GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins

Syntheses participation of accessory proteins. The roles played by many of these accessory proteins are poorly characterized. Klebsiella aerogenes ure ase, a nickel-containing enzyme, provides an ideal system to study metalloc enter assembly. Here, we describe a method for isolating a complex containi ng urease apoprotein and the UreD, UreF, and UreG accessory proteins. We de monstrate that urease apoprotein in this complex is activated to near wild- type enzyme levels when incubated with nickel ions and high (approximate to 100 mM) concentrations of bicarbonate. Significantly, we also observed nic kel-dependent activation at physiologically relevant (approximate to 100 mu M) bicarbonate levels, but only in the presence of GTP, Based on studies i nvolving a nonhydrolyzable analog of GTP, we conclude that nucleotide hydro lysis, not just binding, is required for this process. The critical nucleot ide-binding site was localized to UreG on the basis of experiments using a variant complex. These studies highlight the relevance of the UreD-UreF-Ure G-urease apoprotein complex to nickel metallocenter assembly and explain th e previously identified in vivo energy requirement for urease activation.