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 (Kis= 8 nM). Inhibitor‐1 did not inhibit its own dephosphorylation in the presence of Mn2+. Its Km as substrate (190 nM) as very much higher than its Ki 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. Copyright © 1983, Wiley Blackwell. All rights reserved