Selenophosphate Synthetase: Enzyme Labeling Studies with [γ-32P]ATP, [β-32P]ATP, [8-14C]ATP, and [75Se]Selenide

Abstract

Selenophosphate synthetase catalyzes a reaction in which ATP and selenide are converted to H3SeP03, H3P04, and AMP in a 1:1:1 ratio. Selenophosphate is derived from the gamma phosphoryl group and orthophosphate from the beta phosphoryl group of ATP. In the absence of selenide, a slow reaction in which ATP is converted quantitatively to 2 H3P04and AMP occurs. Labeling experiments carried out to detect a putative enzyme-bound pyrophosphate intermediate in the overall reaction showed that up to 0.6 equivalent of the32P label from [γ-32P]ATP was bound to protein under enzyme turnover conditions, but only a negligible amount of32P from [β-32P]ATP was present. Thus, no Enz-PP intermediate was present in a detectable amount under the experimental conditions used. Isolated enzyme samples contained75Se from75Se-labeled selenide and [14C]AMP from [8-14C]ATP in amounts similar to the bound32P from [γ-32P]ATP, suggesting that two of the final products, selenophosphate and AMP, were the radioactive compounds detected in these experiments.