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 (PP1(cat)) hydro lyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala (K-M = 3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (P-i, K-i = 1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of O-18-label from O-18-lab elled water into P-i whether, or not, the product alcohol is present. The d ephosphorylated product alcohol of phosphorylated histone, an alternative s ubstrate for the enzyme, serves as a competitive inhibitor for phosphopepti de hydrolysis (K-i = 60 muM) and co-mediates O-18-label exchange into P-i i n a concentration-dependent manner (K-M = 64 muM). These results indicate t hat hydrolysis occurs through the direct attack of an activated water molec ule on the phosphate ester moiety of the substrate in a ternary complex mec hanism. (C) 2001 Elsevier Science Ltd. All rights reserved.