Related Experiment Video
Updated: Aug 24, 2026

Colorimetric Analysis of Alkaline Phosphatase Activity in S. aureus Biofilm
Published on: April 12, 2019
Phosphate binding in the active site of alkaline phosphatase and the interactions of 2-nitrosoacetophenone with
Le Zhang1, René Buchet, Gérard Azzar
1Universite Claude Bernard Lyon I, UFR Chimie-Biochimie UMR CNRS 5013, 69622 Villeurbanne Cedex, France.
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.
Related Concept Videos
Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Phosphorylation
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Allosteric Regulation
Phosphate Buffer
Sodium dihydrogen phosphate does not fully dissociate in neutral or acidic solutions. When a strong base, such as sodium hydroxide (NaOH), is introduced into the solution, sodium dihydrogen phosphate...
Indirect-Acting Cholinergic Agonists: Mechanism of Action
Reversible inhibitors like edrophonium bind to a specific part of the enzyme called the anionic catalytic site. They form noncovalent bonds, which means they are not strongly attached to the enzyme. This creates a temporary and less stable enzyme–inhibitor complex, leading to...

