The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris

Abstract

The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with L-phenylalanine and L-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, L-tryptophan was a potent allosteric activator (K a =1.5 μM), while feedback inhibition was effected by L-tyrosine (K i =15 μM) or by L-phenylalanine (Ki=15 μM). At pH 6.1, all three effectors acted competitively, influencing the apparent K m for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. L-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of L-tyrosine and L-tryptophan were only modestly different, in striking contrast to results obtained with L-phenylalanine. At pH 7.2 (i) the K i for L-phenylalanine was elevated over 30-fold to 500 μM, (ii) the kinetics of inhibition became non-competitive, and (iii) L-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. L-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K i =0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n'=1.8.