A study on the kinetic mechanism of apoenzyme reconstitution from Aerococcus viridans lactate oxidase
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Updated: Aug 31, 2026

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition
Published on: October 3, 2018
Sergio A Streitenberger1, José A López-Mas, Alvaro Sánchez-Ferrer
1Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus de Espinardo, E-30071 Murcia, Spain.
Abstract:
The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k3 and k-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.
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