Regulation of Aspartate Family Amino Acid Biosynthesis in Brevibacterium flavum

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Abstract
Threonine dehydratase [L-threonine hydro-lyase (deaminating), EC 4.2.1.16] was purified about 100-fold from sonic extracts of Brevibacterium flavum and properties of the enzyme were examined. The relationship between the activity and the concentrations of the enzyme did not show linearity. Thus, the specific activity decreased as the enzyme concentration was reduced. However, the relationship be tween the activity and the incubation time at a fixed enzyme concentration was linear. The enzyme reaction was specific to L-threonine, and neither D-threonine nor L-serine replaced it for the reaction. Pyridoxal phosphate (PAL-P)** activated the enzyme about twice. The optimum pH of the reaction was 8.0 with Tris-HCl buffer. Potassium phosphate activated the enzyme significantly and shifted the .optimum pH to 9. The saturation curve of the substrate, L-threonine, was sigmoidal and the Hill coefficient was calculated to be 3.1, indicating that this enzyme is allosteric, having at least two threonine binding sites. The enzyme activity was specifically inhibited by the end product, L-isoleucine, and was markedly activated by L-valine. In the presence of 10 mM threonine, isoleucine concentration giving 50% inhibition was 0.34 mM, while 5mM valine activated the enzyme about 30 times. In the presence of valine, the substrate saturation curve became hyperbolic. Vm. was not altered by the presence of these effectors. The relationship between isoleucine and valine was competitive. The inhibition by isoleucine was not so affected by pH of the medium, while the rate of activation by valine markedly depended on the pH, showing about 30-fold activation at pH 7.5 and 13-fold at pH 9.0, with potassium phosphate buffer. Isoleucine strongly protected the enzyme against heat inactivation. However, valine did not show any protective effect. From the gel filtration experiments, the molecular weight was estimated to be about 2×105, which was not affected in the presence or absence of isoleucine or valine.

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