Fluorescence energy transfer experiments with Escherichia coli carbamoyl-phosphate synthetase

Abstract

Fluorescence energy transfer experiments were used to measure distance between 3 fluorescently labeled SH sites on E. coli carbamoyl-phosphate synthetase, an unsymmetrical dimer. When 5 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 .ANG.. The distance between site 1 and site 3 (large subunit) was .apprx. 27 .ANG. and between site 2 and site 3 was .apprx. 21 .ANG.. A similar approach was employed to determine distances between each SH 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 .gtoreq. 21 .ANG. 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 relatinships among the SH sites. IMP and UMP produced a slight decrease in fluorescence between sites while glutamine and ATP produced a slight increase in fluorescence.