PURIFICATION OF THE ESCHERICHIA-COLI REGULATORY PROTEIN TYRR AND ANALYSIS OF ITS INTERACTIONS WITH ATP, TYROSINE, PHENYLALANINE, AND TRYPTOPHAN

A plasmid that directs the overexpression of the Escherichia coli regu latory protein TyrR was constructed. Cell extracts of an E. coli strai n harboring the plasmid were used to develop a two-step procedure for purifying homogenous TyrR. The weight-average molecular weight of the pure protein was determined by sedimentation equilibrium analyses to b e 110,000 +/- 5,000, indicating that native TyrR is a homodimer. The b inding of ligands to TyrR was investigated by the techniques of sedime ntation velocity meniscus depletion and steady state dialysis. One mol of ATP bound per mol of TyrR subunit with half-maximal saturation at 5-7 mu M ATP. ATP binding curves exhibited positive cooperativity, wit h a value of 1.3 for the Hill constant, n(H). The binding was not sign ificantly affected by the presence of either 500 mu M tyrosine or 2 mM phenylalanine. Binding of tyrosine to TyrR (40 mu M subunit) could no t be detected in the absence of ATP, indicating that the TyrR-tyrosine complex has a dissociation constant (K-d) in excess of 180 mu M. Howe ver, binding was observed in the presence of saturating ATP (200 mu M) , where 1 mol of tyrosine bound per mol of TyrR subunit with half-maxi mal saturation at 50 mu M tyrosine. The binding exhibited positive coo perativity (n(H) of 1.2). There was no detectable binding of either ph enylalanine or tryptophan to TyrR (40 mu M) in the absence or presence of 200 mu M ATP, indicating that any binding of these amino acids to TyrR or TyrR.ATP also has a Rd in excess of 180 mu M. Each of these am ino acids was found to inhibit the binding of tyrosine by TyrR.ATP whe n present in large molar excess (20 mu M tyrosine and 2 or 10 mM pheny lalanine or tryptophan), indicating that TyrR binds each of these amin o acids, albeit more weakly than it binds tyrosine.