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

Frank M. Raushel, Paul M. Anderson, Joseph J. Villafranca
1983 Biochemistry  
Two paramagnetic probes, viz., Mn2+ and Cr3+-ATP, were used to map distances to various loci on carbamoyl-phosphate synthetase by using N M R measurements. The paramagnetic influence of Mn2+ on the 'H 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 lo4 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
more » ... e 13C-5 atom of glutamate (8.6 A), to the monovalent cation site (-8 A), and to the phos-Carbamoyl-phosphate synthetase from Escherichia coli catalyzes the formation of an important metabolite, carbamoyl phosphate from C 0 2 , ATP, and a nitrogen source (NH, or glutamine depending on the organism). Carbamoyl phosphate is an important precursor for pyrimidine and arginine biosynthesis, and carbamoyl-phosphate synthetase is allosterically regulated in most organisms. In E. coli, IMP stimulates activity while UMP inhibits catalysis. Ornithine acts as a potent activator of this enzyme and can overcome inhibition by UMP (Trotta et al., 1973). Thus, regulation of the activity of carbamoyl-phosphate synthetase is crucial for the biosynthesis of nucleic acids and proteins. In higher organisms this enzyme is also important in expulsion of nitrogen via the urea cycle which has arginine as an essential component.
doi:10.1021/bi00277a020 pmid:6342670 fatcat:bu3yjb6s7rgyxostmd3emn326m