title: Kinetic analysis of metal binding to the amino-terminal domain of ZntA by monitoring metal-thiolate charge-transfer complexes.
abstract: ZntA, a P(1B)-ATPase transporter from Escherichia coli, mediates resistance specifically to Pb(2+), Zn(2+), and Cd(2+) by active efflux. ZntA has a hydrophilic N-terminal domain that binds one metal ion. This domain, approximately 120 residues long, contains the GXXCXXC motif that has been shown to be the binding site for metal ions such as Cu(+) and Zn(2+) in P(1B)-type ATPases, and an additional cysteine-rich motif, CCCDGAC. We report here that binding of Pb(2+) and Cd(2+) to this domain produces changes in the absorbance spectrum in the 250-400 nm range indicative of metal-thiolate charge-transfer complexes. The spectral changes indicate that only two cysteines are ligands to Cd(2+), but three or more cysteines are involved in binding Pb(2+); this confirms earlier results that the GXXCXXC sequence is not sufficient to bind Pb(2+), which likely involves residues from the CCCDGAC motif. The absorbance changes were used to measure metal binding kinetics of the N-terminal domain using stopped-flow techniques. Binding was described by simple second-order kinetics with a rate constant, k(on), of approximately 10(6)-10(7) M(-)(1) s(-)(1), at 4 degrees C. The activation energy of binding is similar for both Pb(2+) and Cd(2+); however, the entropy change is greater for Pb(2+). The surprisingly large rate constant for metal binding to the N-terminal domain of ZntA, compared to its low turnover rate, indicates that this step is not rate limiting in the overall transport mechanism. These results, in conjunction with earlier studies, suggest that metal binding to the transmembrane site in ZntA or metal release from the transporter is the slow step in the reaction cycle.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
authors.forename: Sabari J
authors.initials: SJ
authors.lastname: Dutta
authors.forename: Junbo
authors.initials: J
authors.lastname: Liu
authors.forename: Bharati
authors.initials: B
authors.lastname: Mitra
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D007700:Kinetics; D007854:Lead / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013696:Temperature
chemical_list: D008670:Metals; D013438:Sulfhydryl Compounds; D002104:Cadmium; D007854:Lead; D003300:Copper; D000251:Adenosine Triphosphatases; C109898:Zn(II)-translocating P-type ATPase; D003545:Cysteine