Abstract
Tyrosine and tryptophan are the regulators of the dimeric yeast chorismate mutase. Biochemical studies reveal two binding sites per molecule for both effectors, tyrosine or tryptophan. A single binding site is built up by helix 8 and helices 4 and 5 of two different subunits. The binding sites have been analyzed in the active enzyme by site directed mutagenesis of critical codons of the coding gene, ARO7. Gly-141 and Ser-142, which both reside on helix 8, are involved in the binding of tyrosine or tryptophan presumably by interacting specifically with the amino- and carboxylate-groups of these amino acid effectors. Interaction with Thr-145 of helix 8 is required for a strong tyrosine binding to the allosteric site. Replacement of Arg-75, which connects helices 4 and 5 or of Arg-76, which is part of helix 5 by alanine residues, resulted in unregulated enzymes. These two residues are bonded to the carboxylate group and phenolic hydroxyl group of tyrosine, respectively, but do not interact with tryptophan by hydrogen bonding in the crystal structures. Phenylalanine, which has low binding affinity slightly activated the chorismate mutase. A T145V mutant chorismate mutase, however, showed increased activation by phenylalanine. Our results support a mechanism by which tyrosine contracts the allosteric site by interacting with its phenolic hydroxyl group. Tryptophan works in an inverse way by opening the allosteric site through the steric size of its side chain.
Highlights
Chorismate is the last common precursor of the amino acids tyrosine, phenylalanine, and tryptophan
Phenylalanine is not an inhibitor of yeast chorismate mutase. This is in contrast to the homologous plastidic chorismate mutase from Arabidopsis thaliana, which is inhibited by both amino acids, tyrosine and phenylalanine [7]
We investigated the effects of the amino acid replacements on regulation of the enzymes and on binding of tyrosine and tryptophan, respectively
Summary
Materials—Chorismic acid as barium salt was purchased from Sigma. Ethylamino-Sepharose was prepared following the protocol for activation of Sepharose CL-4B [13] and by coupling of the ligand, ethylamino-HCl, to the activated matrix. Reaction volumes of 250 l containing 100 mM Tris, pH 7.6, 2 mM EDTA, 20 mM dithiothreitol, optionally 0.1 mM tyrosine, 0.1 mM phenylalanine or 0.01 mM tryptophan, chorismate mutase enzyme, and chorismate in a range from 0.25 to 10 mM were used. The maximum velocity Vmax, the Hill-coefficient nH, and the substrate concentration at half maximal velocity [S]0.5 or Km were determined using a computer program which was applying the Quasi-Newton method (Davidon-Fletcher-Powell algorithm) to fit optimal curves to the data [20]. Binding Assays—The binding of tyrosine, phenylalanine, and tryptophan to chorismate mutase was assayed with an equilibrium dialysis apparatus with 200 l of half cell volumes and Spectra/Por membrane discs with a molecular cut-off of 12,000 –14,000 Da (Spectrum, Houston, Texas). The specific molar activity of ligand solution was calculated from its concentration corrected to the added 14C-labeled compound and from its activity determined by counting unequilibrated samples. All data obtained were standardized to the G141S mutant enzyme which showed no detectable effector binding activity
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