EVIDENCE FOR 2 AROMATIC AMINO ACID-BINDING SITES, ONE ATP-DEPENDENT AND THE OTHER ATP-INDEPENDENT, IN THE ESCHERICHIA-COLI REGULATORY PROTEIN TYRR

In Escherichia coli, genetic regulation of aromatic amino acid biosynt hesis and uptake is effected by the protein TyrR, which acts via ligan d-mediated repression and activation, Characterization of the interact ions of tyrosine, phenylalanine and tryptophan with TyrR revealed the presence of two separate aromatic amino acid-binding sites, one ATP-de pendent, the other ATP-independent, Binding to the ATP-dependent site induces the self-association of TyrR. Using sedimentation equilibrium analyses, dissociation constants for this site in the dimeric and hexa meric forms of TyrR were determined to be 330 mu M and 24 mu M, respec tively, for tyrosine, and 55 mM and 3.7 mM, respectively, for phenylal anine. Tryptophan bound with a strength similar to that of phenylalani ne, and both phenylalanine and tryptophan competed with the binding of tyrosine, The ATP-independent site, which has not been observed previ ously, was characterized by ultraviolet (u.v.) difference spectroscopy and a sedimentation-velocity meniscus-depletion method. Phenylalanine bound co-operatively to this site, exhibiting half-saturation at 260 mu M. Tryptophan competed weakly with phenylalanine, half-saturation o ccurring at 1,2 mM, No binding of tyrosine to this site could be detec ted. We propose that the binding of phenylalanine or tryptophan to thi s ATP-independent site is responsible for phenylalanine- and tryptopha nmediated regulation by TyrR.