Faculty Advisor or Committee Member

José Argüello, Advisor

Identifier

etd-0428103-100252

Abstract

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 (75º 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 µmol/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.

Publisher

Worcester Polytechnic Institute

Degree Name

MS

Department

Chemistry & Biochemistry

Project Type

Thesis

Date Accepted

2003-04-28

Accessibility

Unrestricted

Subjects

Cu2+, ATPase, CPx, CopB, Copper ions, Carrier proteins

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