A nuclear magnetic resonance study of the topography of binding sites of Escherichia coli carbamoyl-phosphate synthetase.

Two paramagnetic probes, viz., Mn2+ and Cr3+-ATP, were used to map distances to various loci on carbamoyl-phosphate synthetase by using NMR measurements. The paramagnetic influence of Mn2+ on the 1H of L-glutamate and L-ornithine was measured at 200 and 360 MHz. On the basis of these data, a correlation time for the paramagnetic interaction was determined (2 X 10(-9) s) and used to compute distances. These were in the range 7-9 A. Distances were also calculated from Mn2+ to the 13C-5 atom of glutamate (8.6 A), to the monovalent cation site (approximately 8 A), and to the phosphorus atoms of ATP in the Co(NH3)4ATP complex. For studies of the monovalent cation site relaxation rates of 6Li+, 7Li+, and 15NH4+ were measured. With Cr3+ ATP as a paramagnetic substrate analogue, Cr3+ to 13C distances were measured with the substrates HCO3(-) and [5-13C]glutamate. These NMR data provide the first topographical map of the arrangement of substrates, metal ion activators, and allosteric modifiers on the Escherichia coli carbamoyl-phosphate synthetase dimer.