Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding.

19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E. coli fluorotyrosine alkaline phosphatase. The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein. They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate. The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines. Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site. The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH. The local environment of the individual fluorotyrosines is also discussed.