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) have been 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-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 dephospho-enzyme 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 three discrete intermediate complexes by NMR techniques: glucose 1-phosphate bound to the phosphoenzyme, glucose 1,6-bisphosphate bound to the dephosphoenzyme (only one complex of this type was observed), and glucose 6-phosphate bound to the phosphoenzyme. Complete assignments of the phosphorus resonances of these intermediates have been 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000494:pharmacology; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010733:Phosphoglucomutase / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D005958:Glucosephosphates; D002104:Cadmium; D019298:Glucose-6-Phosphate; C031590:glucose-1-phosphate; D010733:Phosphoglucomutase