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Measuring Enzymatic Stability by Isothermal Titration Calorimetry
Published on: March 26, 2019
Relation between dynamics, activity and thermal stability within the cholinesterase family
Marie Trovaslet1, Marcus Trapp, Martin Weik
1Institut de Recherches Biomédicales des Armées, antenne de La Tronche, France.
Chemico-Biological Interactions
|September 4, 2012
Summary
Elastic incoherent neutron scattering reveals that human acetylcholinesterase (hAChE) is more flexible than mouse acetylcholinesterase (mAChE) and human butyrylcholinesterase (hBChE). This increased flexibility correlates with higher enzyme activity, but not thermal stability.
Area of Science:
- Biophysics
- Enzymology
- Structural Biology
Background:
- Incoherent neutron scattering (INS) is a powerful technique for probing molecular dynamics in biological systems.
- Cholinesterases (ChEs) are crucial enzymes involved in neurotransmission, with varying activities and stabilities.
- Understanding the relationship between enzyme dynamics, activity, and stability is key to enzyme function.
Purpose of the Study:
- To investigate the temperature-dependent dynamics of human acetylcholinesterase (hAChE), mouse acetylcholinesterase (mAChE), and human butyrylcholinesterase (hBChE) using elastic incoherent neutron scattering (EINS).
- To correlate enzyme dynamics with catalytic activity and thermal inactivation kinetics.
- To explore the relationship between protein flexibility and crystallizability.
Main Methods:
- Elastic Incoherent Neutron Scattering (EINS) measurements on IN16 spectrometer at ILL to determine atomic mean square displacement (MSD) as a measure of flexibility.
- Frequency Window Model (FWM) analysis to extract activation energies for thermodynamical transitions.
- Enzymatic activity assays using thiocholine esters and thermal inactivation studies.
Main Results:
- hAChE exhibited higher MSD values above 200K compared to mAChE and hBChE, indicating greater flexibility.
- Activation energies increased from hBChE to mAChE to hAChE, inversely correlating with MSD.
- hAChE showed systematically higher catalytic activity than mAChE and hBChE at the same temperatures (280-316K), suggesting a dynamics-activity correlation.
- No direct correlation was found between enzyme dynamics and thermal inactivation kinetics.
Conclusions:
- Enzyme flexibility, as measured by MSD, is directly correlated with catalytic activity within the cholinesterase family.
- The relationship between enzyme dynamics and catalytic stability is complex and not directly explained by flexibility alone.
- Protein flexibility may influence the ability of cholinesterases to form crystals.
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