Allosteric regulation of inducer and operator binding to the lactose repressor.
The effects of cysteine modification and variations in pH on the equilibrium parameters for inducer and operator binding to the lactose repressor protein were examined. Operator binding affinity was minimally affected by increasing the pH from 7.5 to 9.2, whereas inducer binding was decreased for both the unliganded protein and the repressor-operator complex over the same range. Inducer binding to the repressor became more cooperative at high pH. The midpoint for the change in inducer affinity and cooperativity was pH 8.3; this value correlates well with cysteine ionization. The differential between repressor-operator affinity in the presence and absence of inducer was significantly decreased by modification of the protein with methyl methanethiosulfonate (MMTS). In contrast to unreacted protein, the inducer binding parameters for MMTS-modified repressor were largely unaffected by pH variation. The free energy for formation of the completely liganded protein was calculated for two pathways; the delta G values for these two independent routes were equivalent only for stoichiometries of four inducers and two operators per repressor molecule. All of the binding data were analyzed quantitatively by using a Monod-Wyman-Changeux two-state model for allosteric regulation. The observed dependences of the isopropyl beta-D-thiogalactoside binding curves on pH, DNA concentration, and MMTS modification were fitted by varying only the equilibrium constant between the two conformational states of the protein. With this analysis, high pH favors the T (high operator/low inducer affinity) state, while modification of cysteine-281 with MMTS elicits a shift into the R (high inducer/low operator affinity) state.(ABSTRACT TRUNCATED AT 250 WORDS)
D000494:Allosteric Regulation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005809:Genes, Regulator; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D008024:Ligands; D012097:Repressor Proteins; D014157:Transcription Factors; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate