Observing enzyme ternary transition state analogue complexes by ¹⁹F NMR spectroscopy

Ternary transition state analogue (TSA) complexes probing the isomerization of β-d-glucose 1-phosphate (G1P) into d-glucose 6-phosphate (G6P) catalyzed by catalytically active, fluorinated (5-fluorotryptophan), β-phosphoglucomutase (βPGM) have been observed directly by ¹⁹F NMR spectroscopy. In these complexes MgF₃^- and AlF₄^- are surrogates for the transferring phosphate. However, the relevance of these metal fluorides as TSA complexes has been queried. The 1D ¹⁹F spectrum of a ternary TSA complex presented a molar equivalence between fluorinated enzyme, metal fluoride and non-isomerizable fluoromethylenephosphonate substrate analogue. Ring flips of the 5-fluoroindole ring remote from the active site were observed by both ¹⁹F NMR and X-ray crystallography, but did not perturb function. This data unequivocally demonstrates that the concentration of the metal fluoride complexes is equivalent to the concentration of enzyme and ligand in the TSA complex in aqueous solution.