Worcester Polytechnic Institute Electronic Theses and Dissertations Collection

Title page for ETD etd-042905-112044


Document Typethesis
Author NameYang, Ying
URNetd-042905-112044
TitleMechanism of Metal delivery and binding to transport sites of Cu+-transporting ATPases
DegreeMS
DepartmentChemistry & Biochemistry
Advisors
  • Josť M.ArgŁello, Advisor
  • Keywords
  • metal transport
  • heavy metal binding
  • Cu+
  • Cys
  • PIB-type ATPase
  • CopA
  • Date of Presentation/Defense2005-05-04
    Availability unrestricted

    Abstract

    CopA, a thermophilic membrane ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across cellular membranes. CopA contains at least two metal binding domains, a regulatory N-terminal Metal Binding Domain (N-MBD) and an occlusion/coordinating metal binding site in the 6th, 7th and 8th transmembrane segments. Previous studies showed that the presence of millimolar concentration of Cys is essential for CopA activity. The high affinity of CopA for metal in the presence of millimolar concentration of Cys suggests a multifaceted interaction of the enzyme with Cys. To elucidate the role of Cys, we studied its effect on the partial reactions of the catalytic cycle of CopA. We observed that 2-50 mM Cys accelerates enzyme turnover with little effect on the Cu+ affinity of CopA. Cys accelerates enzyme phosphorylation, but has no effect on the dephosphorylation rates. Thus, Cys increases steady state phosphoenzyme levels. Besides, Cys has no significant effect on E1¡êE2 equilibrium. Similar results were observed in truncated CopA lacking the N-MBD suggesting that enzyme activation by Cys is independent of the regulatory metal binding sites. These results and the kinetic analysis of activation curves suggest that while Cu+ is delivered to the transport site as a Cu-Cys complex, Cys in the mM range stimulates the ATPase acting as a non-essential activator.

    Files
  • YYang.pdf

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