Probes were introduced into the active site of adenylosuccinate synthetase from Escherichia coli for fluorescence energy transfer measurements and to measure enzyme conformational changes attending ligand binding. The distances from fluorescent reporter groups covalently bound to a highly reactive cysteinyl residue (Cys[superscript]291) of adenylosuccinate synthetase, to active site binding ligands were determined. Energy transfer from formycin 5[superscript]'-monophosphate, a competitive inhibitor of IMP, to Cys[superscript]291, modified with the fluorescent chromophore N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate, indicated that the chromophores are 17 A apart in the presence or absence of GTP. Analogous experiments using tetramethylrhodamine maleimide to modify Cys[superscript]291 and TbGTP revealed a distance of 9 A in the presence or absence of IMP. These studies support the suggestion that the substrates GTP and IMP add randomly to the enzyme. Transfer of excitation energy also occurred from the protein moiety of the enzyme to the fluorescent label on Cys[superscript]291 with an efficiency of 34%. Dissociation constants of adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. TbGTP and TbGDP were used as native reporter groups, and changes in their fluorescence on complexing with the enzyme and various ligands made it possible to detect conformational changes occurring at the active site. Evidence is presented for abortive complexes of the type: enzyme-TbGTP-adenylosuccinate and enzyme-TbGTP-adenylosuccinate-aspartate. These results suggest that the IMP and aspartate binding sites are spatially separated. It has been shown previously that modification of Cys[superscript]344 affects the activity of the enzyme. When Cys[superscript]344 was replaced by either serine or alanine, the mutant enzymes were found to be as active as the wild-type enzyme. Site-directed mutagenesis of Trp[superscript]309 to Phe and Trp[superscript]378 to Phe were done separately to produce two mutant forms of the enzyme whose k[subscript] cats were 28% less than the wild type while there were no significant differences between the Michaelis constants of the mutant enzymes and the wild type. Some differences were noted in the fluorescence spectra of the mutants indicating differences in the microscopic environments of these amino acids.