title: Phosphate binding in the active site of alkaline phosphatase and the interactions of 2-nitrosoacetophenone with alkaline phosphatase-induced small structural changes.
abstract: To monitor structural changes during the binding of Pi to the active site of mammalian alkaline phosphatase in water medium, reaction-induced infrared spectroscopy was used. The interaction of Pi with alkaline phosphatase was triggered by a photorelease of ATP from the inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP. After photorelease, ATP was sequentially hydrolyzed by alkaline phosphatase giving rise to adenosine and three Pi. Although a phosphodiesterase activity was detected prior the photorelease of ATP, it was possible to monitor the structural effects induced by Pi binding to alkaline phosphatase. Interactions of Pi with alkaline phosphatase were evidenced by weak infrared changes around 1631 and at 1639 cm(-1), suggesting a small distortion of peptide carbonyl backbone. This result indicates that the motion required for the formation of the enzyme-phosphate complex is minimal on the part of alkaline phosphatase, consistent with alkaline phosphatase being an almost perfect enzyme. Photoproduct 2-nitrosoacetophenone may bind to alkaline phosphatase in a site other than the active site of bovine intestinal alkaline phosphatase and than the uncompetitive binding site of L-Phe in bovine intestinal alkaline phosphatase, affecting one-two amino acid residues.
authors.affiliation: Universite Claude Bernard Lyon I, UFR Chimie-Biochimie UMR CNRS 5013, 69622 Villeurbanne Cedex, France.
authors.forename: Le
authors.initials: L
authors.lastname: Zhang
authors.forename: René
authors.initials: R
authors.lastname: Buchet
authors.forename: Gérard
authors.initials: G
authors.lastname: Azzar
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D010710:Phosphates / Q000737:chemistry*; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared
chemical_list: D000596:Amino Acids; D010710:Phosphates; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase