Mechanistic insights revealed through characterization of a novel chromophore in selenophosphate synthetase from Escherichia coli.
The incorporation of selenium into specific proteins and tRNAs requires selenophosphate (SePO3), whose formation is catalyzed by selenophosphate synthetase. In a Mg/ATP-dependent reaction, selenophosphate synthetase catalyzes the phosphorylation of selenide to yield AMP, inorganic phosphate, and SePO3. In this report, a previously unrecognized chromophore covalently attached to selenophosphate synthetase is characterized. The UV/Vis spectrum of selenophosphate synthetase has a feature centered at 315 nm that is irreversibly destroyed by alkylation. Moreover, addition of Zn2+, which is known to inhibit selenophosphate synthetase, reversibly quenches the 315 nm absorption. Since Zn2+ is known to bind to Cys17, these data strongly suggest that this residue participates in the 315 nm absorption. Upon incubation with both Mg2+ and ATP, the lambda(max) of the chromophore shifts to 340 nm, and it is shown that the shift requires binding of nucleotide having a hydrolyzable gamma-phosphoryl group. These data indicate that either the chromophore is directly involved in phosphoryl transfer or indirectly reflects a phosphorylation-dependent conformational change in selenophosphate synthetase. This work provides the first spectroscopic handle on catalytic steps associated with SePO3 synthesis, which will be used to study the molecular structure of the chromophore and its role in the catalytic mechanism of selenophosphate synthetase.
Wolfe
Matt D
MD
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012, USA. wolfem@nhlbi.nih.gov
D000255:Adenosine Triphosphate / Q000378:metabolism; D000478:Alkylation; D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012643:Selenium / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000378:metabolism
D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D010770:Phosphotransferases; C062762:selenophosphate synthetase; D012643:Selenium; D008274:Magnesium; D015032:Zinc