Biochemical analysis of Escherichia coli selenophosphate synthetase mutants. Lysine 20 is essential for catalytic activity and cysteine 17/19 for 8-azido-ATP derivatization.
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.
Kim
I Y
IY
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Veres
Z
Z
Stadtman
T C
TC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000201:enzymology*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases; D013379:Substrate Specificity
D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins; D029721:Drosophila Proteins; D008670:Metals; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C062762:selenophosphate synthetase; D008239:Lysine; D003545:Cysteine