ALPHA-ISOPROPYLMALATE SYNTHETASE OF HYDROGENOMONAS H-16

1. α-Isopropylmalate synthetase, the first enzyme of the leucine biosynthetic pathway, has been investigated in crude extracts of cells of Hydrogenomonas H 16 and of auxotrophic mutants derived therefrom. Quantitative measurements were based upon a fluorometric assay system. The synthesis of this enzyme is controlled by repression and derepression. In cells grown in the presence of l-leucine the enzyme activity amounts to a third compared to cells grown in minimal medium. When auxotrophic mutants requiring both valine and isoleucine for growth were grown with valine as the limiting factor, the enzyme activity was increased 24 fold; in leucine auxotrophic cells grown under limitation by leucine the enzyme activity was increased 21 fold compared to the wildtype grown in minimal fructose medium; the enzyme formed under conditions of derepression is not less stable than the enzyme formed in the wildtype. 2. α-Isopropylmalate synthetase in the crude extract of cells grown under conditions of derepression is non-competitively inhibited, by l-leucine. Maximal inhibition amounts to 93% at pH 7.4. The sensitivity to leucine is maximal at pH 7.2 and negligible at pH 8.4, which is the pH-optimum of catalytic activity. dl-5′,5′,5′-trifluoroleucine the structural analogue of l-leucine inhibits the enzyme by 83%. Other structurally related amino acids have no inhibitory effect. However, they are able to relieve the inhibition by l-leucine when applied in 5 to 10 fold excess. The following amino acids act antagonistically to l-leucine: l-valine, l-isoleucine, dl-α-aminobutyric acid, dl-norvaline, dl-norleucine, and cycloleucine. The antagonistic effect of valine depends on its concentration; at 0.5 mM l-leucine and varying concentrations of l-valine the enzyme activity increase nearly linearly up to 6 mM l-valine and then levels off. A regulatory function is ascribed to this antagonistic effect. 3. α-Ketoisovaleric acid is the normal substrate of α-isopropylmalate synthetase. However, the enzyme catalyzed the synthesis of other α-alkylmalic acids, when several α-keto acids were incubated together with crude cell extract, and acetyl-coenzyme A. The formation of malic acid, α-methylmalic acid, α-ethylmalic acid, α-n-propylmalic acid and α-isobutylmalic acid was followed by the incorporation of radioactivity from 14C-labelled acetic acid. The synthesis of these derivations of malic acid by the crude extract is inhibited by l-leucine. © 1969 Springer-Verlag.