Urease hydrolyzes urea to ammonia and carbamic acid, which then spontaneously decomposes into another ammonia molecule and carbonic acid. The active site contains two nickel atoms bridged by a carbamylated lysine residue. In order to assemble the active site, four accessory proteins, UreD, UreE, UreF, and UreG, are necessary. This dissertation focuses on the roles UreG and UreF play in the activation process by investigating the individual characteristics of these proteins as purified, as well as determining how they interact with the other urease accessory proteins in the Klebsiella aerogenes system. UreG is a GTPase required for the assembly of the urease active site; however, it has no GTPase activity when purified alone. A Strep-tagged version, UreGStr, as well as several site-directed variants were constructed and their effects on urease activation, metal-binding properties, and protein: protein interactions with other urease-related proteins were assessed. The Strep-tag had no effect on the ability of UreG to participate in urease activation, but the K20A, D49A, C72A, H74A, D80A, and S111A variants essentially abolished enzyme activity. UreGStr binds one nickel or zinc ion per monomer (Kd ~ 5 μM for each metal ion). The binding site includes residue Cys72 as shown by the 12-fold increase in the Kd for nickel ions in this variant, as well as a lack of a thiolate-to-nickel charge-transfer band in the UV-visible spectrum. Based on homology to HypB, a hydrogenase maturation protein, His74 is also a likely metal ligand. Pull-down assays in cell-free extracts demonstrated that Asp80 is essential for stabilizing the UreGStr interaction with UreABC--UreD--UreF. In vitro pull-down assays demonstrated that the interaction between UreGStr and UreE is metal-dependent. This result suggests that UreE transfers its bound Ni to UreG in the UreABC--UreD--UreF--UreG complex, where it can then be passed to the nascent active site, possibly via UreD. UreF was proposed previously to be a GTPase activating protein (GAP) for the GTPase UreG. Based on the UreF crystal structure from Helicobacter pylori, sixteen residues in K. aerogenes UreF were chosen for mutagenesis to alanine. When produced in the context of the urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type activity. Using the UreE-F construct and its variants in the same context, UreE-F was demonstrated to co-purify with urease apoprotein, UreD, and in some cases UreG from cell-free extracts. The variants that did not bind UreG correlated with low urease activity mutant cells, and mapped to a distinct surface on the UreF structure, defining the UreG binding site. In contrast to the GAP hypothesis for UreF, the UreABC--UreD--UreF(K165A)--UreGStr complex had higher GTPase activity than the wild-type complex according to urease activation assays on purified protein. Further studies showed that the urease activity and GTPase activity were uncoupled in the K165A UreF-containing complex. Additional experiments with these complexes demonstrated that UreF gates the GTPase activity of UreG in order to enhance the fidelity of urease activation and guard against incorrect metal insertion in the presence of Zn.
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