ENZYME-BOUND INTERMEDIATES IN THE CONVERSION OF GLUCOSE 1-PHOSPHATE TO GLUCOSE-6-PHOSPHATE BY PHOSPHOGLUCOMUTASE - PHOSPHORUS NMR-STUDIES

The interactions between metal ions and the phospho form of rabbit muscle phosphoglucomutase (EC 2.7.5.1) were studied by 31P NMR. In the metal-free enzyme, the width at half-height of the 31P signal is 10 .+-. 1 Hz at 81 MHz. In enzyme.sbd.Cd2+ complexes, the presence of spin-spin coupling with 113Cd2+ (J113Cd-O-31P = 16 Hz) and the absence of such splitting with 114Cd2+ indicate that Cd2+ binds directly to the enzymic phosphate. The absence of detectable splitting on transfer of the phosphate group to the acceptor hydroxyl group of bound glucose 1-phosphate, or glucose 6-phosphate (to give the 113Cd2+ complex of the dephosphoenzyme and glucose 1,6-bisphosphate), indicates that this transfer eliminates the direct metal ion-phosphate interaction. The enzyme-catalyzed reaction is slowed sufficiently by the addition of Li+ to allow studies of 3 discrete intermediate complexes by NMR techniques: glucose 1-phosphate bound to the phosphoenzyme, glucose 1,6-bisphosphate bound to the dephosphoenzyme (only 1 complex of this type was observed) and glucose 6-phosphate bound to the phosphoenzyme. Complete assignments of the P resonances of these intermediates were made by labeling the phosphate ester group of either the enzyme or the sugar with 17O and by NMR polarization transfer studies. The effect of bound metal ions on these resonances also was determined. A 31P NMR titration study of the Li+ complex of the dephosphoenzyme with glucose 1,6-bisphosphate and a 31P NMR polarization transfer experiment indicate that .beta.-glucose 1,6-bisphosphate binds to the enzyme less tightly than .alpha.-glucose 1,6-bisphosphate. The relative mobilities and solvent accessibility of the phosphate ester groups in the free phosphoenzyme and the above complexes were investigated by measurements of 31P NMR line widths as a function of magnetic field strength, nuclear Overhauser effects and spin-lattice relaxation times in 1H2O and 2H2O. The serine phosphate in the free phosphoenzyme is highly accessible to solvent molecules. Binding of Li+ does not affect this solvent accessibility. In a ternary complex (phosphoenzyme, glucose 6-phosphate, metal ion), the enzymic phosphate becomes much less accessible and possibly inaccessible to solvent. The phosphate ester group of the substrate also is partially immobilized, but not to as great an extent as the enzymic phosphate. An analysis was conducted of contributions to the line width of the 31P NMR signal of the phosphoenzyme provided by various relaxation mechanisms, including relaxation induced by 17O substitution.