Kinetic characterization of the calmodulin-activated catalytic subunit of phosphorylase kinase.

The phosphorylase kinase holoenzyme from skeletal muscle is composed of a catalytic and three different regulatory subunits. Analysis of the kinetic mechanism of the holoenzyme is complicated because both the natural substrate phosphorylase b and also phosphorylase kinase itself have allosteric binding sites for adenine nucleotides. In the case of the kinase, these allosteric sites are not on the catalytic subunit. We have investigated the kinetic mechanism of phosphorylase kinase by using its isolated catalytic gamma-subunit (activated by calmodulin) and an alternative peptide substrate (SDQEKRKQISVRGL) corresponding to the convertible region of phosphorylase b, thus eliminating from our system all known allosteric binding sites for nucleotides. This peptide has been previously employed to study the kinetic mechanism of the kinase holoenzyme before the existence of the allosteric sites on the regulatory subunits was suspected [Tabatabai, L. B., & Graves, D. J. (1978) J. Biol. Chem. 253, 2196-2202]. This peptide was determined to be as good an alternative substrate for the isolated catalytic subunit as it was for the holoenzyme. Initial velocity data indicated a sequential kinetic mechanism with apparent Km's for MgATP and peptide of 0.07 and 0.47 mM, respectively. MgADP used as product inhibitor showed competitive inhibition against MgATP and noncompetitive inhibition against peptide, whereas with phosphopeptide as product inhibitor, the inhibition was competitive against both MgATP and peptide. The initial velocity and product inhibition studies were consistent with a rapid equilibrium random mechanism with one abortive complex, enzyme-MgADP-peptide. The substrate-directed, dead-end inhibitors 5'-adenylyl imidodiphosphate and Asp-peptide, in which the convertible Ser of the alternative peptide substrate was replaced with Asp, were competitive inhibitors toward their like substrates and noncompetitive inhibitors toward their unlike substrates, further supporting a random mechanism, which was also the conclusion from the report cited above that used the holoenzyme.