Kinetic studies of inducer binding to lactose repressor protein.
The kinetics of binding of the inducer, isopropyl-beta, D-thiogalactoside to lactose repressor from Escherichia coli was studied by stopped flow rapid mixing techniques. Three different spectral probes for measuring changes in the conformation of the repressor were used: ultraviolet absorbance, fluorescence, and a reporter group, 2-mercuri-4-nitrophenol, in the visible region. Repressor can be reacted with this mercurial to modify two of the three free sulfhydryl groups per monomer without loss of inducer or operator binding activities. The observed first order rate constant for the reaction of repressor with 2-chloromercuri-4-nitrophenol at pH 7.5 and 20 degrees was found to be on the order of 0.1 s-1, an unexpectedly slow rate for this type of reaction. Once bound to repressor, the nitrophenol serves as a chromophoric probe to monitor changes in the surrounding environment. The binding of inducer to repressor causes a change in the absorbance of the bound 2-mercuri-4-nitrophenol moiety and exhibits a second order rate of 3.2 x 10(4) M-1 s-1. Similar rates were obtained when binding of inducer is monitored by changes in either the ultraviolet absorbance of fluorescence of tryptophan residues. Since the same rate of spectral perturbation is observed for different regions of the primary structure of the protein, the conformational change produced in response to inducer binding appears to be translated rapidly throughout the protein molecule.
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004790:Enzyme Induction; D004794:Enzyme Repression; D004926:Escherichia coli / Q000378:metabolism*; D005796:Genes; D007544:Isopropyl Thiogalactoside / Q000378:metabolism*; D007700:Kinetics; D007785:Lactose / Q000096:biosynthesis*; D008628:Mercury; D009942:Organometallic Compounds; D011485:Protein Binding; D013865:Thioglycosides / Q000378:metabolism*
D001426:Bacterial Proteins; D009942:Organometallic Compounds; D013865:Thioglycosides; D007544:Isopropyl Thiogalactoside; D008628:Mercury; D007785:Lactose