Lecture_9_Kinetics_I_2025
Page 1: Introduction
Lecture on Enzyme Kinetics I
Date: 02/13/2025
Instructor: jracca@iu.edu
Page 2: Enzyme Kinetics Overview
Definition: Kinetics describes the rate of a reaction and product formation as a function of time.
Influencing parameters:
Temperature
pH
Substrate concentration
Changes to the enzyme
Page 3: Importance of Studying Enzyme Kinetics
Molecular mechanisms of enzyme-catalyzed reactions.
Impact of small molecules (therapeutics) on enzyme activity.
Understanding enzyme mutations linked to diseases.
Mechanisms to alter enzyme activity.
Page 4: Outline of Topics
Initial velocity and substrate concentration
Derivation of the Michaelis-Menten equation
Description of Michaelis-Menten constant (Km)
Lineweaver-Burk plots
Enzyme turnover and specificity
Page 5: General Equilibrium for Enzymes
Reaction sequence: E + S ⇌ [ES] ⇌ E + P
Page 6: Enzyme Kinetics – Concentrations
Initial mixing of enzyme and substrate leads to a brief pre-steady state with rising [ES].
Page 7: Pre-steady State Kinetics
Very brief and transient time period, challenging to measure.
Page 8: Kinetics in Pre-steady State
Formation of [ES] is rapid.
Substrate concentration ([S]) decreases quickly.
Unbound enzyme concentration also decreases.
Product formation increases, even if low.
Steady state reached when [ES] is constant.
Page 9: Initial Velocity Vo
Vo: initial reaction rate (velocity).
Dependent on substrate concentration; [S] should be saturating ([S] >>> [E]).
Page 10: Relationship Between [S] and Vo
Higher [S] leads to increased reaction velocity.
Page 11: Changes in Initial Rate
Initial rate changes depend on [S]; changes decrease as [S] increases.
Page 12: Initial Velocity and Substrate Concentration
At constant [E]: initial velocity (Vo) depends on substrate concentration ([S]).
Max velocity (Vmax) occurs when [S] is much greater than [E].
Page 13: Michaelis-Menten Kinetics
Reaction sequence: S + E ⇌ [ES] ⇌ E + P.
Rate limiting step is the breakdown of [ES].
Page 14: Saturation Kinetics
At Vmax, [ES] is constant; unbound [E] decreases.
Page 15: Michaelis-Menten Equation
Relates [S] and Vo; rate-limiting step is product formation from [ES].
Page 16: Michaelis-Menten Equation Overview
Quantitative relation: Vo, [S], Vmax, and Km.
Page 17: Deriving the Michaelis-Menten Equation
Assumptions simplify expression; enzyme is not consumed in catalytic reactions.
Page 18: Dropping P → S in Derivation
Initial product formation is low; can ignore reverse reaction k-2.
Page 19: Relation Between Vo and [ES]
Vo depends on breakdown of [ES] to products.
Page 20: Total Enzyme Concentration
Total enzyme [Et]: [Et] = [Eunbound] + [ES].
Page 21: Rates of Formation and Breakdown
Established relationship governing formation and breakdown of [ES].
Page 22: Steady-State Assumption
At steady state, [ES] formation rate = breakdown rate.
Page 23: Solving for [ES]
Derivation involves algebraic manipulations relating [Et] and [S] to [ES].
Page 24: Further Simplification
Reorganizing to define Km in relation to [S] and rate constants.
Page 25: Expressing Velocity and Saturation
Vmax defined at enzyme saturation ([ES]=[Et]).
Page 26: Final Michaelis-Menten Equation
Vo = Vmax[S] / (Km + [S]).
Page 27: Michaelis-Menten Curve
Curve characteristics:
Vo: initial velocity
Vmax: maximum rate
Km: at half Vmax.
Page 28: Defining the Michaelis-Menten Constant (Km)
Km: substrate concentration at half-maximal reaction rate.
Page 29: Numerical Relationship of Km
Practical definition of Km at Vo = 1/2 Vmax.
Page 30: Km and Affinity
Km indicates enzyme-substrate affinity.
Lower Km → higher affinity.
Page 31: Relationship Between Kd and Km
Km is related to the enzyme’s dissociation constant (Kd).
Page 32: Relationship Between Kd and Km
Km measures affinity; Kd measures binding tightness.
Page 33: Some Km Values
Table of Km for various enzymes/substrates.
Page 34: Considerations for Km
Km reflects enzyme dynamics; complex function based on reaction steps.
Page 35: Lineweaver-Burk Equation
Reciprocal of Michaelis-Menten; linearizes the graph for analysis.
Page 36: Lineweaver-Burk Graph
X-intercept: -1/Km; Y-intercept: 1/Vmax.
Page 37: Complicated Reactions with Lineweaver-Burk Equation
Useful for understanding reaction complexities.
Page 38: Enzyme Turnover (kcat)
kcat: number of substrate molecules converted to product per unit time.
Page 39: Determining kcat
Depends on the rate-limiting step of the reaction.
Page 40: Turnover Variability
Table of turnover numbers (kcat) for various enzymes.
Page 41: Enzyme Specificity
Specificity constant: kcat/Km; reflects catalytic effectiveness.
Page 42: Diffusion Controlled Limit
Enzyme reaction rates approach limits set by diffusion (108 to 109 M-1s-1).
Page 43: Catalytic Perfection in Enzymes
List of enzymes approaching diffusion-controlled limits.
Page 44: Simplified Michaelis-Menten Conditions
Assumes single substrate, unregulated, and single subunit.
Page 45: Reality of Enzyme Regulation
Enzymes often highly regulated and multi-subunit, influenced by various mechanisms.
Page 46: Key Takeaways
Understand and apply MM and Lineweaver-Burk equations.
Comprehend assumptions in MM derivation.
Determine Km and Vmax from a Lineweaver-Burk plot.
Recognize the relationship between Km and Kd.
Grasp concepts of enzyme turnover and specificity.
Note this content is part 1 of 2 on enzyme kinetics.