31P nuclear magnetic resonance study of alkaline phosphatase: the role of inorganic phosphate in limiting the enzyme turnover rate at alkaline pH.
31P nuclear magnetic resonance (NMR) was used to directly observe the binding of inorganic phosphate to alkaline phosphatase. Evidencq for the tight binding of 1.5-2.0 mol of inorganic phosphate per dimer of alkaline phosphatase is presented. Two distinct forms of bound phosphate are observed, one predominating above pH 7 and representing the non-covalent E-P1 complex and the other predominating below pH 5 and representing the covalent E-P1 complex. The 31P NMR line width of the E-P1 complex indicates that the dissociation of noncovalent phosphate is the rate-limiting step in the turnover of the enzyme at high pH.
D000469:Alkaline Phosphatase / Q000378:metabolism; D001665:Binding Sites; D002104:Cadmium; D003956:Dialysis; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D010710:Phosphates / Q000494:pharmacology; D010759:Phosphorus Isotopes; D011485:Protein Binding; D011487:Protein Conformation; D015032:Zinc
D008024:Ligands; D010710:Phosphates; D010759:Phosphorus Isotopes; D002104:Cadmium; D000469:Alkaline Phosphatase; D015032:Zinc