Fluorometric studies of aza-epsilon-adenylylated glutamine synthetase from Escherichia coli.
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.
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
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