Title page for ETD etd-0428103-100252

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
José Argüello, Advisor

Keywords
Cu2+
ATPase
CPx
CopB

Date of Presentation/Defense 2000-04-25

Availability unrestricted

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.

Files
ManaCapelli.pdf

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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	José Argüello, Advisor
Keywords	Cu2+ ATPase CPx CopB
Date of Presentation/Defense	2000-04-25
Availability	unrestricted
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.
Files	ManaCapelli.pdf