EXPRESSION, PURIFICATION, CHARACTERIZATION, AND DELETION MUTATIONS OF PHOSPHORYLASE-KINASE GAMMA-SUBUNIT - IDENTIFICATION OF AN INHIBITORY DOMAIN IN THE GAMMA-SUBUNIT

A catalytic fragment, gamma(1-298), derived from limited chymotryptic digestion of phosphorylase b 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 gamma subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E. 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 (gamma(1-353) and gamma(1-341)) or less (gamma(1-331) and gamma(1-276)) specific activity than does the full-length wild-type gamma, gamma(1-386). Three truncated forms, gamma(1-316), gamma(1-300), and gamma(1-290) have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme. All recombinant ys exhibit similar K-m values for both substrates, i.e., about 18 mu M for phosphorylase b and about 75 mu M for MgATP. Three truncated gamma s, gamma(1-316), gamma(1-300), and gamma(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 gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma(1-331). This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma(301-331). gamma(1-290), but not gamma(1-276), which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity. Both gamma(1-290) and gamma(1-300) have several properties similar to full-length wildtype gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.