Glycogen, or animal starch, is a storage form of glucose in mammals. In skeletal muscle, activation of the sympathetic nervous system leads to the rapid breakdown of glycogen to provide energy to sustain contraction, a key component of the fight-or-flight response. In this response, the enzyme glycogen phosphorylase catalyzes the phosphorolysis of glycogen to produce glucose-1-P, but only after it has first been activated through phosphorylation by the regulatory enzyme phosphorylase kinase (PhK). For over 50 years this has been thought to be the only function of PhK and its only catalytic activity. We have now discovered that in addition to its kinase activity, PhK is also a glycoside hydrolase (GH) that can directly hydrolyze glycogen to release free glucose. It is this GH activity, which is carried on a different subunit than the kinase activity in the (???)4 PhK complex, that we propose to thoroughly characterize through 4 broad Specific Aims as the necessary first step in elucidating its biological role in health and disease, which is our long-term goal. (1) The physiological role of PhK's GH activity will be assessed by comparing the concentrations of glycogen, glucose and other metabolites in control muscle cells in culture against those found in mutated cells in which the GH activity of PhK has been ablated. This approach will utilize gene editing and unbiased metabolic screens. (2) The ? subunit carries GH activity, and to ablate that activity in order to achieve Aim 1, the two likely GH catalytic residues will be mutated and the resultant protein evaluated. Likewise, mutations in the GH domains of the ? subunit that cause glycogen storage disease (GSD) will be produced to determine if the GSD is caused by perturbed GH activity, as opposed to kinase activity. These studies will exploit a baculovirus- mediated expression system developed in our laboratory. (3) Through standard enzyme assays, we will screen probable mechanisms for how the GH activity might be regulated. PhK's multiple subunits have sites for allosteric activators and phosphorylation and participate in an extensive interaction network that controls its kinase activity. We will determine if the GH activity is similarly allosterically activated. (4) Using standard biochemical and carbohydrate chemistry techniques, we will first characterize the enzymology of this GH activity, particularly its substrae specificity, as glycogen is the only glycoside that has been tested as a substrate and there may be other glycosides that PhK hydrolyzes. We will also determine if the kinase and GH activities can occur simultaneously and how they affect each other. Disease symptoms associated with PhK deficiency (Type IX GSD) or elevation (psoriatic epidermis) has always been considered assuming that PhK acts only as a kinase. The possibility that PhK may also function biologically as a GH that directly hydrolyzes glycogen to form glucose requires re-evaluation of the biological effects with which it has been associated, especially given that both its kinase and GH activities result in the catabolism of glycogen. A biological GH function for PhK would also necessitate reconsideration of the clinical management of GSD's in general, not just Type IX.
Glycogen is the storage form of glucose in mammals and can be readily mobilized when needed. A key enzyme for this mobilization is phosphorylase kinase (PhK), which activates an enzyme that catabolizes glycogen. We have now found, however, that PhK itself can directly hydrolyze glycogen, releasing free glucose. This challenges our textbook view of PhK and necessitates reconsideration of the clinical management of all glycogen storage diseases.