F-19 NMR INVESTIGATIONS OF THE CATALYTIC MECHANISM OF PHOSPHOGLUCOMUTASE USING FLUORINATED SUBSTRATES AND INHIBITORS

The complexes of phosphoglucomutase with a number of fluorinated substrate analogues have been investigated by F-19 NMR and the effects of the binding of Li+ and Cd2+ to these complexes determined. Very large downfield chemical shift changes (-14 to -19 ppm) accompanied binding of the inhibitors 6-deoxy-6-fluoro-alpha-D-glucopyranosyl phosphate and alpha-glucosyl fluoride 6-phosphate to the phosphoenzyme. Smaller shift changes were observed for ligands substituted with fluorine at other positions. Addition of Li+ to enzyme/fluorinated ligand complexes caused a 10(2)- to 10(3)-fold decrease in ligand dissociation constants as witnessed by the change from intermediate to slow-exchange conditions in the NMR spectra. Measurement of the F-19 NMR spectra of complexes of the Li+-enzyme with each of the fluoroglucose I-phosphates and 6-phosphates has provided some insight into the environment of each of these fluorines (thus also parent hydroxyls) in each of the complexes. Results obtained argue strongly against a single sugar binding mode for the glucose 1- and 6-phosphates. Two enzyme-bound species were detected in the F-19 NMR spectra of the complexes formed by reaction of the Cd2+-phosphoenzyme complex with the 2- and 3-fluoroglucose phosphates. These are tentatively assigned as the fluoroglucose 1,6-bisphosphate species bound in two different modes to the dephosphoenzyme. Only one bound species was observed in the case of the 4-fluoroglucose phosphates. The results from this investigation, and those above, are consistent with an exchange type of mechanism {[}Ray, W. J., Mildvan, A. S., & Long, J. W. (1973) Biochemistry 12, 3724] for the enzyme in which there are two distinct glucose ring binding sites.