Abstract
A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A divalent metal ion, Mg2+, and a monovalent cation, K+, NH4+, or Rb+, are required for selenophosphate synthetase activity [Veres, Z., Kim, I. Y., Scholz, T. D. & Stadtman, T. C. (1994) J. Biol. Chem. 269, 10597-10603]. Na+ and Li+ are ineffective as activators and in the presence of K+ are inhibitory. Mn-ATP, although not able to replace Mg-ATP for catalytic activity, binds to the enzyme provided an active monovalent cation is present. No Mn-ATP is bound when K+ is replaced with Na+. The requirement for K+, both for Mn-ATP binding and for catalytic activity of the synthetase, indicates a specific monovalent cation-induced conformational state of the enzyme. Previously we reported that activity of the enzyme is markedly inhibited by micromolar levels of Zn2+ in the presence of millimolar levels of Mg2+ [Kim, I. Y., Veres, Z. & Stadtman, T. C. (1993) J. Biol. Chem. 268, 27020-27025]. Binding of Mn-ATP also is decreased upon addition of Zn2+, indicating that the inhibitory effect of Zn2+ is exerted at the substrate-binding step of the overall selenophosphate synthetase reaction. When a cysteine residue at position 17 or 19 is replaced with serine, Mn-ATP binding to these mutant enzymes is unaffected by Zn2+ addition. Direct involvement of these cysteine residues in the zinc binding site was shown by use of 65ZnCl2. Radioactive Zn2+ bound to wild-type enzyme and was retained after gel filtration, but under the same conditions the catalytically inactive Cys-17 mutant protein and the catalytically active Cys-19 mutant enzyme were unlabeled.