title: Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens.
abstract: TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P(1B)-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E(A)=38kJ⋅mol(-1)) over the whole concentration range while it increased from 17 to 42kJ⋅mol(-1) with rising copper concentration and decreased from 39 to 23kJ⋅mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.
authors.affiliation: Universität Konstanz, Mathematisch-Naturwissenschaftliche Sektion, Fachbereich Biologie, Konstanz, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Leitenmaier
authors.forename: Annelie
authors.initials: A
authors.lastname: Witt
authors.forename: Annabell
authors.initials: A
authors.lastname: Witzke
authors.forename: Anastasia
authors.initials: A
authors.lastname: Stemke
authors.forename: Wolfram
authors.initials: W
authors.lastname: Meyer-Klaucke
authors.forename: Peter M H
authors.initials: PM
authors.lastname: Kroneck
authors.forename: Hendrik
authors.initials: H
authors.lastname: Küpper
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D001703:Biophysics; D002104:Cadmium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000378:metabolism*
chemical_list: D002104:Cadmium; D000251:Adenosine Triphosphatases; D015032:Zinc