ISOTOPE-EXCHANGE STUDIES ON THE ESCHERICHIA-COLI SELENOPHOSPHATE SYNTHETASE MECHANISM

Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide, In the absence of selenide, ATP is converted quanti tatively to AMP and two orthophosphates in a very slow partial reactio n, A monophosphorylated enzyme derivative containing the gamma-phospho ryl group of ATP has been implicated as an intermediate from the resul ts of positional isotope exchange studies, Conservation of the phospha te bond energy in the final selenophosphate product is indicated by it s ability to phosphorylate alcohols and amines to form O-phosphoryl- a nd N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-C-14]A DP or [8-C-14]AMP and unlabeled ATP were carried out, and P-31 NMR ana lysis of reaction mixtures enriched in (H2O)-O-18 was performed, A slo w enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction, Under these con ditions ADP is slowly converted to AMP. Incorporation of O-18 from (H2 O)-O-18 exclusively into orthophosphate in the overall selenide-depend ent reaction indicates that the beta-phosphoryl group of the enzyme-bo und ADP is attacked by water with liberation of orthophosphate and for mation of AMP, Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyro phosphorylated enzyme intermediate that was postulated earlier can be excluded.