Probing of the substrate binding domain of lactose permease by a proton pulse.
The lactose permease of Escherichia coli coupled proton transfer across the bacterial inner membrane with the uptake of beta-galactosides. In the present study we have used the cysteine-less C148 mutant that was selectively labeled by fluorescein maleimide on the C148 residue, which is an active component of the substrate transporting cavity. Measurements of the protonation dynamics of the bound pH indicator in the time resolved domain allowed us to probe the binding site by a free diffusing proton. The measured signal was reconstructed by numeric integration of differential rate equations that comply with the detailed balance principle and account for all proton transfer reactions taking place in the reaction mixture. This analysis yields the rate constants and pK values of all residues participating in the fast proton transfer reaction between the bulk and the protein's surface, revealing the exposed residues that react with free protons in a diffusion controlled reaction and how they transfer protons among themselves. The magnitudes of these rate constants were finally evaluated by comparison with the rate predicted by the Debye-Smoluchowski equation. The analysis of the kinetic and pK values indicated that the protein-fluorescein adduct assumes two conformation states. One is dominant above pH 7.4, while the other exists only below 7.1. In the high pH range, the enzyme assumes a constrained configuration and the rate constant of the reaction of a free diffusing proton with the bound dye is 10 times slower than a diffusion controlled reaction. In this state, the carboxylate moiety of residue E126 is in close proximity to the dye and exchanges a proton with it at a very fast rate. Below pH 7.1, the substrate binding domain is in a relaxed configuration and freely accessed by bulk protons, and the rate of proton exchange between the dye and E126 is 100,000 times slower. The relevance of these observations to the catalytic cycle is discussed.
Nachliel
E
E
Laser Laboratory for Fast Reaction in Biology, Department of Biochemistry, Tel Aviv University, Tel Aviv, Israel.
Gutman
M
M
D001665:Binding Sites; D002384:Catalysis; D003593:Cytoplasm / Q000737:chemistry; D029968:Escherichia coli Proteins; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D019897:Periplasm / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D013379:Substrate Specificity; D027981:Symporters; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; C037041:fluorescein 5-maleimide; C021499:lactose permease