ABSTRACT
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 (SPS(238)), 262 (SPS(262)), and 332 (SPS(332)) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS(262) and SPS(332)) have been purified to homogeneity. SPS(238) 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 SPS(332) mutant and one TNP-ATP binding site for SPS(238) mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [(14)C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS(332), SPS(262), and SPS(238) 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; SPS(238) likely has one ATP-binding site removed by truncation.
