Spectroscopic investigation of the reaction mechanism of CopB-B, the catalytic fragment from an archaeal thermophilic ATP-driven heavy metal transporter.
The mechanism of ATP hydrolysis of a shortened variant of the heavy metal-translocating P-type ATPase CopB of Sulfolobus solfataricus was studied. The catalytic fragment, named CopB-B, comprises the nucleotide binding and phosphorylation domains. We demonstrated stoichiometric high-affinity binding of one nucleotide to the protein (K(diss) 1-20 microm). Mg is not necessary for nucleotide association but is essential for the phosphatase activity. Binding and hydrolysis of ATP released photolytically from the caged precursor nitrophenylethyl-ATP was measured at 30 degrees C by infrared spectroscopy, demonstrating that phosphate groups are not involved in nucleotide binding. The hydrolytic kinetics was biphasic, and provides evidence for at least one reaction intermediate. Modelling of the forward reaction gave rise to three kinetic states connected by two intrinsic rate constants. The lower kinetic constant (k(1) = 4.7 x 10(-3) s(-1) at 30 degrees C) represents the first and rate-limiting reaction, probably reflecting the transition between the open and closed conformations of the domain pair. The subsequent step has a faster rate (k(2) = 17 x 10(-3) s(-1) at 30 degrees C), leading to product formation. Although the latter appears to be a single step, it probably comprises several reactions with presently unresolved intermediates. Based on these data, we suggest a model of the hydrolytic mechanism.
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, Germany.
Christian
C
Völlmecke
Carsten
C
Kötting
Klaus
K
Gerwert
Mathias
M
Lübben
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D010782:Photolysis; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D016944:Sulfolobus / Q000201:enzymology*
D019843:Archaeal Proteins; D027682:Cation Transport Proteins; C489503:CopB protein, Archaea; D000079922:Copper Transport Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases