Wk 3&4 lecture 2 Enzyme Kinetics

Enzyme Overview

Definition of Enzymes: Biological catalysts that speed up chemical reactions without being consumed in the process.
Specificity: Enzymes bind specifically to their substrates, which allows for highly regulated metabolic processes.
Thermodynamics: Enzymes lower the activation energy required for a reaction without changing the free energy of the overall reaction.

Definition of Kinetics: The study of reaction rates and how they are affected by various factors.
Metabolic Pathways: The action of enzymes is crucial for the flow and movement of molecules; enzymes serve as facilitators, similar to traffic controllers.

Michaelis-Menten Kinetics: Developed by Leonor Michaelis and Maude Menten to mathematically describe enzyme kinetics.
- Contribution of Maud Menten: One of the first women to earn a medical doctorate in Canada, she collaborated on this fundamental work in enzyme kinetics while pursuing her PhD.

Basic Reaction Model: Enzyme (E) + Substrate (S) ⇌ Enzyme-Substrate Complex (ES) → Product (P).
Reaction Rate (Velocity): The rate at which the substrate is converted to product can be described in two ways:
- Amount of substrate (S) that disappears in a given time.
- Amount of product (P) that is formed in that same time.
Example Analogy: Using Play-Doh to visually represent the transformation of substrates into products; as substrate is converted, the same amount of product is formed over time.

Measurement of Velocity (v): Expressed as: v = -d[S]/dt (substrate loss) = d[P]/dt (product formation).
Kinetics of Enzyme Action: The velocity of enzyme action depends on substrate concentration and is fastest at higher concentrations—akin to a turnstile at a busy station.
- Velocity is proportional to substrate concentration (V = k[S]).

First-Order Reactions: For a reaction with a single substrate S, the rate is directly proportional to [S].
- Equation: V = k[S].
Second-Order Reactions: Involves two different substrates (A and B), whereby velocity depends on the concentrations of both.
- Equation: V = k[A][B].
Pseudo First-Order Reactions: Occur when one substrate concentration is high enough that the rate appears dependent on only the other substrate.

Testing Enzyme Activity: Using varying concentrations of substrate to assess reaction rates and product formation visually, often tracking color development (e.g., a color change from colorless to blue).
Effects of Substrate Concentration: Initial velocity (V0) increases with substrate concentration due to formation of the enzyme-substrate complex reaching a saturation point. Hyperbolic relationship between V0 and substrate concentration.

Vmax: Maximum reaction velocity occurs when all enzyme active sites are saturated with substrate.
Hyperbolic Curve: Plotting initial velocity against substrate concentration results in a hyperbolic curve approaching Vmax.
Steady State: The concentration of the enzyme-substrate complex (ES) remains relatively constant during the reaction after the initial phase.

Formula: V0 = Vmax[S] / (Km + [S]) where:
- Km (Michaelis constant) = [S] at which V0 = Vmax/2.
- Vmax is the rate at which the enzyme is saturated with substrate.

Kilometers (Km): A measure of enzyme efficiency; it indicates the substrate concentration required for the enzyme to achieve half-maximal velocity.
Turnover Number (kcat): Number of substrate molecules converted to product per second when the enzyme is saturated.

Double Reciprocal Plot: 1/V0 = (Km/Vmax)(1/[S]) + 1/Vmax, turning the hyperbola into a straight line, allowing easier determination of Km and Vmax.
Graphical Interpretation: The y-intercept represents 1/Vmax while the x-intercept represents -1/Km.

Ratio of kcat / Km: Indicates how efficiently the enzyme converts substrate to product.
- A higher ratio means better efficiency at lower substrate concentrations.
Contextual Comparison: Different enzymes exhibit varying Km and kcat values influencing their catalytic efficiency.
Impact of Environmental Factors: pH, temperature, and ionic strength can influence Km and kcat, respectively, and thus enzyme activity.

Enzyme kinetics are fundamental for understanding metabolic processes.
The Michaelis-Menten model provides a basic framework for enzyme activity analysis.
Vmax and Km are critical parameters for characterizing enzyme function and efficiency.
Understanding these principles is crucial for the application of metabolic studies and enzyme-targeted therapies in health and disease.