A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A cysteine residue (Cys-17) that is essential for catalytic activity of the enzyme (Kim, I.Y., Veres, Z., and Stadtman, T. C. (1992) J. Biol. Chem. 267, 19650-19654) is located in a glycine-rich segment near the N terminus of the protein. The possibility that this peptide sequence (HGAGCGCK) defines the ATP-binding site of the enzyme, as does a conserved ATP or GTP binding sequence (GXXXXGKS/T) found in several other proteins, was tested by site-specific mutagenesis. Thus His-13 and Gly-18 were changed to Asn and Val, respectively, and Lys-20 to Arg or Gln. Catalytic activity was markedly decreased by mutation of Lys-20 to Arg and abolished by mutation of Lys-20 to Gln. The mutation of Cys-19 and His-13 did not substantially alter the ATP Km and Vmax values, whereas the Gly-18 mutation resulted in a 4-fold increase in the ATP Km value compared with that of the wild type. ATP binding properties of the mutant enzymes were determined using Mn-[32P]ATP or Mn-[14C]ATP and gel filtration. Photoaffinity labeling of the proteins with [gamma-32P]8-azido-ATP showed that all mutant enzymes could be labeled with the ATP analog except those in which Cys-17 or Cys-19 were replaced with serine.