Studies of ligand binding to Escherichia coli adenylosuccinate synthetase.
Dissociation constants of Escherichia coli 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. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not quench the fluorescence to any great extent, and their dissociation constants could not be determined. These ligand binding studies were generally supportive of the kinetic mechanism proposed earlier for the enzyme. Cys291 was modified with the fluorescent chromophores N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate and tetramethylrhodamine maleimide in order to measure enzyme conformational changes attending ligand binding. The excitation and emission spectra of these fluorophores are not altered by the addition of active site binding ligands. 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.
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
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D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000265:Adenylosuccinate Synthase / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D006160:Guanosine Triphosphate / Q000378:metabolism; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D008024:Ligands; D009282:Naphthalenesulfonates; D012235:Rhodamines; D007291:Inosine Monophosphate; C012168:adenylosuccinate; D000249:Adenosine Monophosphate; C006421:1,5-I-AEDANS; D006160:Guanosine Triphosphate; D000265:Adenylosuccinate Synthase; D003545:Cysteine