Abstract
The reactions of the Escherichia coli D-galactosebinding protein with the ligands D-galactose and Dglucose have been examined by stopped flow rapid mixing techniques. The change in tryptophan fluorescence that occurs with galactose binding is described by a rapid, second order process; no additional phases of fluorescence change were observed. The resultant time courses were fitted to a simple equilibrium model and the rate constants for galactose binding were calculated to be k,, = 3.3 x lo’ M“ s-l and koa = 4.6 s-l in 10 nm Tris-HC1, pH 7.4 at 20°C. In addition, the dissociation rate constants for glucose (1.4 s-’) and galactose (4.5 s-’) were determined directly from displacement reactions at high concentrations of the complimentary sugar. In both cases, the time courses were exponential in form and consistent with the first order release of sugar from a single binding site. We have also utilized a novel kinetic approach to determine the association rate constant for glucose binding (3 to 4 % lo7 M-’ s-’). For all of these experiments, the data are consistent with a simple mechanism of sugar binding to a single site on the galactose-binding protein. We have also established that purified galactose-binding protein contains an approximately stoichiometric amount of bound glucose which can be readily removed by dialysis against 2 M guanidine HC1. The presence of bound glucose may account for the heterogeneous galactose equilibrium binding curves which have been reported in the past by other investigators.
Highlights
The reactionsofthe Escherichia coli D-galactose- = M)
Established that purified galactose-binding protein coWn-e have addressed this problem and have established, in tains an approximately stoichiometric amooufnbtound contrast to the findings of Silhavy and Boos [13], that the glucosewhichcan be readilyremovedby dialysis against 2 M guanidine HC1T. he presence of bound glucose may account for the heterogeneous galactose equilibrium binding curves which have been reported in the past by other investigators
LA), anddidnotexhibit increasing rates with increasing galactose concentration. This first order behavior suggested that the apparent roaftceombination with galactose is limited by either a slow protein isomerization or the displacementof an endogenously bound ligand molecule. In order totest the latter idea, a sample of purified GBP was dialyzed against a buffered solution of 2 M guanidine HC1 at pH 8.2 to remove the presumed “hidden ligand.”
Summary
WAVELENGTH (nm) of purified galactose-bindingprotein (3.0PM) with D-galactose (3.0 to 100 PM) was monitored by following the decrease in fluorescence emission at wavelengths greater than 350 nm. LA), anddidnotexhibit increasing rates with increasing galactose concentration This first order behavior suggested that the apparent roaftceombination with galactose is limited by either a slow protein isomerization or the displacementof an endogenously bound ligand molecule. In order totest the latter idea, a sample of purified GBP was dialyzed against a buffered solution of 2 M guanidine HC1 at pH 8.2 to remove the presumed “hidden ligand.”. The reactionexhibited kinetic behavior which corresponded exactlyto thatobserved for the reaction of galactose with the initial sampleof purified GBP (Fig. 1B) This result strongly supports the notion that the hidden ligand is glucose. Clealculations [16, 18]based upon the assumption thaitsotopic dilution is occurring indicate that 88 & 23%of purified GBP contains bound glucose.
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