title: Cooperative binding is not required for activation of muscle phosphorylase.
abstract: Muscle and liver glycogen phosphorylase isozymes differ in their responsiveness to the activating ligand AMP. The muscle enzyme, which supplies glucose in response to strenuous activity, binds AMP cooperatively, and its enzymatic activity becomes greatly enhanced. The liver isozyme regulates the level of blood glucose, and AMP is not the primary activator. In muscle glycogen phosphorylase, the residue proline 48 links two secondary structural elements that bind AMP. This amino acid residue is replaced with a threonine in the liver isozyme; unlike the muscle enzyme, liver binds AMP noncooperatively, and the enzymatic activity is not greatly increased. We have substituted proline 48 in the muscle enzyme with threonine, alanine, and glycine and characterized the recombinant enzymes kinetically and structurally to determine if proline at this position is critical for cooperative AMP binding and activation. Importantly, all of the engineered enzymes were fully activated by phosphorylation, indicating that enzymatic activity was not compromised. Only the mutant enzyme with alanine at position 48 responds like the wild-type enzyme to the presence of AMP, indicating that proline is not absolutely required for full cooperative activation. The substitution of either threonine or glycine at this position, however, creates enzymes that no longer bind AMP cooperatively. The enzyme with threonine at position 48 further mimics the liver enzyme, in that the maximal enzymatic activity is also reduced. Significantly, the glycine substitution caused the enzyme to be fully activated by AMP, although binding was not cooperative. The hyperactivation of the glycine mutant by AMP suggests that the total free energy of activation has decreased.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94141-0448.
authors.forename: M F
authors.initials: MF
authors.lastname: Browner
authors.forename: P K
authors.initials: PK
authors.lastname: Hwang
authors.forename: R J
authors.initials: RJ
authors.lastname: Fletterick
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D006005:Phosphorylases / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D014961:X-Ray Diffraction
chemical_list: D000249:Adenosine Monophosphate; D006005:Phosphorylases