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.