Urease is a fast enzyme, presenting k _cat of the order of 10³–10⁴ s⁻¹. To enable the visualization of its mechanism we explored the technique of cryo-FTIR. By means of using a ruthenium complex as a caged-urea, upon light irradiation urea was released and we could see the coordination of the urea to the active site.

Abstract

Caged compounds are a valuable tool to photocontrol the release of enzymatic substrates in a reaction. For example, caged urea molecules were proposed to control urea delivery to urease reactions and aid the determination of the catalytic mechanism of this enzyme. Owing to the success of several caged compounds based on ruthenium complexes, we synthesized a new inorganic caged urea (RuBpy-urea) and monitored the release of free urea by infrared spectroscopy upon irradiation. Transient infrared absorption (TRIR) indicates diffusion-controlled release of urea, and kinetics studies after photolysis demonstrate the functionality of the new caged molecule for photocontrolled delivery of urea to urease. Coupling photocontrolled delivery of urea to cryo-FTIR (fourier transform infrared spectroscopy) revealed that inhibition of urease results in different FTIR spectrum than the one observed for the noninhibited urease.