The Enzymology of Prephenate Dehydrogenase in Bacillus subtilis

Prephenate dehydrogenase (prephenate:NAD oxido-reductase (decarboxylating)) was examined in crude extracts of strains 168 and 23 of Bacillus subtilis by using a fluorometric assay procedure. Both strains had prephenate dehydrogenases with similar kinetic and allosteric characteristics. The enzyme had an absolute requirement for NAD as a cofactor. The intrinsic Km for prephenate was 2 mm; for NAD the Km equaled 1 mm. Optimal catalysis of the reaction was achieved at a temperature of 32°, pH 8.4. l-Tyrosine, a competitive inhibitor of prephenate dehydrogenase activity, accentuated strongly the cooperativity of binding of prephenate molecules to the enzyme. l-Tyrosine also protected the enzyme from temperature inactivation. d-Tyrosine and l-phenylalanine were also competitive inhibitors of prephenate dehydrogenase. Inhibitions by l-tryptophan and p-hydroxyphenylpyruvate were mixed with respect to prephenate. Further repression of the synthesis of prephenate dehydrogenase by l-tyrosine beyond the existing level of repression in minimal medium was small in both wild type strains. A 2-fold decrement in specific activity resulted when cells were cultured in broth or in minimal media containing l-tyrosine or combinations of aromatic amino acids. Prephenate dehydrogenase from a regulatory mutant, insensitive to feedback inhibition by l-tyrosine, was also insensitive to inhibition by d-tyrosine, l-phenylalanine, and l-tryptophan.