A Kinetic Analysis of the Effects of Inhibitor-1 and Inhibitor-2 on the Activity of Protein Phosphatase-1

Abstract

The steady-state interaction between protein phosphatase-1 and its two inhibitor proteins was studied in vitro at low enzyme concentrations where the assumptions of the Michaelis-Menten equation appeared to be valid. Under these conditions, and in the absence of divalent cations, inhibitor-1 behaved as a mixed inhibitor using phosphorylase α as a substrate, whereas inhibitor-2 was a competitive inhibitor. The results demonstrate that inhibitio-1 and inhibitor-2 do not interact with protein phosphatase-1 in an identical manner. Inhibitor-1 was only a substrate for protein phosphatase-1 in the presence of Mn²⁺, and its dephosphorylation was inhibited competitively by inhibitor-2 (K_is= 8 nM). Inhibitor-1 did not inhibit its own dephosphorylation in the presence of Mn²⁺. Its K_m as substrate (190 nM) as very much higher than its K_i as an inhibitor (1.5 - 7.5 nM). The results are consitent with a modle in which a single binding site for inhibitor-1 is present on protein phosphatase-1, distinct from the binding site for phosphorylase α. It is envisaged that the binding of inhibitor-1 to this site not only inhibits the dephosphorylation of other substrates but permits access of its phosphothreonine to the same catalytic group(s) responsible for the dephosphorylation of other substrates. G-substrate, a protein phosphorylated exlusively on threonine residues, did not inhibit the dephosphorylation of phosphorylase α and its dephosphorylation was potently inhibited by inhibitor-1 or inhibitor-2. The role of the phosphothreonine residue in inhibitor-1 is discussed in the light of these results.