Electrostatic changes in phosphorylase kinase induced by its obligatory allosteric activator Ca2+

Skeletal muscle phosphorylase kinase (PhK) is a 1.3-MDa hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase b. PhK has an absolute requirement for Ca2+ ions, which couples the cascade activation of glycogenolysis with muscle contraction. Ca2+ activates PhK by binding to its nondissociable calmodulin subunits; however, specific changes in the structure of the PhK complex associated with its activation by Ca2+ have been poorly understood. We present herein the first comparative investigation of the physical characteristics of highly purified hexadecameric PhK in the absence and presence of Ca2+ ions using a battery of biophysical probes as a function of temperature. Ca2+- induced differences in the tertiary and secondary structure of PhK measured by fluorescence, UV absorption, FTIR, and CD spectroscopies as low resolution probes of PhK's structure were subtle. In contrast, the surface electrostatic properties of solvent accessible charged and polar groups were altered upon the binding of Ca2+ ions to PhK, which substantially affected both its diffusion rate and electrophoretic mobility, as measured by dynamic light scattering and zeta potential analyses, respectively. Overall, the observed physicochemical effects of Ca2+ binding to PhK were numerous, including a decrease in its electrostatic surface charge that reduced particle mobility without inducing a large alteration in secondary structure content or hydrophobic tertiary interactions. Without exception, for all analyses in which the temperature was varied, the presence of Ca2+ rendered the enzyme increasingly labile to thermal perturbation.