The fast release of sticky protons: kinetics of substrate binding and proton release in a multidrug transporter.
EmrE is an Escherichia coli H(+)-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP(+), a high-affinity substrate, is 2 x 10(7) M(-1).s(-1), a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the k(off) of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs.
Adam
Yoav
Y
Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Tayer
Naama
N
Rotem
Dvir
D
Schreiber
Gideon
G
Schuldiner
Shimon
S
D000596:Amino Acids / Q000378:metabolism; D017920:Antiporters / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011522:Protons; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D017920:Antiporters; D029968:Escherichia coli Proteins; D011522:Protons; C075436:EmrE protein, E coli