Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism of the lactose permease from Escherichiacoli.
A mechanistic model for lactose/H(+) symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover, in the ground state, the symported H(+) is shared between His-322 (helix X) and Glu-269 (helix VIII), whereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H(+) to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302-->Ala and Arg-302-->Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentration gradient, translocation modes that involve net H(+) movement. In marked contrast, the mutants catalyze equilibrium exchange of lactose and bind ligand with high affinity. These characteristics are remarkably analogous to those of permease mutants with neutral replacements for Glu-325, a residue that plays a direct role in H(+) translocation. These observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover.
Sahin-Toth
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Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
Kaback
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HR
D001120:Arginine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001692:Biological Transport; D001693:Biological Transport, Active; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009004:Monosaccharide Transport Proteins; D016296:Mutagenesis; D009598:Nitrophenylgalactosides / Q000378:metabolism; D011522:Protons; D013379:Substrate Specificity; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D018698:Glutamic Acid; C021499:lactose permease; D001120:Arginine; D007785:Lactose