Properties of a Mr = 38,000 phosphoprotein phosphatase. Modulation by divalent cations, ATP, and fluoride.

A Mr = 38,000 phosphoprotein phosphatase (EC 3.1.3.16) was purified to near homogeneity from bovine cardiac muscle. The enzyme, classified as a type 2 phosphatase, was not inhibited by the heat-stable protein, inhibitor-2. Activity on peptide substrates was stimulated considerably by Mn2+ ions. The individual and combined effects of divalent cations, ATP, and fluoride were studied in detail employing the phosphonanopeptide Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Gln-Leu as the substrate. ATP and fluoride inhibited enzyme activity completely in the absence of divalent cations. Mg2+ either reduced or completely prevented this inhibition depending upon whether Mn2+ was present. Quantitative analysis of the results revealed that ATP and fluoride do not inhibit by chelation of an essential metal (e.g. Mn2+). Rather, a plausible model for the combined effects of Mg2+, Mn2+, ATP, and fluoride on phosphatase activity must assume that each of these factors acts by binding to individual sites on the enzyme and not to each other.