MECHANISM OF INORGANIC-PHOSPHATE INTERACTION WITH PHOSPHATE-BINDING PROTEIN FROM ESCHERICHIA-COLI

The mechanism of P-i interaction with phosphate binding protein of Esc herichia coli has been investigated using the A197C mutant protein lab eled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescenc e change on binding P-i. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal a mounts of two diastereoisomers due to the chiral center formed on reac tion of the cysteine with the maleimide. These diastereoisomers could not be separated, but P-i binding data suggest that they differ in aff inity and fluorescence change. When P-i binds to MDCC-PBP, the fluores cence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of P-i binding show saturation of the rate at high P-i concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protei n that closes the cleft containing the P-i binding site. Cleft closure has a rate constant of 317 s(-1) (pH 7.0, 5 degrees C), and opening h as a rate constant of 4.5 s(-1) The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closur e as the steady state fluorescence emission (lambda(max), and intensit y) on P-i binding is mimicked by the addition of ethanol to aqueous so lutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of P-i were 0.3 and 2.4 ns, respectively, consistent wit h the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding P-i as meas ured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but wit h greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of P-i and increases strongly upon saturation by P-i. These data are also consistent with an open to clos ed conformational model.