Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli.
The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium; D008345:Manganese; D008433:Mathematics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000249:Adenosine Monophosphate; D008345:Manganese; C033181:aza-epsilon-AMP; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium