The ultimate objective of this research is to provide a dtailed mechanistic understanding of bacterial urease, a nickel-containing enzyme. Elucidation of the urease mechanism potentially could lead to the develepment of specific enzyme inhibitors. Such inhibitors may have utility in several areas of human health, animal husbandry, and agronomy. For example, urinary stones which are induced by ureolytic bacteria infecting the urinary tract may be eliminated through the use of urease inhibitors; thus, the incidence of hospitalization for this conditon (1 per 1000 adults per year) may be significantly reduced. The experiments in this proposal work toward the goal of elucidating unrease action by focusing on the following specific aims. Urease will be isolated and chaacterized from Proteus mirabilis, the bacterium most often associated with urinary stone formation, Selenomonas ruminantium, an important ureolytic rumen microbe, Sporosarcina ureae, a soil bacterium which possesses high levels of urease, and Klebsiella aerogenes, a ureolytic bacterium which can be easily manipulated genetically. The urease from each organism will be analyzed for stability, native and subunit molecular weight, number of nickel per subunit, amino acid composition, amino terminal sequence, and kinetic parameters. The nickel-containing urease which is most readily purified in large quantities and has the largest potential for experimental flexibility will be chosen for in-depth studies. The nickel-site will be extracted and characterized, the nickel ligands will be identified, and reconstitution experiments will be performed. Potential inhibitors and inactivators of urease will be screened, and covalent inactivators will be used to identify active-site amino acid residues.
