title: Fluorometric studies of aza-epsilon-adenylylated glutamine synthetase from Escherichia coli.
abstract: Glutamine synthetase in Escherichia coli is regulated by adenylation and deadenylation reactions. The adenylation reaction converts the divalent cation requirement of the enzyme from Mg2+ to Mn2+. Previously, the catalytic action of unadenylated glutamine synthetase was elucidated by monitoring the intrinsic tryptophan fluorescence change accompanying substrate binding. However, due to the lack of changes in the tryptophan fluorescence, a similar study could not be done with the adenylated enzyme. In this study, therefore, an extrinsic fluor is introduced into the adenylated glutamine synthetase by adenylating the enzyme with 2-aza-1,N6-ethenoadenosine triphosphate, a fluorescent analog of ATP. The modified enzyme (aza-epsilon-glutamine synthetase) exhibits catalytic and kinetic properties similar to those of the naturally adenylated enzyme. The results of fluorometric studies on this aza-epsilon-glutamine synthetase indicated that L-glutamate and ATP bind to both Mn2+ and Mg2+ forms of the enzyme in a random order, but only the Mn2+ form is capable of forming a highly reactive enzyme-bound intermediate which is a prerequisite for the reaction with NH4+ to form products. The extrinsic fluorescence changes are also used to determine the binding constants of various substrates and inhibitors of both the biosynthetic and gamma-glutamyl transfer reactions.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology*; D004985:Ethenoadenosine Triphosphate / Q000031:analogs & derivatives; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D004985:Ethenoadenosine Triphosphate; D005971:Glutamates; D000644:Quaternary Ammonium Compounds; D018698:Glutamic Acid; D008345:Manganese; C012758:aza-epsilon-ATP; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium