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 (SPS ₂₆₂), and 332 (SPS₃₃₂) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS ₂₆₂ 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.