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 regulatory protein TyrR was constructed. Cell extracts of an E. coli strain harboring the plasmid were used to develop a two-step procedure for purifying homogeneous TyrR. The weight-average molecular weight of the pure protein was determined by sedimentation equilibrium analyses to be 110,000 +/- 5,000, indicating that native TyrR is a homodimer. The binding of ligands to TyrR was investigated by the techniques of sedimentation velocity meniscus depletion and steady state dialysis. One mol of ATP bound per mol of TyrR subunit with half-maximal saturation at 5-7 microM ATP. ATP binding curves exhibited positive cooperativity, with a value of 1.3 for the Hill constant, nH. The binding was not significantly affected by the presence of either 500 microM tyrosine or 2 mM phenylalanine. Binding of tyrosine to TyrR (40 microM subunit) could not be detected in the absence of ATP, indicating that the TyrR-tyrosine complex has a dissociation constant (Kd) in excess of 180 microM. However, binding was observed in the presence of saturating ATP (200 microM), where 1 mol of tyrosine bound per mol of TyrR subunit with half-maximal saturation at 50 microM tyrosine. The binding exhibited positive cooperativity (nH of 1.2). There was no detectable binding of either phenylalanine or tryptophan to TyrR (40 microM) in the absence or presence of 200 microM ATP, indicating that any binding of these amino acids to TyrR or TyrR.ATP also has a Kd in excess of 180 microM. Each of these amino acids was found to inhibit the binding of tyrosine by TyrR.ATP when present in large molar excess (20 microM tyrosine and 2 or 10 mM phenylalanine or tryptophan), indicating that TyrR binds each of these amino acids, albeit more weakly than it binds tyrosine.
Argaet
V P
VP
Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia.
Wilson
T J
TJ
Davidson
B E
BE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D002474:Cell-Free System; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005809:Genes, Regulator; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010649:Phenylalanine / Q000378:metabolism* / Q000494:pharmacology; D010957:Plasmids; D012097:Repressor Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism* / Q000494:pharmacology
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012097:Repressor Proteins; D014157:Transcription Factors; C069256:TyrR protein, E coli; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D000255:Adenosine Triphosphate