Competitive Inhibition:

    • Inhibitor resembles substrate and competes for the active site.

    • Example: Tamiflu targets neuraminidase in influenza.

  2. Non-competitive Inhibition:

    • Inhibitor binds to a different site (allosteric site), changing enzyme conformation without competing for the active site.

    • Example: Ibuprofen inhibits cyclooxygenase (Cox2).

Differentiating Inhibition Effects

  • Graphs Simulating Kinetic Inhibition:

    • Lineweaver-Burk plots help derive Vmax and Km values amid inhibition scenarios.

    • Competitive inhibition increases Km, with Vmax remaining constant.

    • Non-competitive inhibition lowers Vmax while Km remains unchanged.

Feedback Regulation

  • Example: CTP inhibits aspartate transcarbamoylase (ATCase) to regulate pyrimidine biosynthesis, ensuring balanced production.

  • Negative Feedback: End products may inhibit upstream enzymes in metabolic pathways.

Cooperative Binding in Allosteric Enzymes

  • Cooperative enzymes exhibit a sigmoidal kinetic curve, indicating binding at one active site influences others.

  • As substrate concentration increases, the enzyme transitions from T (inactive) to R (active) state, enhancing activity by decreasing Km.

Summary of Enzyme Kinetics

  • Interaction Dynamics: Complex spatial and energetic aspects govern enzyme-substrate interactions, influencing efficiency and regulation.

  • Importance of Models: Understanding these mechanisms explains enzyme specificity, regulation, and biological significance in metabolic pathways.