In Vivo and in Vitro Kinetics of Metal Transfer by the Klebsiella aerogenes Urease Nickel Metallochaperone, UreE
2000; Elsevier BV; Volume: 275; Issue: 15 Linguagem: Inglês
10.1074/jbc.275.15.10731
ISSN1083-351X
AutoresGerard J. Colpas, Robert P. Hausinger,
Tópico(s)Corrosion Behavior and Inhibition
ResumoThe urease accessory protein encoded byureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni2+; however, a truncated form of this protein (H144*UreE) binds only 2 Ni2+ per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410–5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni2+ to spectinomycin-treated Escherichia colicells 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 vivourease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing aureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni2+ per dimer with elevatedK d values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni2+ per dimer with unperturbed K d values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999)Biochemistry 38, 4078–4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni2+ withk 1 ∼18 and k 2 ∼100m−1 s−1, and with dissociation rates k −1 ∼3 × 10−3 andk −2 ∼10−4 s−1. Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni2+ do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation. The urease accessory protein encoded byureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni2+; however, a truncated form of this protein (H144*UreE) binds only 2 Ni2+ per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410–5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni2+ to spectinomycin-treated Escherichia colicells 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 vivourease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing aureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni2+ per dimer with elevatedK d values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni2+ per dimer with unperturbed K d values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999)Biochemistry 38, 4078–4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni2+ withk 1 ∼18 and k 2 ∼100m−1 s−1, and with dissociation rates k −1 ∼3 × 10−3 andk −2 ∼10−4 s−1. Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni2+ do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation. product derived from the ureE gene containing a stop codon at the position corresponding to His-144 isopropyl-β-d-thiogalactopyranoside 4-(2-pyridylazo)resorcinol Metallochaperones are intracellular metal-binding proteins that deliver specific cations to target metalloproteins while protecting the cell from reactivity of free metal ions (1.Hausinger R.P. Hausinger R.P. Eichorn G.L. Marzilli L.G. Mechanisms of Metallocenter Assembly. VCH Publishers, Inc., New York1996Google Scholar). For example, yeast contains Atx1, CCS, and Cox17 metallochaperones that specifically deliver Cu2+ to Ccc2 (for later transport to Fet3) (2.Lin S.J. Pufahl A. Dancis A. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 1997; 272: 9215-9220Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 3.Rosenzweig A.C. Huffman D.L. Hou M.Y. Wernimont A.K. Pufahl R.A. O'Halloran T.V. 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Science. 1999; 284: 805-808Crossref PubMed Scopus (1367) Google Scholar), and cytochrome c oxidase (9.Srinivasan C. Posewitz M.C. George G.N. Winge D.R. Biochemistry. 1998; 37: 7572-7577Crossref PubMed Scopus (100) Google Scholar). These proteins use cysteines in 2- and 3-coordinate Cu2+-binding modes, allowing for ready accessibility of the metal ion to the target proteins (4.Pufahl R.A. Singer C.P. Peariso K.L. Lin S.-J. Schmidt P.J. Fahrni C.J. Culotta V.C. Penner-Hahn J.E. O'Halloran T.V.O. Science. 1997; 278: 853-856Crossref PubMed Scopus (593) Google Scholar). Recently, CopZ has been identified and proposed to direct Cu(I) to a Cu2+-responsive repressor (CopY) involved in regulation of bacterial Cu2+ transport proteins (10.Cobine P. Wickramsinghe W.A. Harrison M.D. Weber T. Solioz M. Dameron C.T. FEBS Lett. 1999; 445: 27-30Crossref PubMed Scopus (136) Google Scholar, 11.Wimmer R. Herrmann T. Solio M. Wüthrich K. J. Biol. Chem. 1999; 274: 22597-22603Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Also in bacteria, three distinct Ni2+ metallochaperones have been tentatively identified (12.Hausinger R.P. J. Biol. Inorg. Chem. 1997; 2: 279-286Crossref Scopus (73) Google Scholar). HypB, essential for Ni2+insertion into hydrogenase by a process that requires GTP hydrolysis (13.Maier T. Jacobi A. Sauter M. Böck A. J. Bacteriol. 