Overview
This lecture covers enzyme kinetics, focusing on how substrate concentration affects reaction velocity and the importance of measuring initial velocity in enzymatic reactions.
Enzyme-Substrate Complex and Reaction Stages
- Enzymes bind substrates to form an enzyme-substrate (ES) complex.
- ES complex changes to ES* (transition state), then forms enzyme-product (EP) complex.
- The reaction ends with release of free enzyme and product.
Simplified Enzyme Kinetics Model
- The reaction is often simplified to: Enzyme + Substrate ↔ ES → Enzyme + Product.
- The rate constant k_cat measures how quickly product forms from ES.
Substrate Concentration and Enzyme Saturation
- At low substrate concentration, few enzyme-substrate complexes form, leading to low reaction velocity.
- At intermediate substrate concentration, more enzymes are bound to substrate and velocity increases.
- At high substrate concentration, all enzymes are saturated with substrate, achieving maximum reaction velocity.
Measuring Reaction Velocity
- Reaction velocity (v) is measured as the amount of product formed per unit time (molarity/time).
- Experimental setup: keep enzyme amount constant, vary substrate concentration in different test tubes, and measure product formed after a fixed time.
- Plotting velocity vs. substrate concentration shows low velocity at low substrate, rising to high velocity at high substrate.
Initial Velocity and Data Interpretation
- Another plot: product concentration (or velocity) vs. reaction time.
- Early phase: linear increase (initial velocity, v₀).
- Later phase: curve flattens as reverse reaction starts due to product accumulation.
- Initial velocity (v₀) is used for kinetic studies to avoid measuring the reverse reaction.
Key Terms & Definitions
- Enzyme-Substrate (ES) Complex — Temporary molecule formed when an enzyme binds its substrate.
- k_cat — Catalytic constant; rate at which the enzyme converts ES to product.
- Saturation — Condition where all enzyme molecules are bound to substrate.
- Initial Velocity (v₀) — The rate of product formation at the very start of the reaction.
- Substrate Concentration — Amount of reactant available for the enzyme.
Action Items / Next Steps
- Review definitions and kinetic principles discussed.
- Prepare for questions on measuring initial velocity and interpreting enzyme kinetics graphs.