The protein inhibitor of adenosine 3′,5′-monophosphate-dependent protein kinases the NH 2-terminal portion of the peptide chain contains the inhibitory site

Abstract

The protein inhibitor of adenosine 3′,5′-monophosphate-dependent protein kinases from skeletal muscle was subjected to various chemical and enzymatic treatments in an attempt to delineate the part of the molecule responsible for the interaction with the catalytic subunit of the kinase. Only a small portion of the chain seems to be required since thermolysin and staphylococcal protease digestions do not abolish the inhibitory properties. This inhibitory site must contain the essential arginyl side chain(s), whereas lysyl and carboxylic side chains do not appear to be involved in the interaction with the catalytic subunit. Digestion of the COOH-terminus of the inhibitor by carboxypeptidase Y results in a doubling of the K i value. On the other hand, an inhibitory pentadecapeptide ( K i = 25 nM), presumably NH 2-terminal in the entire molecule, could be isolated from a staphylococcal protease digest by means of gel filtration followed by ion exchange on phosphocellulose. The purified inhibitory peptide contains two out of the four arginyl residues present in the entire molecule. The remarkable affinity and specificity of the protein kinase inhibitor for the catalytic subunit of adenosine 3′,5′-monophosphate-dependent protein kinases may thus be tentatively explained on the basic of a two-prong attachment of the inhibitor. The NH 2-terminal portion of the chain would bind at the substate binding site, whereas the COOH-terminal part would be held elsewhere.