The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris |
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Authors: | S. K. Goers R. A. Jensen |
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Affiliation: | (1) Center for Somatic cell Genetics and Biochemistry, Department of Biological Sciences, State University of New York, 13901 Binghamton, NY, USA;(2) Present address: General Foods Corp., 555 S. Broadway T23-1, 10591 Tarrytown, NY, USA |
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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 (Ka=1.5 M), while feedback inhibition was effected by l-tyrosine (Ki=15 M) or by l-phenylalanine (Ki=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent Km 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 Ki for l-phenylalanine was elevated over 30-fold to 500 M, (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 (Ki=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.Abbreviation DAHP synthase 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase |
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Keywords: | Allosteric regulation Amino acid (aromatic) Caffeic acid Chorismate mutase Nicotiana (chorismate mutase) |
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