Biochemical and Structural Studies on Urease Inhibition. A Nickel-Dependent Virulence Factor

Abstract

Urease is a nickel-dependent enzyme that catalyses the rapid hydrolysis of urea, determining an overall increase of pH and causing negative consequences for human health as well as agriculture. For these reasons, the scientific community has devoted intense efforts in the last several decades for the development of efficient and specific inhibitors of urease able to counteract its negative effects. In this study, a combination of kinetic experiments, carried out using calorimetry and spectrophotometry, and X-ray protein crystallography has been applied to the urease system to determine the inhibition mode of several known urease inhibitors: i) fluoride, ii) sulphite, iii) 1,4-benzoquinone (BQ), iv) catechol (CAT) and v) N-(n-butyl)thiophosphoric triamide (NBPT). Both fluoride and sulphite show a pH-dependent inhibition on urease, directly binding to the two Ni(II) ions in the enzyme active site. Unlike the previous cases, BQ and CAT act as time-dependent urease inhibitors covalently binding to a conserved cysteine residue located on a flexible flap controlling the access of the substrate to the active site cavity. NBPT, a commercial product extensively used in agriculture as a nitrogen stabilizer, acts as a slow-binding inhibitor of urease. In particular, it directly interacts with the nickel ions in the urease active site, undergoing an in situ hydrolysis that generates a tetrahedral moiety blocking the active site and precluding the enzyme from further substrate hydrolysis. All the results shown in this work will be useful to develop, through a structure-based drug design procedure, novel and more efficient urease inhibitors, necessary to modulate its activity and to counterbalance its negative effects.