TAILIEUCHUNG - Báo cáo khoa học: 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 ofSulfolobus 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 (Kdiss 1–20lm). Mg is not necessary for nucleotide association but is essential for the phospha-tase activity. | Spectroscopic investigation of the reaction mechanism of CopB-B the catalytic fragment from an archaeal thermophilic ATP-driven heavy metal transporter Christian Vollmecke Carsten Kotting Klaus Gerwert and Mathias Liibben Lehrstuhlfur Biophysik Ruhr-Universitat Bochum Germany Keywords fluorescence spectroscopy Fourier-transform infrared spectroscopy heavy metal translocation P-type ATPase reaction mechanism Correspondence M. Lubben Lehrstuhl fur Biophysik Ruhr-Universitat Bochum Universitatsstr. 150 D-44780 Bochum Germany Fax 49 234 32 14626 Tel 49 234 32 24465 E-mail luebben@ Received 14 May 2009 revised 24 July 2009 accepted 21 August 2009 doi 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 Kdiss 1-20 pM . 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 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 k1 X 10-3 s-1 at 30 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 k2 17 X 10-3 s-1 at 30 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

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