1993; 175: 630-635Crossref PubMed Google Scholar, 14.Maier T. Lottspeich F. Böck A. Eur. J. Biochem. 1995; 230: 133-138Crossref PubMed Scopus (126) Google Scholar), binds 18 Ni2+/dimer (K d = 2.3 μm) as isolated from Bradyrhizobium japonicum(15.Fu C. Olsen J.W. Maier R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2333-2337Crossref PubMed Scopus (103) Google Scholar). Similarly, Rhodospirillum rubrum CooJ, which assists with CO dehydrogenase activation (16.Kerby R.L. Ludden P.W. Roberts G.P. J. Bacteriol. 1997; 179: 2259-2266Crossref PubMed Google Scholar), binds 4 Ni2+/monomer (K d = 4.3 μm) (17.Watt R.K. Ludden P.W. J. Biol. Chem. 1998; 273: 10019-10025Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Finally, of particular interest to the work described here is Klebsiella aerogenes UreE, the putative urease metallochaperone (18.Lee M.H. Mulrooney S.B. Renner M.J. Markowicz Y. Hausinger R.P. J. Bacteriol. 1992; 174: 4324-4330Crossref PubMed Google Scholar) that binds 5–6 Ni2+/dimer (average K d = ∼10 μm) (19.Lee M.H. Pankratz H.S. Wang S. Scott R.A. Finnegan M.G. Johnson M.K. Ippolito J.A. Christianson D.W. Hausinger R.P. Protein Sci. 1993; 2: 1042-1052Crossref PubMed Scopus (124) Google Scholar). In each case, mutations within the corresponding genes are phenotypically suppressed by increasing the Ni2+ concentration in the medium (16.Kerby R.L. Ludden P.W. Roberts G.P. J. Bacteriol. 1997; 179: 2259-2266Crossref PubMed Google Scholar, 18.Lee M.H. Mulrooney S.B. Renner M.J. Markowicz Y. Hausinger R.P. J. Bacteriol. 1992; 174: 4324-4330Crossref PubMed Google Scholar, 20.Waugh R. Boxer D.H. Biochimie (Paris). 1986; 68: 157-166Crossref PubMed Scopus (92) Google Scholar). Although all three proteins possess histidine-rich regions, they exhibit no overall sequence similarity. The metal-binding properties of K. aerogenes UreE have been extensively characterized. Spectroscopic studies with wild-type protein reveal pseudo-octahedral Ni2+-binding sites with an average of 3–5 histidine donors, implicating a role for the carboxyl terminus where 10 of the last 15 residues are histidine (19.Lee M.H. Pankratz H.S. Wang S. Scott R.A. Finnegan M.G. Johnson M.K. Ippolito J.A. Christianson D.W. Hausinger R.P. Protein Sci. 1993; 2: 1042-1052Crossref PubMed Scopus (124) Google Scholar). Consistent with the lack of sequence conservation for this feature among other urease-containing microorganisms, a truncated form of K. aerogenes UreE lacking the His-rich region, termed H144*UreE,1 is competent for activating urease in vivo (21.Brayman T.G. Hausinger R.P. J. Bacteriol. 1996; 178: 5410-5416Crossref PubMed Google Scholar). The truncated protein possesses two internal Ni2+-binding sites per dimer; thus, the internal ligands, not the histidine residues at the carboxyl terminus, are necessary for UreE to assist in K. aerogenesurease activation. Competition experiments indicate that Cu2+, Zn2+, and Co2+ bind (to varying degrees) to H144*UreE (21.Brayman T.G. Hausinger R.P. J. Bacteriol. 1996; 178: 5410-5416Crossref PubMed Google Scholar). Spectroscopic studies of Ni2+, Cu2+, and Co2+ binding to H144*UreE and several mutant proteins reveal two distinct binding sites per homodimer, each of which is symmetrically located at the dimer interface (22.Colpas G.J. Brayman T.G. McCracken J. Pressler M.A. Babcock G.T. Ming L.-J. Colangelo C.M. Scott R.A. Hausinger R.P. J. Biol. Inorg. Chem. 1998; 3: 150-160Crossref Scopus (37) Google Scholar, 23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). The two Ni2+ sites exhibit pseudo-octahedral geometry, with one Ni2+ possessing four histidine donor ligands and a second Ni2+ coordinated to two histidines. The Ni2+ dependence of in vivoactivation, characterized in stationary phase cells, indicates that one site (involving the pairs of His-96 and His-112 residues along with two other N or O donors) is critical to the role of UreE in urease activation, whereas the second site (including the pair of His-110 residues and four other N/O donors) is less significant to this process (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). These same experiments show that Asp-111 is important for proper function, but it is unclear whether this residue serves as a ligand or affects Ni2+ binding by secondary interactions. Co2+ binds in a manner similar to Ni2+, whereas Cu2+ binding is distinct. All of the bound Cu2+has tetragonal coordination with two histidine donors each, but at higher Cu2+ concentrations a cysteine donor (Cys-79) coordinates the metal. The differences in coordination environments observed for each type of metal ion are proposed to function in metal specificity so that only Ni2+ is used for urease activation (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). The studies described in this article examine the role of UreE in urease maturation. Two mutant proteins altered at highly conserved residues (H96A/H144*UreE and D111A/H144*UreE) are shown to reduce dramatically the rate of cellular urease activation, even though they bind 2 Ni2+ per dimer with K d values close to control H144*UreE. The kinetics of metal binding/dissociation for these mutant proteins are compared with the more extensively investigated kinetics for H144*UreE. Significantly, the ability of purified proteins to bind and release Ni2+ does not correlate with their ability to function in urease activation. Related to this finding, H144*UreE is shown to inhibit in vitroactivation of urease apoprotein even though it facilitates metallocenter assembly in the cell. These studies emphasize the importance of other accessory proteins in aiding UreE-dependent activation of urease. Escherichia coli HMS174(DE3) cells containing pKK17, pKK17ΔureE-1, pKK17H144*, and variants of pKK17H144* (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar) were used to monitor the kinetic effects of UreE variants on cellular urease activity. These plasmids contain the K. aerogenes ureDABCEFG gene cluster in which ureE encodes wild-type, deleted, truncated, and mutated/truncated versions of UreE, respectively. Fresh cultures were inoculated into LB medium (50 μl into 25 ml) lacking Ni2+ but supplemented with 100 μg·ml−1 ampicillin. Cells were grown for 2 h at 37 °C, induced with 1 mm IPTG for 2 h, and adjusted to 0.2 mg·ml−1 spectinomycin (Sigma) to halt protein synthesis. After 30 min, NiCl2 was added (final concentration of 200 μm), and at selected time points 1-ml aliquots were collected and immediately frozen. Upon thawing, each sample was centrifuged, resuspended in 1.5 ml of 20 mmpotassium phosphate buffer containing 0.5 mm EDTA and 1 mm 2-mercaptoethanol, disrupted in a French pressure cell, and centrifuged to obtain soluble cell extracts for the urease activity assay. Urease activity was measured by quantifying the rate of ammonia release from urea by formation of indophenol, which was measured at 625 nm (24.Weatherburn M.W. Anal. Chem. 1967; 39: 971-974Crossref Scopus (3115) Google Scholar). Assay buffer contained 25 mm HEPES (pH 7.75), 50 mm urea, and 0.5 mm EDTA. One unit of activity was defined as the amount of enzyme required to hydrolyze 1 μmol of urea per min at 37 °C. Protein concentrations were determined according to Lowry et al. (25.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) or by using a commercial protein assay (Bio-Rad) with bovine serum albumin as the standard. E. coliHMS174(DE3) or E. coli BL21(DE3) cells (Novagen) carrying derivatives of pET21 (Novagen) were used for overproduction of recombinant H144*UreE and H144*UreE variants (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). These cultures were grown at 37 °C in TB medium containing 100 μg·ml−1ampicillin (Amersham Pharmacia Biotech) to A 600∼1, induced with 0.5 mm IPTG (Amersham Pharmacia Biotech), and harvested after 4–6 h. H144*UreE and H144*UreE variant proteins were purified according to previously published procedures (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). E. coli DH5α cells containing pKAU17 (26.Mulrooney S.B. Pankratz H.S. Hausinger R.P. J. Gen. Microbiol. 1989; 135: 1769-1776PubMed Google Scholar) were used for production of urease apoprotein. These cultures were grown at 37 °C overnight in LB medium supplemented with 100 μg·ml−1 ampicillin, and the desired protein was purified by previously published procedures (27.Lee M.H. Mulrooney S.B. Hausinger R.P. J. Bacteriol. 1990; 172: 4427-4431Crossref PubMed Google Scholar). Kinetic measurements involving metal interactions with purified H144*UreE and its variants were performed on an OLIS-RSM rapid scan stopped-flow spectrophotometer with a 0.4-cm path length flow cell (Olis Co.). All reactions were studied at room temperature in 10 mmTris-HCl (pH 7.5) buffer with 100 mm NaCl added. Dissociation rates typically were measured upon mixing equal volumes of 40 μm metal chloride plus 20 μm H144*UreE (dimer) solution with 200 μm 4-(2-pyridylazo)resorcinol (PAR) (Aldrich) solution. PAR binds divalent cations with stability constants ranging from 1011 to 1013m (28.Corsini A. Yih I.M. Fernando Q. Freiser H. Anal. Chem. 1962; 34: 1090-1093Crossref Scopus (69) Google Scholar), so essentially all protein-associated metal became bound to PAR upon reaching equilibrium. The concentration of PAR was determined to be sufficient for establishing pseudo-first-order metal-release reactions (29.Furahashi S. Tanaka M. Inorg. Chem. 1969; 10: 2159-2165Crossref Scopus (56) Google Scholar, 30.Hunt J.B. Neece S.H. Ginsburg A. Anal. Biochem. 1985; 146: 150-157Crossref PubMed Scopus (209) Google Scholar), and a 2-fold increase in PAR concentration did not affect the measured rates. Additional studies examined the effects of additives (200 μm imidazole, 100 μm histidine, or 100 μm cysteine) on metal transfer to PAR. Data were analyzed by doing single wavelength fits to the M(II)(PAR)2 absorption at 500 nm (where M indicates metal and ε = 7.0 × 104m−1 cm−1 for Ni2+, 6.6 × 104m−1cm−1 for Zn2+, 6.0 × 104m−1 cm−1 for Co2+, and 6.5 × 104m−1cm−1 for Cu2+) (31.Tanaka M. Funahashi S. Shirai K. Anal. Chim. Acta. 1967; 39: 437-445Crossref Scopus (41) Google Scholar). The fits generated by the Olis fitting program for the dissociation studies are provided as Table S1 in the Supplemental Material. The calculated amplitudes were underestimated in these fits because the window of time points did not begin at 0. For the selected dissociation results described in the text, k obs values and associated amplitudes were determined by performing one or more single exponential fits to the data using the program KaleidaGraph (Synergy Software) after extrapolating to zero time. For binding studies, equal volumes of H144*UreE apoprotein and metal chloride were mixed. Data were analyzed by doing single wavelength fits at 370 nm for Ni2+ (ε = 75m−1 cm−1 per Ni2+bound or 150 m−1 cm−1 per H144*UreE dimer) (22.Colpas G.J. Brayman T.G. McCracken J. Pressler M.A. Babcock G.T. Ming L.-J. Colangelo C.M. Scott R.A. Hausinger R.P. J. Biol. Inorg. Chem. 1998; 3: 150-160Crossref Scopus (37) Google Scholar) or at 365 nm for Cu2+ (ε = 5000 m−1 cm−1 per H144*UreE dimer) (22.Colpas G.J. Brayman T.G. McCracken J. Pressler M.A. Babcock G.T. Ming L.-J. Colangelo C.M. Scott R.A. Hausinger R.P. J. Biol. Inorg. Chem. 1998; 3: 150-160Crossref Scopus (37) Google Scholar). The fits generated by the Olis fitting program are provided as Table S2 in the Supplemental Material. For the selected studies described in the text, data were analyzed by using KaleidaGraph as described above. In addition, iterative simulations were carried out using program A, provided by Dr. David Ballou at the University of Michigan, to obtain rate constants. This program, written by Chung-Jen Chiu, Rong Chang, Joel Dinverno, and David Ballou is an implementation of a 4th order Runge-Kutta algorithm for numerical integration of differential equations (32.Press W.H. Teukolsky S.A. Vetterling W.T. Flannery B.P. Numerical Recipes in C: The Art of Scientific Computing. 2nd Ed. Cambridge University Press, New York1992: 683-688Google Scholar). Equations Equation 1, Equation 2, Equation 3, Equation 4, Equation 5, Equation 6 were used for simulating the Ni2+ data (where H144*UreE is divided into two pools,X and Y, that bind Ni2+differently). d[Ni]/dt=−k1[Ni][X]+k−1[Ni−X]−k2[Ni][Ni−X]+k−2[Ni2X]−ka[Ni][Y]+k−a[Ni−Y]Equation 1 d[X]/dt=−k1[Ni][X]+k−1[Ni−X]Equation 2 d[Ni−X]/dt=k1[Ni][X]−k−1[Ni−X]−k2[Ni][Ni−X]Equation 3 +k−2[Ni2−X] d[Ni2−X]/dt=k2[Ni][Ni−X]−k−2[Ni2−X]Equation 4 d[Y]/dt=−ka[Ni][Y]+k−a[Ni−Y]Equation 5 d[Ni−Y]/dt=ka[Ni][Y]−k−a[Ni−Y]Equation 6 Similarly, Equations Equation 7, Equation 8, Equation 9, Equation 10, Equation 11 were used for simulating the Cu2+ data (where the protein is represented byZ = X + Y). d[Cu]/dt=−k1[Cu][Z]+k−1[Cu−Z]−k2[Cu][Cu−Z]Equation 7 +k−2[Cu2−Z]−k3[Cu][Cu2−Z]+k−3[Cu3−Z] d[Z]/dt=−k1[Cu][Z]+k−1[Cu−Z]Equation 8 d[Cu−Z]/dt=k1[Cu][Z]−k−1[Cu−Z]−k2[Cu][Cu−Z]Equation 9 +k−2[Cu2−Z] d[Cu2−Z]/dt=k2[Cu][Cu−Z]−k−2[Cu2−Z]−k3[Cu][Cu2−Z]+k−3[Cu3−Z]Equation 10 d[Cu3−Z]/dt=k3[Cu][Cu2−Z]−k−3[Cu3−Z]Equation 11 The effects of H144*UreE on production of active urease was examined under urease apoprotein activation conditions (33.Park I.