Nuclear magnetic resonance study of the topography of binding sites of Escherichia coli carbamoyl-phosphate synthetase

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Abstract
Two paramagnetic probes, i.e., 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 .times. 10-9 s) and used to compute distances. These were in the range 7-9 .ANG.. Distances were also calulated from Mn2+ to the 13C-5 atom of glutamate (8.6 .ANG.), to the monovalent cation site (.apprx. 8 .ANG.) and to the P 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 analog, 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 E. coli carbamoyl-phosphate synthetase dimer.