Carbamoyl phosphate synthetase (CPS) from E. coli is potentially overlaid with a network of allosterism, interconnecting active sites, effector binding sites, and aggregate interfaces to control its mechanisms of catalytic synchronization, regulation, and oligomerization, respectively. To characterize these conformational changes, a tryptophan-free variant of CPS was genetically engineered by substituting six native tryptophans with tyrosines. Each tryptophan was then reinserted, singly, as a specific fluorescence probe of its corresponding microenvironment. The amino acid substitutions themselves result in little apparent disruption of the protein; variants maintain catalytic and allosteric functionality, and the fluorescence properties of each tryptophan, while unique, are additive to wild-type CPS. Whereas the collective, intrinsic fluorescence response of E. coli CPS is largely insensitive to ligand binding, changes of the individual probes in intensity, lifetime, anisotropy, and accessibility to acrylamide quenching highlight the dynamic interplay between several protein domains, as well as between subunits. W213 within the carboxy phosphate domain, for example, exhibits an almost 40% increase in intensity upon saturation with ATP; W437 of the oligomerization domain, in contrast, is essentially silent in its fluorescence to the binding of ligands. Nucleotide and bicarbonate association within the large subunit induce fluorescence changes in both W170 and W175 of the small subunit, indicative of the type of long-range interactions purportedly synchronizing the carboxy phosphate and amidotransferase domains of the enzyme to initiate catalysis. ATP and ADP engender different fluorescence responses in most tryptophans, perhaps reflecting coordinating, conformational changes accompanying the cycling of reactants and products during catalysis. † This work was supported by NIH grant P20 RR016478 (to JLJ) from the INBRE Program of the National Center for Research Resources at Southwestern Oklahoma State University, and by NIH grant GM33216 and Welch Foundation grant A1543 (to GDR) at Texas A&M University. Molecular graphics images were produced using the UCSF package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, which is supported by NIH grant P41 RR-01081. *Corresponding authors. Phone: (580) Fax: (580) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCarbamoyl phosphate synthetase (CPS) 1 from E. coli coordinates five substrates (2 ATP, bicarbonate, glutamine, and water) through a series reactions involving at least three unstable intermediates during the synthesis of its ultimate product, carbamoyl phosphate (1,2). Moreover, crystal structure coordinates have revealed that the reaction mechanism involves three distinct active sites separated by almost 100Å, but connected by a series of intra-and inter-molecular tunnels that presumably shuttle unstable intermediates from site to site (3-5).In the minimal mecha...