EF

Enzymes, Part 2

Lecture Overview

  • Date: March 6th, 2025

  • Reading Material: Biochemistry: Concepts and Connections, Chapter 8, Pages 251-277

  • Focus on enzyme-related topics, specifically concerning acetylcholinesterase inhibitors and enzyme inhibition types.

Acetylcholinesterase Inhibitors

Relevant Diseases

  • Toxins and enzyme inhibition serve important therapeutic roles.

  • Exploration of how enzyme inhibition can act as a target for therapies.

Basic Kinetics of Enzymes

  • Introduction to the Michaelis-Menten equation for understanding enzyme kinetics.

  • Discussion on different types of enzyme inhibition:

    • Competitive inhibitors

    • Uncompetitive inhibitors

    • Non-competitive inhibitors

Acetylcholine (ACh)

Definition and Function

  • Acetylcholine is an excitatory neurotransmitter mainly present in the peripheral nervous system with lesser amounts in the central nervous system.

  • Functions primarily at the neuromuscular junction to enable communication between nervous system and muscle cells by stimulating muscle cell sodium channels.

The Neuromuscular Junction (NMJ)

Components of NMJ

  • Three main components:

    1. Nerve cell (motor neuron)

    2. Muscle fiber

    3. Schwann cell

Acetylcholinesterase – The Enzyme

Function and Importance

  • Type: Hydrolase (breaks a bond using water).

  • It hydrolyzes ACh into acetate and choline.

  • Necessary for regulation of ACh levels at NMJ; excessive ACh can lead to symptoms like sweating, nausea, diarrhea, and respiratory issues.

Toxins and Enzyme Inhibition

Sarin Gas Mechanism

  • An irreversible inhibitor that binds covalently to acetylcholinesterase, rendering it ineffective.

  • Known for its use in lethal attacks (e.g., the 1995 Tokyo subway incident).

Therapeutic Use of ACh Inhibitors

Alzheimer’s Disease

  • ACh esterase inhibitors temporarily improve memory and function in Alzheimer's.

  • They increase ACh levels in the brain areas affected by the disease, although not a cure and with side effects.

Understanding Basic Enzyme Kinetics

Michaelis-Menten Equation

  • Describes the reaction dynamics:

    • E + S ⇌ ES → P + E

    • Rate of reaction can be defined as: v = kcat[ES]

Steady State of Reactions

  • A steady state forms between bound (ES complex) and free enzyme until substrate is mainly consumed.

  • Defined mathematically: K_m = K_{-1} / K_{1} [S] / [E].

Kinetics Derivation

Detailed Steps

  • The rate constants are named:

  • K1 (binding to ES),

  • K-1 (unbinding back to E + S),

  • Kcat (product release).

  • Establishes relationship: K1 = K-1 + Kcat to derive K_m, defined as the substrate concentration at half maximum velocity.

Km vs Kcat

Definitions

  • K_m: Substrate concentration for half-max velocity; reflects enzyme affinity. Lower K_m indicates higher affinity.

  • Kcat: Represents rate constant upon substrate binding; provides insights into enzyme catalytic speed under saturation.

Lineweaver-Burk Plots (L-B Plots)

Purpose and Interpretation

  • Useful for visualizing first-order reactions in a linear manner.

  • Critical parameters include:

    • Slope of the line = K_m / Vmax

    • Changes in Km indicate alterations in substrate binding affinity.

Types of Enzyme Inhibitors

General Classes

  • Reversible Inhibitors: Bind non-covalently and are generally temporary.

  • Irreversible Inhibitors: Form covalent bonds and permanently deactivate enzymes.

Competitive Inhibitors

  • Bind to active site and prevent substrate binding, decreasing reaction rate.

Uncompetitive Inhibitors

  • Bind to ES complex, reducing both Km and Vmax, which is different from non-competitive inhibition.

Non-competitive Inhibitors

  • Bind to both free enzyme and enzyme-substrate complex.

Regulation of Enzyme Activity

Need for Regulation

  • Enzymes need regulation to control product formation rates; mechanisms include:

    • Substrate-level control

    • Feedback control

Substrate Control Examples

  • Excess substrate slows product formation; excess product acts as an inhibitor (e.g., hexokinase and glucose-6-phosphate).

Feedback Control Overview

  • Product binds to initial enzyme in a metabolic pathway to regulate production effectively.

Covalent Modifications

Regulation Techniques

  • Not as common, but essential (e.g., phosphorylation of proteins).

  • Example: Activation of pancreatic enzymes from zymogens to active forms in the intestine.

Summary of Key Concepts

  • The function of acetylcholine and acetylcholinesterase.

  • Understanding the Michaelis-Menten equation and Lineweaver-Burk plots.

  • Different types of enzyme inhibitors and their graphical interpretation.

  • Regulation of enzyme activity through products.