Fluorescence energy transfer experiments with Escherichia coli carbamoyl-phosphate synthetase.
Fluorescence energy transfer experiments were used to measure distances between three fluorescently labeled sulfhydryl sites on Escherichia coli carbamoyl-phosphate synthetase, an unsymmetrical dimer. When five different combinations of fluorescent donor-acceptor pairs are used, the distance between site 1, located on the large subunit, and site 2, located on the small subunit, is in the range of 27-33 A. Similarly, the distance between site 1 and site 3 (large subunit) was approximately 27 A and between site 2 and site 3 was approximately 21 A. A similar approach was employed to determine distances between each sulfhydryl group and the ATP site(s), and in all cases no fluorescence quenching was observed using Cr3+ATP or Co(NH3)4ATP as substrate analogues. A lower limit could be calculated from these data, resulting in a distance of greater than or equal to 21 A from each sulfhydryl site to the ATP site. Additional experiments were performed to evaluate if the substrates ATP, HCO3(-), or glutamine or the allosteric modifiers ornithine, IMP, and UMP altered the distance relationships among the sulfhydryl sites. IMP and UMP produced a slight decrease in fluorescence between sites while glutamine and ATP produced a slight increase in fluorescence.
D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia)