Concerted Feedback Inhibition

Concerted feedback inhibition of aspartokinase activity from Pseudomonas fluorescens by the combination of l-threonine and l-lysine, or by l-threonine and l-methionine was freely and rapidly reversible. Inhibition by l-threonine, or by the concerted pairs of amino acids was kinetically mixed with respect to l-aspartate and noncompetitive with regard to ATP. In the presence of l-threonine the Stokes radius of the enzyme increased from 44 to 49 A; a much larger value of about 56 A was estimated when both l-threonine and l-lysine were added to the enzyme. The same amino acids increased the Stokes radius of the enzyme at 15° in buffer solutions containing ATP, l-aspartate, and Mg2+, the substrates of the phosphorylation reaction, indicating that the larger molecular species were catalytically inactive. Although in phosphate buffer at 25° the combination of l-threonine plus l-methionine did not show an additional increment in the molecular radius over that seen in l-threonine alone, the same combination induced a large change (from 49 to 54 A) in the buffer containing the substrates at 15°. l-Threonine, and a combination of l-threonine plus l-lysine also increased the s20,w value of the aspartokinase by about 50%; whereas, a 10% increase in the S value was noted in l-lysine alone. The cumulative results strongly suggest that the inhibitory amino acids caused oligomerization of the enzyme. Since the inhibition of enzyme activity accompanied the structural alterations of the protein and a good correlation was seen between the degree of inhibition and the extent of enzyme oligomerization under similar experimental conditions, it is proposed that the mechanism of inhibition by l-threonine and by the concerted pairs of amino acid end products may involve modifier-induced formation of inactive enzyme oligomers.