title: Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.
abstract: It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.
authors.affiliation: Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece. nikos@apollon.servicenet.ariadne-t.gr
authors.forename: N G
authors.initials: NG
authors.lastname: Oikonomakos
authors.forename: S E
authors.initials: SE
authors.lastname: Zographos
authors.forename: K E
authors.initials: KE
authors.lastname: Tsitsanou
authors.forename: L N
authors.initials: LN
authors.lastname: Johnson
authors.forename: K R
authors.initials: KR
authors.lastname: Acharya
mesh_terms: D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation; D005459:Fluorides / Q000378:metabolism; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D017905:Phosphites / Q000378:metabolism; D010763:Phosphorylase b / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits
chemical_list: D010710:Phosphates; D017905:Phosphites; C012980:fluorophosphate; D010763:Phosphorylase b; D005459:Fluorides