title: Mutations in paired alpha-helices at the subunit interface of glycogen phosphorylase alter homotropic and heterotropic cooperativity.
abstract: Allosteric switching between inactive and active conformational states in muscle glycogen phosphorylase alters the pattern of van der Waals contacts and hydrogen bonds between two helices located at the dimer interface of the enzyme. Alanine was substituted for residues N270, N274, and R277 to perturb helix interactions, which differ in inactive and active conformations. In addition, the entire alpha-helix in each subunit was exchanged with the analogous region from yeast phosphorylase. The N274A mutant shows increased affinity and reduced cooperativity for the activator, AMP, and reduced cooperativity for the substrate, glucose 1-phosphate. The N270A and R277A mutants, in contrast, show reduced binding and cooperativity for AMP and relatively little change in binding or cooperativity for glucose 1-phosphate. The substitution of the helix from the yeast enzyme results in an 8-fold reduction in Vmax, a loss in cooperativity for both AMP and glucose 1-phosphate, but little change in the affinities of either ligand. Crystallographic analyses of the N274A and R277A mutants show that these substitutions cause only small changes in the structure of the unliganded, inactive form of phosphorylase. The substitution at N274 eliminates intersubunit interactions which selectively stabilize the enzyme in an inactive conformation. The kinetic results indicate that the mutations at N270 and R277, on the contrary, perturb packing interactions at the dimer interface of the activated enzyme and weaken binding of AMP. The relatively modest effects of the replacement with the helices from the yeast enzyme indicate that the helices are not crucial for catalytic function.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Buchbinder
authors.forename: J J
authors.initials: JJ
authors.lastname: Guinovart
authors.forename: R J
authors.initials: RJ
authors.lastname: Fletterick
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D006860:Hydrogen Bonding; D007700:Kinetics; D006005:Phosphorylases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits
chemical_list: D006005:Phosphorylases