Glutamate Formation by a New In Virto Enzyme System Consisting of Purified Clutamine Synthetase and Glutamate Synthase

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After the addition of ammonia to the culture medium, the concentration of glutamine in B. flavum cells increased in 20 s with a decrease in glutamate. In the subsequent 30 s, the glutamine concentration deceassed again with an increase in glutamate. An enzyme system, which consisted of purified glutamine synthetase (GS) and glutamate synthase (GOGAT) with ATP-and NADPH-regenerating systems, was made up to study the functions of the GS/GOGAT pathway : concentrations of the substrates and of the enzymes were decided on according to the intracellular conditions. Changes in the concentrations of amino acids caused by the addition of ammonia to the system were very similar to those of intracellular glutamate and glutamine when ammonia was added to the bacterial culture. The time required for the complete formation of glutamate from 0.5 mM ammonia was about 4-tims shorter in the GS-GOGAT system than in the system using purified glutamate dehydrogenase (GDH) and the NADPH-regenerating system. The glutamate synthase reaction in the GS/GOGAT system was inhibited by some amino acids much more markedly than in the standard assay mixture consisting of glutamine, α-ketoglutarate and NADPH. These results gave further evidence elucidating the operation of the GS/GOGAT pathway in ammonia assimilation, and suggested that a reconstructed enzyme system is useful for studying phosiological mechanisms.
社団法人日本生物工学会の論文
1989-06-25