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Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions
Published on: April 4, 2014
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Measuring Rapid Time-Scale Reaction Kinetics Using Isothermal Titration Calorimetry
Justin M Di Trani1, Nicolas Moitessier1, Anthony K Mittermaier1
1Department of Chemistry, McGill University , H3A 0B8 Montreal, Canada.
Analytical Chemistry
|June 8, 2017
Summary
Isothermal titration calorimetry (ITC) can now measure rapid enzyme kinetics in seconds. A new ITC empirical response model (ITC-ERM) accurately analyzes these fast reactions, revealing hidden non-Michaelis-Menten kinetics.
Area of Science:
- Biophysical chemistry
- Enzyme kinetics
- Biochemical thermodynamics
Background:
- Isothermal titration calorimetry (ITC) is a versatile technique for studying biological interactions and enzymatic catalysis.
- Traditional ITC kinetics assays are limited to long reaction timescales (minutes to hours) due to instrument response limitations.
- Previous attempts to analyze rapid ITC kinetics have faced challenges with instrument response and data interpretation.
Purpose of the Study:
- To evaluate the capabilities and limitations of commercial ITC instruments for rapid kinetics measurements.
- To develop a method for accurately analyzing ITC data from fast enzymatic reactions.
- To explore the potential of rapid ITC kinetics for uncovering novel enzymatic behaviors.
Main Methods:
- Determined the time resolution of commercial ITC instruments, finding it to be approximately 0.2 seconds.
- Developed an ITC empirical response model (ITC-ERM) to accurately represent ITC peak shapes across various reaction durations.
- Applied the ITC-ERM to study the kinetics of trypsin and prolyl oligopeptidase at rapid timescales.
Main Results:
- Successfully performed ITC kinetics assays with reaction durations as short as tens of seconds.
- Demonstrated that standard ITC assumptions lead to significant deviations in peak shapes for short reaction times.
- The ITC-ERM accurately reproduced measured ITC peak shapes for all reaction durations.
- Discovered non-Michaelis-Menten kinetics for prolyl oligopeptidase in short-time scale measurements using ITC-ERM, which were not apparent in long-time scale experiments or standard short-time analyses.
Conclusions:
- Commercial ITC instruments possess a time resolution suitable for studying rapid enzymatic reactions.
- The developed ITC-ERM is crucial for accurate analysis of fast ITC kinetics data.
- Rapid ITC kinetics measurements, when analyzed with ITC-ERM, can reveal complex enzymatic behaviors like non-Michaelis-Menten kinetics that are otherwise obscured.
- This approach offers new possibilities for understanding biological dynamics at a faster timescale.
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