Protein phosphatase 1 catalyses the direct hydrolytic cleavage of phosphate monoester in a ternary complex mechanism

The catalytic subunit of the Ser/Thr protein phosphatase 1 (PP1cat) hydrolyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala (KM=3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (Pi, Ki=1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of 18O-label from 18O-labelled water into Piwhether, or not, the product alcohol is present. The dephosphorylated product alcohol of phosphorylated histone, an alternative substrate for the enzyme, serves as a competitive inhibitor for phosphopeptide hydrolysis (Ki=60 μM) and co-mediates 18O-label exchange into Pi in a concentration-dependent manner (KM=64 μM). These results indicate that hydrolysis occurs through the direct attack of an activated water molecule on the phosphate ester moiety of the substrate in a ternary complex mechanism.