| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2003: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
Urease is a Ni-containing enzyme that catalyzes the hydrolysis of urea to form ammonia and carbamate. It possesses a dinuclear Ni active site with the protein providing a bridging carbamylated lysine residue as well as an aspartyl and four histidyl ligands. Meanwhile some metalloenzymes that have dinuclear metal ions in the active site have been known. For example, tyrosinase is believed to have coupled dinuclear copper active site similar to those of hemocyanin and catechol oxidase, where each of two copper ions is coordinated to three histidine imidazoles. Methane monooxygenase has dinuclear iron active site where each iron ion is coordinated by six atoms, they include one nitorogen derived from His and five oxygen comprised of glutamic acid residues, bridging hydroxides, and a terminal water. Manganese containing catalase has also dinuclear manganese active site. In this research project, we prepared some metal substituted ureases, and investigated the oxidase activities of the meta
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l substituted ureases. As the results, we found that the copper-substituted urease showed tyrosinase activity, which is the oxidation of phenols to o-quinones. Although native urease (Ni) and apo protein (apo) did not catalyze any reactions, Cu-urease (Cu) was observed to have mono- and di-phenolase activities. Furthermore, K217A mutant of Cu-urease, which has no bridging amino acid residue of metal ions in the active site showed higher tyrosinase activity than wild-type Cu-urease. The structure of active site in the mutant would similar as that in copper-containing catechol oxidase. We also found that iron substituted urease was observed to catalyze the peroxidation of guaiacol, although apo did not catalyze any oxidation reactions. The result suggests that iron substituted urease could be a good model for methane monooxygenase which has dinuclear iron active site. Furthermore, we found that manganese substituted urease had catalase activity that hydrogen peroxide was disproportioned into dioxygen and water. These findings suggest that the function of metalloenzyme can be changed by metal substitution. Less
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