Binding of ATP and its derivatives to selenophosphate synthetase from Escherichia coli

Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS238), 262 (SPS262), and 332 (SPS332) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS262 and SPS332) have been purified to homogeneity. SPS238 has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS332 mutant and one TNP-ATP binding site for SPS238 mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [14C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS332, SPS262, and SPS238 were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS238 likely has one ATP-binding site removed by truncation.