Characterization of metal-substituted Klebsiella aerogenes urease

Abstract

 Urease possesses a dinuclear Ni active site with the protein providing a bridging carbamylated lysine residue as well as an aspartyl and four histidyl ligands. The apoprotein can be activated in vitro by incubation with bicarbonate/CO₂ and Ni(II); however, only ∼15% forms active enzyme (Ni-CO₂-urease^A), with the remainder forming inactive carbamylated Ni-containing protein (Ni-CO₂-urease^B). In the absence of CO₂, apoprotein plus Ni(II) forms a distinct inactive Ni-containing species (Ni-urease). The studies described here were carried out to better define the metal-binding sites for the inactive Ni-urease and Ni-CO₂-urease^B species, and to examine the properties of various forms of Co-, Mn-, and Cu-substituted ureases. X-ray absorption spectroscopy (XAS) indicated that the two Ni atoms present in the Ni-urease metallocenter are coordinated by an average of two histidines and 3–4 N/O ligands, consistent with binding to the usual enzyme ligands with the lysine carbamate replaced by solvent. Neither XAS nor electronic spectroscopy provided evidence for thiolate ligation in the inactive Ni-containing species. By contrast, comparative studies of Co-CO₂-urease and its C319A variant by electronic spectroscopy were consistent with a portion of the two Co being coordinated by Cys319. Whereas the inactive Co-CO₂-urease possesses a single histidyl ligand per metal, the species formed using C319A apoprotein more nearly resembles the native metallocenter and exhibits low levels of activity. Activity is also associated with one of two species of Mn-CO₂-urease. A crystal structure of the inactive Mn-CO₂-urease species shows a metallocenter very similar in structure to that of native urease, but with a disordering of the Asp360 ligand and movement in the Mn-coordinated solvent molecules. Cu(II) was bound to many sites on the protein in addition to the usual metallocenter, but most of the adventitious metal was removed by treatment with EDTA. Cu-treated urease was irreversibly inactivated, even in the C319A variant, and was not further characterized. Metal speciation between Ni, Co, and Mn most affected the higher of two pK _a values for urease activity, consistent with this pK _a being associated with the metal-bound hydrolytic water molecule. Our results highlight the importance of precisely positioned protein ligands and solvent structure for urease activity.