Regulation of Nicotinamide Adenine Dinucleotide Phosphate-specific Glutamate Dehydrogenase from Brevibacterium flavum, a Glutamate-producing Bacterium*

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Abstract
Glutamate dehydrogenase [EC 1.4.1.4] was sonically extraced from Brevibacterium flavum, a glutamate-producing bacterium, and purified 65-fold. The enzyme was specific for NADP(H), and showed homotropic interactions with respect to α-keto-glutarate and L-glutamate. The enzyme showed two peaks of activity in the chro-matogram obtained by Sephadex G-200 gel-filtration. However, when the gel filtration was carried out with the buffer containing 0.1 M KCI, only one peak corresponding to a smaller molecular weight species was observed. In the presence of KC1, the enzyme did not exhibit the homotropic interactions described above and was markedly activated at lower concentrations of α-ketoglutarate and glutamate. The result of kinetic analysis indicated that the enzymatic reaction proceeded in a sequential order of addition of substrates. In the forward reaction (glutamate formation), NADPH, α-ketoglutarate, and ammonium ion react in this order with the enzyme, and glutamate and then NADP+ is released from the enzyme. The Michaelis and dissociation constants for these five reactants were determined. The enzyme was inhibited by the reaction products, glutamate in the forward reaction and ammonium ion and α-ketoglutrate in the reverse reaction, but not by other TCA**-cycle intermediates and amino acids. The control of activity by this product inhibition was interpreted as a mechanism of biological regulation of glutamate synthesis and degradation.

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