-S. Hausinger R.P. Science. 1995; 267: 1156-1158Crossref PubMed Scopus (147) Google Scholar). Purified urease apoprotein (2.5 μm) samples were assayed every 30 min for 4–6 h while being incubated at 37 °C in 50 mm NaHCO3, 100 μm NiCl2, 150 mm NaCl, and 25 mm HEPES buffer (pH 8.3) containing varying levels of H144*UreE. The in vivo and in vitro kinetics of metal transfer were studied for K. aerogenes UreE, a putative metallochaperone thought to deliver Ni2+ to urease. Previous studies had measured the effects of UreE variants on cellular urease activity levels for cultures in stationary phase and had examined purified UreE variants for Ni2+ binding stoichiometry and affinity under equilibrium conditions (21.Brayman T.G. Hausinger R.P. J. Bacteriol. 1996; 178: 5410-5416Crossref PubMed Google Scholar, 23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). The present work examined the rates of urease activation in cells and the rates of metal binding/dissociation with purified UreE variants. In addition, the effects of UreE addition on urease apoprotein activation were examined. The in vivorates of urease metallocenter assembly were compared for E. coli HMS174(DE3) cells expressing the K. aerogenesurease gene cluster with variations in ureE. The relative amounts of urease and UreE synthesized was unaffected by the mutations, as determined by SDS-polyacrylamide gel electrophoresis (data not shown). Using spectinomycin-treated cells that were incapable of new protein synthesis, the development of urease activity from previously synthesized apourease and accessory proteins was examined upon addition of 200 μm NiCl2 (Fig.1). For cells that synthesized wild-type UreE (filled squares), urease activity was detected within 10 min of Ni2+ addition (panel A) and continued to increase over the 4-h time course of the experiment (panel B). Very similar results were found for cells containing H144*UreE (filled circles), supporting our proposal that the carboxyl-terminal histidine-rich region is not essential for urease activation (21.Brayman T.G. Hausinger R.P. J. Bacteriol. 1996; 178: 5410-5416Crossref PubMed Google Scholar). In contrast, when ureE was deleted a delay in urease activation was observed (>30 min, filled triangles, panel A) and the specific activity gradually increased over the entire course of the study (∼25% of the control rate, panel B). The lag period observed in the deletion strain highlights the importance of UreE for the rapid transfer of Ni2+ into preformed urease apoprotein. On the other hand, these studies reinforce previous conclusions that UreE is not required for urease activation (18.Lee M.H. Mulrooney S.B. Renner M.J. Markowicz Y. Hausinger R.P. J. Bacteriol. 1992; 174: 4324-4330Crossref PubMed Google Scholar, 23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). With the exception of cells containing H96A/H144*UreE, all cultures possessing H144*UreE variants demonstrated intermediate abilities to activate urease with greatest inhibition being noted for cells producing the H110A, D111A, and H112A variants. These findings underscore the complexity of assigning specific functions to structural features in the metal-binding protein, but we note that His-110 and His-112 were previously suggested to be Ni2+ ligands, and Asp-111 was proposed to be functionally important (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). The cultures synthesizing H96A/H144*UreE are of special interest because their levels of urease activity were less that those observed in theureE deletion mutant. It was not surprising to find that cells producing H110A/H144*UreE and H112A/H144*UreE exhibited reductions in their in vivo urease activation rates. Previous studies had shown that these purified proteins bound only one Ni2+ per dimer with elevatedK d values (16 and 36 μm) compared with a control H144*UreE sample