Expression, purification, characterization, and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

Abstract

A catalytic fragment,γ _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the γ subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the γ subunit, full-length wild-type and seven truncated forms of γ were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar (γ _1-353 andγ _1-341) or less (γ _1-331 andγ _1-276) specific activity than does the full-length wild-type γ,γ _1-386. Three truncated forms,γ _1-316,γ _1-300, andγ _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type γ and the nonactivated holoenzyme. All recombinant γs exhibit similarK _m values for both substrates, i.e., about 18μM for phosphorylaseb and about 75 μM for MgATP. Three truncated γs,γ _1-316,γ _1-300, andγ _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type γ and a 3.5- to 4.5-fold greater efficiency than that of the truncatedγ _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of γ, which is located atγ _301-331·γ _1-290, but notγ _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the γ subunit to exhibit phosphotransferase activity. Bothγ _1-290 andγ _1-300 have several properties similar to full-length wild-type γ, including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.