Concerted Inhibition and Its Reversal by End Products of Aspartate kinase in Brevibacterium flavum*

Abstract

Aspartate kinase [ATP: L-aspartate 4-phosphotransferase, EC 2. 7.2. 4] has been partially purified about 10 fold from sonic extracts of Brevibacterium flavum. This enzyme requires ATP and a divalent cation for its activity and SO₄ ion stimulates the reaction significantly. While L-lysine or L-threonine at 1 mM gave only 10—20% inhibition, simultaneous addition of the two amino acids at 1 mM each produced over 90% inhibition. L-Isoleu-cine or L-valine had an activating effect and recovered the activity from the concerted inhibition caused by L-lysine plus L-threonine. From kinetic analysis, the aspartate kinase reaction was explained as a Michaelis type rapid equilibrium system with random addition of the two substrates (L-aspartate and ATP). The inhibition by L-lysine was competitive to L-aspartate, of mixed type to ATP, while the inhibition by L-threoninc was partially competitive to both L-aspartate and ATP. The concerted inhibition was explained by an increased affinity of one inhibitor to the enzyme which was caused by binding of another inhibitor. Thus, when L-threonine had been bound to the enzyme or the enzyme-ATP complex, L-lysine showed approximately 17 and 100 times higher affinity to these enzyme complexes, respectively. The activation by L-isoleucine was K type. The relationship between L-isoIeucine and L-lysine was competitive. The reversal of the concerted inhibition by L-isoleucine was explained by a competitive relationship between L-isolcucine and L-lysine.