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 NMR spectroscopy was used to study a fully active Escherichia coli fluorotyrosine alkaline phosphatase (EC-3.1.3.1). The fluorotyrosine resonances provide sensitive probes of the conformational stages of the protein. They were used to follow the addition of Zn or Co to the apoprotein and the titration of the protein with Pi. or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate. Two molecules of Pi / 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 1 specific tyrosine/subunit may be near the metal site. The 19F NMR results, combined with 31P NMR results, lead directly to the conclusion that dissociation of noncovalently bound Pi. from the enzyme is the rate-limiting process in enzyme catalysis at high pH. The local environment of the individual fluorotyrosines is also discussed.