This chapter describes a procedure for the chemical synthesis of O,O,O trimethylsilylselenophosphate and its hydrolysis under anaerobic conditions in aqueous buffers. Selenophosphate, either 75Se-labeled or nonradioactive, is synthesized under strictly anaerobic conditions from ATP and selenide by selenophosphate synthetase, isolated from an overproducing Excherichia coli strain. Selenophosphate synthetase (seID gene product) is a 37-kDa protein that contains 7 cysteine residues, at least one of which is essential for catalytic activity. The enzyme does not use sulfide as substrate, and other common nucleoside triphosphates do not replace ATP. Although the selenophosphate product formed by the enzyme is extremely oxygen labile, selenophosphate synthetase is not inactivated by exposure to oxygen. No loss of activity was detected after passage of a stream of oxygen through a solution of the enzyme in pH 7.2 buffer for 10 min in the absence of added reducing agent. Although sensitive to treatment with H202, a relatively high concentration (3 mM) was required to destroy 50% of the activity during 30 min.
ASJC Scopus subject areas
- Molecular Biology