Multinuclear magnetic resonance studies of metal ion binding sites of phosphoglucomutase

Abstract

Metal binding at the activating site of rabbit muscle phosphoglucomutase was studied by 31P, 7Li, and 113Cd NMR spectroscopy. A 7Li NMR signal of the binary Li+ complex of the phosphoenzyme was not observed probably because of rapid transverse relaxation of the bound ion due to chemical exchange with free Li+. The phosphoenzyme-Li+-G6-P ternary complex is more stable, kinetically, and yields a well-resolved peak from bound Li+ at -0.24 ppm from LiCl with a line width of 5 Hz and a T1 relaxation time of 0.51 .+-. 0.07 s at 78 MHz. When glucose 1-phosphate was bound, instead, the chemical shift of bound 7Li+ was -0.13 ppm; and in the Li+ complex of the dephosphoenzyme and glucose bisphosphate a partially broadened 7Li+ peak appeared at -0.08 ppm. Thus, the bound metal ion has a somewhat different environment in each of these 3 ternary complexes. The 113Cd NMR signal of the binary Cd2+ complex of the phosphoenzyme appears at 22 ppm relative to Cd(ClO4)2 with a line width of 20 Hz at 44.4 MHz. Binding of substrate and formation of the Cd2+ complex of the dephosphoenzyme and glucose bisphosphate broaden the 113Cd NMR signal to 70 Hz and shift it to 75 ppm. The 53 ppm downfield shift upon the addition of substrate along with 1H NMR data suggests that 1 oxygen ligand to Cd2+ in the binary complex is replaced by a nitrogen ligand at some intermediate point in the enzymne reaction. In addition to the binding of Li+ and Cd2+ at the activating site for metal ions, weaker binding of either metal ion at an ancillary site was observed in the presence of bound substrate. Binding of either Li+ or Cd2+ at the weak site of the Cd2+-dephosphoenzyme-bisphosphate complex results in similar chemical shift changes in the 31P NMR peaks of the bisphosphate, presumably by virtue of a conformational change in the enzyme. No effect of ancillary binding was observed on the enzymic phosphate in the absence of bound substrate. Signals from Li+ or Cd2+ bound at the weak site were not observed in the 7Li NMR or 113Cd NMR studies, apparently because of rapid transverse relaxation resulting from a chemical exchange process. Although the binding of the monovalent Li+ at the activating site for divalent metal ions may be peculiar to phosphoglucomutase, the use of Li+ as a probe of the binding site for bivalent metal ions in other enzymic systems might prove fruitful.