Genetically Desensitized Aspartate Kinase to the Concerted Feedback Inhibition in Brevibacterium flavum

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Abstract
Aspartate kinases [ATP: L-aspartate 4-phosphotransferase, EC 2. 7. 2. 4] were partially purified from Brevibacterium flavum and its mutant, which is resistant to L-threonine plus S-(2-aminoethyl)-L-cysteine (AEC), * a lysine analogue, and produces a large amount of L-lysine, and their properties were compared with each other. Both enzymes showed a homotropic interaction of the substrate, aspartate, which disappeared in the presence of the activator, threonine or ammonium sulfate. The degree of activation by ammonium sulfate was larger with the parental enzyme than with the mutant enzyme. In the presence of ammonium sulfate, double reciprocal plots of the reaction rate against one substrate concentration at various fixed concentrations of another substrate were linear and met at a point with both enzymes. Moreover, ADP, one of the reaction products inhibited the enzymes competitively to both substrates, aspartate and ATP, suggesting rapid equilibrium random Bi Bi mechanism for both enzymatic reactions. Kms for aspartate and ATP were similar with both enzymes. Threonine slightly activated the mutant enzyme but partially competitively inhibited the parental enzyme in the presence of ammonium sulfate, while, in the absence of the salt, it was an activator for both enzymes. Gel filtration experiments showed dimer formation by the addition of threonine in the presence of ammonium sulfate. Regardless of the presence of ammonium sulfate, lysine inhibited both enzymes to the same degree at a high concentration. In contrast to the parental enzyme, concerted inhibition by lysine plus threonine was not observed with the mutant enzyme. Furthermore, a simultaneous addition of threonine diminished the inhibitory effect of lysine. Isoleucine only slightly activated the mutant enzyme, while about twice activation was observed with the parental enzyme. In conclusion, a genetic alteration occurred in aspartate kinase of an analogue-resistant mutant affected all tested actions of the allosteric effectors, threonine, isoleucine, and ammonium sulfate but not those of the substrates and the competitive inhibitor, lysine. Effect of AEC was similar to those of lysine with both enzymes. Thus, the specific growth inhibition of the parental strain by this analogue plus threonine which was reversed by the addition of lysine, seems to be caused by the concerted inhibition of the aspartate kinase. The resistance of the mutant to these amino acids as well as lysine overproduction in the mutant can be well explained by the lack of the concerted inhibition in the mutant enzyme.

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