In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE

The urease accessory protein encoded by ureE from Klebsiella aerogenes is p roposed to deliver Ni(II) to the urease apoprotein during enzyme activation . Native UreE possesses a histidine-rich region at its carboxyl terminus th at binds several equivalents of Ni2+; however, a truncated form of this pro tein (H144*UreE) binds only 2 Ni2+ per dimer and is functionally active (Br ayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416), Th e urease activation kinetics were studied in vivo by monitoring the develop ment of urease activity upon adding Ni2+ to spectinomycin-treated Escherich ia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes obse rved for the H96A, H110A, D111A, and H112A substitutions. Notably, urease a ctivity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni2+ per dimer with elevated K-d values compared with contro l H144*UreE, whereas the H96A and D111A variants bound 2 Ni2+ per dimer wit h unperturbed R, values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Ha usinger, R. P. (1999) Biochemistry 38, 4078-4088). To understand why cells containing the latter two proteins showed reduced rates of urease activatio n, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was show n to sequentially bind two Ni2+ with k(1) similar to 18 and k(2) similar to 100 M-1 s(-1) and with dissociation rates k(-1) similar to 3 x 10(-3) and k(-2) similar to 10(-4) s(-1). Similar apparent rates of binding and dissoc iation were noted for the two mutant proteins, suggesting that altered H144 *UreE interactions with Ni2+ do not account for the changes in cellular ure ase activation. These conclusions are further supported by in vitro experim ents demonstrating that addition of H144*UreE to urease apoprotein. activat ion mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting U reE-dependent urease maturation.