that bound two Ni2+ per dimer (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). In contrast, reduced cellular urease activation levels cannot be explained by the Ni2+ binding properties of H96A/144*UreE or D111A/H144*UreE. The number of Ni2+ bound and the average Ni2+ binding affinities for these purified proteins were essentially identical to results obtained with H144*UreE (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). The significance of these results is reflected in the high conservation of His-96 and Asp-111 among UreE sequences from different microorganisms (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar). To define better how altering these two residues can have such dramatic effects within the cell while having no affect on equilibrium Ni2+ binding, we carried out kinetic measurements of their metal binding and dissociation rates. These results were compared with those for H144*UreE which was subjected to more extensive kinetic analysis. The rates of metal ion release from H144*UreE and its variants were examined by using UV-visible stopped-flow spectroscopy to monitor chromophore development at 500 nm arising from metal chelation by PAR. These studies assume that PAR bound only the free metal ion and did not actively remove protein-bound metal. The observed rates of chromophore development for release of protein-bound metals (k obs) were considered to be equivalent to the dissociation rates (k off) because the affinity of the metals for PAR at this concentration far exceeded the affinity for protein (28.Corsini A. Yih I.M. Fernando Q. Freiser H. Anal. Chem. 1962; 34: 1090-1093Crossref Scopus (69) Google Scholar). Dissociation of Ni2+ from Ni2+-H144*UreE or its H96A and D111A variants was monitored by PAR chromophore development as shown in Fig. 2. In each case, Ni2+ was present at 2-fold the concentration of protein (10 μm dimer after mixing). Given the previously establishedK d values of ∼10 μm for these proteins (23.Colpas G.J. Brayman T.G. Ming L.-J. Hausinger R.P. Biochemistry. 1999; 38: 4078-4088Crossref PubMed Scopus (80) Google Scholar), approximately half the metal would be protein-bound with the rest free in solution. Mixing with PAR led to an immediate burst in chromophore development, associated with chelation of the free metal to form the Ni(PAR)2 complex, followed by a slower increase in absorbance. As shown in Table I, the initial phase occurred at a rate (k obs−1 ∼0.2 s−1) matching that observed for protein-free control experiments, whereas the slower changes (k obs−2; e.g. 0.002 s−1for the control sample) represent the dissociation rates for the protein-bound metal. The D111A and H96A variants released Ni2+ 2- and 3-fold more rapidly than the H144*UreE, demonstrating that the in vivo effects of these proteins do not arise from a slow rate of Ni2+ dissociation.Table IRates of metal dissociation for H144*UreE and variantsMetalProteinAdditivek obsAmplitudeμmμmμms −1ΔANi2+(20)H144*UreE (10)None0.200.2620.002150.173Ni2+(20)H96A/H144*UreE (10)None0.10.2650.00580.250Ni2+(20)D111A/H144*UreE (10)None0.160.3290.00440.215Ni2+(20)H144*UreE (10)ImidazoleaThe additive was included with the PAR solution. (200)0.250.3610.00420.176Ni2+(20)H144*UreE (10)HistidineaThe additive was included with the PAR solution. (100)0.360.1010.0260.2520.00600.158Ni2+ (20)NoneImidazolebThe additive was substituted for the protein solution.(200)0.270.5570.0310.271Ni2+ (20)NoneHistidinebThe additive was substituted for the protein solution.(100)0.400.2260.0270.375Ni2+ (20)NoneCysteinebThe additive was substituted for the protein solution.(100)0.590.2550.00520.272Ni2+ (10)His144*UreE (5)None0.140.1650.00680.081Zn2+ (10)His144*UreE (5)None250.140.270.026Co2+(10)H144*UreE (5)None2.10.1240.0510.026Cu2+(10)H144*UreE (5)None0.430.0570.0200.036Ni2+(20)NoneNone0.120.599Buffered solutions containing varied concentrations of the indicated metal plus H144*UreE or its H96A and D111A variants were mixed in a stopped-flow spectrophotometer with equal volumes of a buffered solution containing PAR at 5-fold excess over metal. The values shown are the final concentrations after mixing. In selected experiments, additives were included in the PAR solution or substituted for the protein. The changes in absorbance
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