ABSTRACT
Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate, which enters glycolysis to fulfill the energetic requirements of the organism. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. To support inhibitor design, we determined the crystal structures of the active and inactive forms of human liver glycogen phosphorylase a. During activation, forty residues of the catalytic site undergo order/disorder transitions, changes in secondary structure, or packing to reorganize the catalytic site for substrate binding and catalysis. Knowing the inactive and active conformations of the liver enzyme and how each differs from its counterpart in muscle phosphorylase provides the basis for designing inhibitors that bind preferentially to the inactive conformation of the liver isozyme.
Subject(s)
Liver/enzymology , Phosphorylases/chemistry , Phosphorylases/metabolism , Adenosine Monophosphate/metabolism , Animals , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Diabetes Mellitus/drug therapy , Diabetes Mellitus/metabolism , Drug Design , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Models, Molecular , Muscles/enzymology , Phosphorylases/genetics , Protein Conformation , Protein Structure, Secondary , RabbitsABSTRACT
The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit. However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.
