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J. Biol. Chem., Vol. 279, Issue 47, 49214-49221, November 19, 2004

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Sugar Recognition by the Lactose Permease of Escherichia coli^*

José Luis Vázquez-Ibar, Lan Guan, Adam B. Weinglass, Gill Verner, Ruth Gordillo¶, and H. Ronald Kaback||

From the Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, Howard Hughes Medical Institute and the Chemistry and Biochemistry Department, UCLA, Los Angeles, California 90095-1662

Biochemical, luminescence and mass spectroscopy approaches indicate that Trp-151 (helix V) plays an important role in hydrophobic stacking with the galactopyranosyl ring of substrate and that Glu-269 (helix VIII) is essential for substrate affinity and specificity. The x-ray structure of the lactose permease (LacY) with bound substrate is consistent with these conclusions and suggests that a possible H-bond between Glu-269 and Trp-151 may play a critical role in the architecture of the binding site. We have now probed this relationship by exploiting the intrinsic luminescence of a single Trp-151 LacY with various replacements for Glu-269. Mutations at position 269 dramatically alter the environment of Trp-151 in a manner that correlates with binding affinity of LacY substrates. Furthermore, chemical modification of Trp-151 with N-bromosuccinimide indicates that Glu-269 forms an H-bond with the indole N. It is concluded that 1) an H-bond between the indole N and Glu-269 optimizes the formation of the substrate binding site in the inward facing conformation of LacY, and 2) the disposition of the residues implicated in sugar binding in different conformers suggests that sugar binding by LacY involves induced fit.

Received for publication, July 2, 2004 , and in revised form, August 25, 2004.

^* This work was supported in part by Grant DK51131:09 from the National Institutes of Health (to H. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a Postdoctoral Fellowship from the Ministerio de Educacion Cultura y Deporte, Spain.

^|| To whom correspondence should be addressed: HHMI/UCLA, 5-748 MacDonald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. Tel.: 310-206-5053; Fax: 310-206-8623; E-mail: RonaldK{at}HHMI.UCLA.edu.

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