PMID:8262938
| Citation |
Kim, IY, Veres, Z and Stadtman, TC (1993) Biochemical analysis of Escherichia coli selenophosphate synthetase mutants. Lysine 20 is essential for catalytic activity and cysteine 17/19 for 8-azido-ATP derivatization. J. Biol. Chem. 268:27020-5 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/chemistry; Adenosine Triphosphate/metabolism; Affinity Labels; Amino Acid Sequence; Azides/chemistry; Bacterial Proteins/antagonists & inhibitors; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Catalysis; Cysteine/genetics; Cysteine/metabolism; Drosophila Proteins; Escherichia coli/enzymology; Lysine/genetics; Lysine/metabolism; Metals/pharmacology; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphotransferases; Substrate Specificity |
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