Chapter 6 Pt. 2 (February 26,2025)

Overview of Enzyme Kinetics

• Enzymes interact with substrates to produce products quickly, allowing biological reactions to occur efficiently.

• The fundamental equation represents an enzyme (E) plus a substrate (S) forming an enzyme-substrate complex (ES), which then dissociates to form product (P) while regenerating the enzyme:E + S ⇌ ES → E + P

• Enzyme is not consumed during the reaction, allowing it to act on multiple substrate molecules.

Understanding Enzyme Saturation Kinetics

• Focus: Enzyme kinetics, a classic area of study in biochemistry, which started in the early 20th century.

• Key concept: Enzyme kinetics helps elucidate the role of enzymes in accelerating reactions and understanding their behavior under different conditions.

• Enzyme Mass: Enzymes are significantly larger than substrates by mass.

• Concentrations:

  • Enzymes usually operate at nanomolar concentrations.

  • Substrate concentrations can be millimolar or higher, making them in excess relative to enzymes.

Factors Affecting Reaction Rates

• Changes in temperature, pH, and substrate concentration can influence reaction rates significantly.

• Understanding variable effects on enzyme productivity is crucial:

  • Higher enzyme concentration increases reaction speed linearly until substrate becomes limiting.

  • Saturation Kinetics: After a certain substrate concentration, the reaction rate levels out due to maximum enzyme activity.

Reaction Equilibrium and Thermodynamics

• Enzymes can accelerate spontaneous (exergonic) reactions, making them occur faster without changing the equilibrium state or the free energy (9;ΔG9;).

• Negative ΔG indicates a spontaneous reaction, leading to complete substrate conversion into product.

• High substrate levels push the reaction towards product formation, while product accumulation can reverse the reaction back towards equilibrium.

Measuring Enzyme Activity

• Laboratory setups involve measuring the rate of product formation or substrate consumption in controlled conditions.

• The important measurement is the initial velocity (v₀) during the linear portion of the reaction curve before substrate concentration drops.

• To analyze enzymatic reactions:

  • Preferably, keep substrate concentration high to limit it as a variable while adjusting enzyme concentration for optimal measurements.

Kinetics Data and Graphing

• The relationship between substrate concentration and reaction velocity produces a hyperbolic curve, characteristic of saturation kinetics:

  • Increases in substrate lead to increased velocities until a maximum (Vmax) is reached.

  • Michaelis-Menten Equation:v₀ = (Vmax [S]) / (Km + [S])

  • Km (Michaelis constant) indicates the substrate concentration at which the reaction rate is half of Vmax.

    • Lower Km = higher affinity of the enzyme for the substrate.

Inhibition Types

• Competitive Inhibition:

  • Inhibitor competes with substrate for the active site.

  • Vmax remains unchanged; Km increases.

• Noncompetitive Inhibition:

  • Inhibitor binds to an allosteric site, changing the enzyme shape and lowering its ability to convert substrate to product.

  • Vmax decreases; Km remains unchanged.

• Mixed Inhibition:

  • Displays characteristics of both competitive and noncompetitive inhibition, affecting both Vmax and Km.

Allosteric Regulation

• Allosteric activation and inhibition can modulate enzyme activity based on the concentration of substrates or products, allowing for negative feedback in enzymatic pathways.

• Common in metabolic pathways to ensure efficiency and resource management within the cell.

Environmental Influences on Enzyme Activity

• Most enzymes function optimally at around pH 7, but some enzymes like pepsin function in highly acidic environments (pH around 2).

• Temperature increases generally increase reaction speeds until exceeding optimal levels, which can denature the enzyme.