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 quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-14C]ADP or [8-14C]AMP and unlabeled ATP were carried out, and 31P NMR analysis of reaction mixtures enriched in H218O was performed. A slow 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 conditions ADP is slowly converted to AMP. Incorporation of 18O from H218O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation 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 pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.
Walker
H
H
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Ferretti
J A
JA
Stadtman
T C
TC
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002852:Chromatography, Ion Exchange; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010103:Oxygen Isotopes; D010758:Phosphorus; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D014867:Water
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D029721:Drosophila Proteins; D010103:Oxygen Isotopes; D014867:Water; D010758:Phosphorus; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C062762:selenophosphate synthetase