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 P i to the active site of mammalian alkaline phosphatase in water medium, reaction-induced infrared spectroscopy was used. The interaction of P i 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 P i. Although a phosphodiesterase activity was detected prior the photorelease of ATP, it was possible to monitor the structural effects induced by P i binding to alkaline phosphatase. Interactions of P i 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.