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

Phosphoserine phosphatase belongs to a new class of phosphotransferases for ming an acylphosphate during catalysis and sharing three motifs with P-type ATPases and haloacid dehalogenases, The phosphorylated residue was identif ied as the first aspartate in the first motif (DXDXT) by mass spectrometry analysis of peptides derived from the phosphorylated enzyme treated with Na BH4 or alkaline [O-18]H2O. Incubation of native phosphoserine phosphatase w ith phosphoserine in [O-18]H2O did not result in O-18 incorporation in resi due Asp-20, indicating that the phosphoaspartate is hydrolyzed, as in P-typ e 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, an d D183E), Other mutations inactivated the enzyme by >80% (S109A and G180A) or even by greater than or equal to 99% (D179N, D183N, K158A, and K158R), M utations G178A and D179N decreased the affinity for phosphoserine, suggesti ng that these residues participate in the binding of the substrate. Mutatio ns of Asp-179 decreased the affinity for Mg2+, indicating that this residue interacts with the cation, Thus, investigated residues appear to play an i mportant role in the reaction mechanism of phosphoserine phosphatase, as is known for equivalent residues in P-type ATPases and haloacid dehalogenases .