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Measuring Enzymatic Stability by Isothermal Titration Calorimetry
Published on: March 26, 2019
Structural stability of an enzyme biocatalyst
P A Dalby1, J P Aucamp, R George
1The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, UCL (University College London), Torrington Place, London WC1E 7JE, U.K. p.dalby@ucl.ac.uk
Biochemical Society Transactions
|November 23, 2007
Summary
Transketolase (TK) inactivation in biocatalysis involves complex denaturation pathways. Understanding these mechanisms, including cofactor interactions and intermediate states, is key to improving enzyme stability and process efficiency.
Area of Science:
- Biochemistry
- Enzyme Kinetics
- Protein Chemistry
Background:
- Transketolase (TK) is prone to inactivation during biocatalysis through various mechanisms like oxidation, inhibition, and cofactor dissociation.
- The precise role of protein denaturation in these inactivation pathways remains incompletely understood.
Purpose of the Study:
- To characterize the urea-induced reversible denaturation of Escherichia coli TK apo- and holo-enzyme forms.
- To investigate the reconstitution pathway of holo-TK from apoenzyme and cofactors.
- To explore the impact of oxidizing conditions on TK deactivation mechanisms.
Main Methods:
- Urea-induced denaturation studies on E. coli TK apo- and holo-enzymes.
- Enzyme reconstitution assays.
- Analysis of intermediate states during denaturation and reconstitution.
- Preliminary studies under oxidizing conditions.
Main Results:
- Identified an unusual intermediate state where cofactors are bound but the enzyme is inactive, occurring in both reconstitution and denaturation pathways.
- Demonstrated convergence of holo- and apoenzyme denaturation pathways at a partially denatured apo-homodimer intermediate.
- Preliminary findings suggest increased complexity in deactivation mechanisms under oxidizing conditions.
Conclusions:
- Protein denaturation significantly contributes to TK inactivation, involving distinct intermediate states.
- The study elucidates complex denaturation and reconstitution pathways for TK, highlighting cofactor-bound inactive intermediates.
- Further research into oxidative stress is needed to fully understand biocatalytic enzyme deactivation.
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