Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases

J Biol Chem. 1999 Nov 26;274(48):33985-90. doi: 10.1074/jbc.274.48.33985.


Phosphoserine phosphatase belongs to a new class of phosphotransferases forming an acylphosphate during catalysis and sharing three motifs with P-type ATPases and haloacid dehalogenases. The phosphorylated residue was identified as the first aspartate in the first motif (DXDXT) by mass spectrometry analysis of peptides derived from the phosphorylated enzyme treated with NaBH(4) or alkaline [(18)O]H(2)O. Incubation of native phosphoserine phosphatase with phosphoserine in [(18)O]H(2)O did not result in (18)O incorporation in residue Asp-20, indicating that the phosphoaspartate is hydrolyzed, as in P-type ATPases, by attack of the phosphorus atom. Mutagenesis studies bearing on conserved residues indicated that four conservative changes either did not affect (S109T) or caused a moderate decrease in activity (G178A, D179E, and D183E). Other mutations inactivated the enzyme by >80% (S109A and G180A) or even by >/=99% (D179N, D183N, K158A, and K158R). Mutations G178A and D179N decreased the affinity for phosphoserine, suggesting that these residues participate in the binding of the substrate. Mutations of Asp-179 decreased the affinity for Mg(2+), indicating that this residue interacts with the cation. Thus, investigated residues appear to play an important role in the reaction mechanism of phosphoserine phosphatase, as is known for equivalent residues in P-type ATPases and haloacid dehalogenases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism*
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Aspartic Acid / metabolism
  • Borohydrides / pharmacology
  • Catalysis
  • Humans
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Mass Spectrometry / methods
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction / drug effects
  • Oxygen Isotopes
  • Phosphoproteins / metabolism
  • Phosphoric Monoester Hydrolases / chemistry
  • Phosphoric Monoester Hydrolases / genetics
  • Phosphoric Monoester Hydrolases / metabolism*
  • Phosphorylation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Trypsin / metabolism
  • Water / metabolism


  • Borohydrides
  • Oxygen Isotopes
  • Phosphoproteins
  • Recombinant Proteins
  • Water
  • Aspartic Acid
  • Phosphoric Monoester Hydrolases
  • phosphoserine phosphatase
  • Trypsin
  • Adenosine Triphosphatases