Selenophosphate synthetase from Escherichia coli produces selenophosphate from ATP and selenide. Selenophosphate is a biologically active selenium donor that is required for the specific insertion of selenocysteine into proteins as directed by the UGA codon, and is also essential for a posttranscriptional tRNA modification of some tRNAs involving substitution of selenium for sulfur. Indirect evidence suggests a multistep reaction involving the formation of an enzyme-pyrophosphate intermediate. However, previous attempts to detect the postulated enzyme-pyrophosphate derivative were not successful. To analyze the catalytical reaction mechanism, selenophosphate synthetase was incubated with [g-P-32]ATP in the presence of selenide and then separated from reagents by gel filtration. After treatment of P-32-labeled protein with iodine, a known reagent to induce cleavage of thiophosphates, the amount of P-32 incorporated was decreased from 0.61 to 0.40 equivalent/mole. The bell-shaped pH lability profile of the phospho-enzyme resembles that of a thio-phosphate ester linkage. These results suggest that one modified form of the enzyme is a thiophosphate derivative. In contrast to the P-32-labeled enzyme, the protein labeled with C-14 (0.40 equivalent/mole) by reaction with [8-14-C]ATP was unaffected by treatment with iodine. Se-75-labeled enzyme formed by reaction with KSe-75H was isolated under the same conditions as P-32- labeled protein. Cys-17, which is located in a glycine-rich region of the polypeptide and is essential for the catalytic reaction, is required both for P-32 and Se-75 labelling. Attempts to identify the phosphorylation and selenium binding site(s) by digesting P-32 or Se-75- labeled enzyme with proteases, however, were unsuccessful because of the extreme lability of the intermediate(s). In situ P-31 and Se-77 NMR studies to characterize the intermediate(s) and crystallization of this enzyme are in progress.