Kinetics of jack bean urease inhibition by 2,3-dichloro-1,4-naphthoquinone. Elucidation of the mechanism: redox cycling and sulfhydryl arylation

Abstract

The inhibition of jack bean urease by 2,3-dichloro-1,4-naphthoquinone (DCNQ) was studied at ambient temperature in 20 mM phosphate buffer, pH 7.8. The process was investigated by incubation procedure in the absence of substrate. It was found that DCNQ acted as a time- and concentration-dependent inactivator of urease. The time course of the reaction displayed a biphasic mode. Each phase followed a pseudo-first-order kinetics, however the inactivation rate at the first phase was significantly faster than at the next one. The biphasity indicated the complex mechanism of DCNQ action on urease. Quinones action on proteins has been elucidated as at least two processes: direct arylation of essential protein thiols and/or indirect oxidation of essential thiols by reactive oxygen species (ROS) realising during quinone reduction to semiquinones. The next evidence of the studied mechanism was provided by the reactivation experiment that showed the participation of reversible and irreversible processes in the inactivation. The application of dithiothreitol (DTT) into DCNQ blocked-urease solution resulted in an effective enzyme activity regain which quickly returned to 70 ± 10%. The irreversible inactivation of urease was attributed to DCNQ arylation of thiol residues in the protein. On the other hand, it was assumed that the reversible inactivation was a result of the action of ROS such as H₂O₂. Presence of H₂O₂ in the incubation system was proved by an experiment with the use of catalase. The enzyme by the elimination of H₂O₂ decreased DCNQ inactivating influence on urease. The comparison of participation of the fast and slow phase in the inactivation with the percentage of the process reversibility was assumed that the fast period was a result of the arylation mechanism while the slow phase was related to the oxidative influence of H₂O₂.

Keywords::

• Urease
• inactivation
• 2,3-dichloro-1,4-naphthoquinone

Acknowledgements

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.