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

Abstract

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 gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM 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 (Vmax/Km) 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 wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.

Neural regulation of the formation of skeletal muscle phosphorylase kinase holoenzyme in adult and developing rat muscle

“…It is further implied by these data that there is close integration in the expression of the catalytic and regulatory subunits of phosphorylase kinase, so that free γ subunit does not arise. Free γ subunit is highly active [5,53]. If it were to arise in the absence of the regulatory subunits it would result in the rapid phosphorylation of phosphorylase and lead to unregulated and rapid glycogenolysis.…”

Proximal regions of the catalytic γ and regulatory β subunits on the interior lobe face of phosphorylase kinase are structurally coupled to each other and with enzyme activation

“…Although there is not direct evidence that this latter region of the g subunit interacts with the catalytic cleft so as to alter its activity, Owen et al (1995) have pointed out that the I-helix does interact with residues 134 to 138 of the E-helix and that Phe286 and Phe287, which are located in the short helix just C-terminal to the I-helix and are also within the epitope-containing region for the anti-g mAb, help to maintain structure in this region of the molecule through buried side-chain interactions that closely mimic those of the corresponding residues (Trp296 and Phe297) in protein kinase A. They further pointed out that, in a study by Huang et al (1993), truncation of the g subunit at residue 276, at the immediate boundary of the epitope-containing region 277-290, resulted in a decrease in activity to 1/25th that of the catalytic domain truncated at residue 300, suggesting a potential role in the expression of catalytic activity for some of the residues within the stretch from 277 to 300. On the other hand, in assays with full-length isolated g subunit or the gd complex, we did not detect activation by the Fab'-SH fragment of the anti-g mAb (data not shown), which would seem to be more consistent with indirect coupling than direct.…”

The crystal structure of human muscle glycogen phosphorylase a with bound glucose and AMP: An intermediate conformation with T‐state and R‐state features

“…Protein was purified via Cu metal affinity, Q Sepharose, and 5′‐AMP Sepharose chromatography. The purified GP b was incubated with phosphorylase kinase γ subunit 1–30023 at 5 μg/mg GP in the presence of 2.5 mM ATP and 5 mM MgCl 2 . The phosphorylated GP a sample was loaded onto a Superdex 200 column equilibrated with 50 mM B‐glycerophosphate, 100 mM NaCl, 0.2 mM EDTA, 1 mM DTT, pH 7.5.…”