Document Type thesis Author Name Mana Capelli, Sebastian C. URN etd-0428103-100252 Title CopB from Archaeoglobus fulgidus: a thermophilic Cu2+ transporting CPx-ATPase Degree MS Department Chemistry & Biochemistry Advisors JosÚ ArgŘello, Advisor Keywords Cu2+ ATPase CPx CopB Date of Presentation/Defense 2000-04-25 Availability unrestricted
In this work we present the first characterization of a Cu2+-transporting ATPase. The thermophilic bacteria Archaeoglobus fulgidus contains two genes, CopA and CopB, encoding for CPx-ATPases. CopB belongs to the subgroup IB-4 of the CPX-ATPases. These enzymes are characterized by a CPH motif in the 6th transmembrane domain and a His-rich N-terminus metal binding domain (MBD). CopB was heterologously expressed in E. coli. Membranes were prepared and used to measure activity. CopB was active at high temperature (75┬║ C), high ionic strength and pH 5.7. The enzyme was activated by Cu2+, and in to a lesser extent by Ag+ and Cu+. CopB showed a Vmax = 5 ┬Ámol/mg/h and a high apparent affinity (K1/2 = 0.28 ┬▒ 0.09 μM) for Cu2+. Uptake of 64Cu2+ into everted vesicles was also measured in order to show that Cu2+ is not only activating the enzyme but being transported. Compared with CopB-WT, CopB-T (lacking the N-terminus MBD) did not show any difference in its activation by the different metal ions, demonstrating that the cytoplasmic MBD has no role in the metal selectivity. CopB-T also showed a 40 % decrease in the ATPase activity. CopB-WT and CopB-T presented similar levels of phosphorylation. However, CopB-T exhibited a reduced rate of dephosphorylation (slower transition from the E2P to the E2 conformation). These observations suggest a regulatory role for the cytoplasmic MBD.
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