BIOSYNTHESIS OF ISOLEUCINE AND VALINE IN HYDROGENOMONAS H 16

Threonine desaminase and acetohydroxyacid synthase in cell-free extracts of Hydrogenomonas H 16 and mutants derived therefrom have been studied with respect to their regulatory properties. 1. The activity of threonine desaminase has been determined employing two combined optical tests using the oxidation of NADH2 as indicator system: by trapping the ammonia produced through glutamate dehydrogenase and α-ketoglutarate and by reducing the α-ketobutyrate produced through lactate dehydrogenase. Optimum pH has been found to be 9.0 in tris-buffer. The desaminase reaction is completely inhibited already by 0.3 mM L-isoleucine. The inhibition is partially relieved by L-valine. The substrate (threonine) saturation curve has paraboloid shape; in the presence of isoleucine (0.05 mM) its shape is sigmoid. The Hill-coefficient in the absence of the inhibitor is n=1.1 and in the presence of L-isoleucine is n=2.4 (pH 8.3). 2. A prototrophic revertant has been isolated from an isoleucine requiring mutant defective in threonine desaminase. This revertant excretes isoleucine. The threonine desaminase of this revertant differs from the wildtype enzyme by a diminished affinity for isoleucine; half maximal inhibition occurs at 2.6 mM isoleucine in contrast to 0.2 mM at the wildtype enzyme. 3. The acetohydroxyacid synthase has its pH optimum at 9.0. At this pH the inhibition by L-valine amounts to 25%. At pH 7.4 a 50% inhibition was observed at a 0.1 mM concentration of valine. By varying the magnesium concentration an 80% inhibition was observed at 0.2 mM valine. The sensitivity of the enzyme is specific for L-valine; L-isoleucine and L-leucine neither decrease the activity nor act antagonistically. 4. When an isoleucine-valine-auxotrophic mutant was grown in a chemostat and when growth was limited by the concentration of valine, the formation of both enzymes, threonine desaminase and acetohydroxyacid synthase, became derepressed. When growth was limited by the concentration of isoleucine only threonine desaminase became derepressed. © 1969 Springer-Verlag.