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Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements
Journal article   Peer reviewed

Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements

Nancy Carrasco, Irene B Puttner, Lisa M Antes, Jonathan A Lee, J. Douglas Larigan, Julius S Lolkema, Paul D Roepe and H. Ronald Kaback
Biochemistry (Easton), Vol.28(6), pp.2533-2539
03/01/1989
DOI: 10.1021/bi00432a028
PMID: 2567181
url
https://research.rug.nl/en/publications/5cd441f9-7653-4825-ab7e-7b5dae35fd58View
Open Access

Abstract

lac permease with Ala in place of Glu325 was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The reconstituted molecule is completely unable to catalyze lactose/H+ symport but catalyzes exchange and counterflow at least as well as wild-type permease. In addition, Ala325 permease catalyzes downhill lactose influx without concomitant H+ translocation and binds p-nitrophenyl alpha-D-galactopyranoside with a KD only slightly higher than that of wild-type permease. Studies with right-side-out membrane vesicles demonstrate that replacement of Glu325 with Gln, His, Val, Cys, or Trp results in behavior similar to that observed with Ala in place of Glu325. On the other hand, permease with Asp in place of Glu325 catalyzes lactose/H+ symport about 20% as well as wild-type permease. The results indicate that an acidic residue at position 325 is essential for lactose/H+ symport and that hydrogen bonding at this position is insufficient. Taken together with previous results and those presented in the following paper [Lee, J. A., Püttner, I. B., & Kaback, H. R. (1989) Biochemistry (third paper of three in this issue)], the findings are consistent with the idea that Arg302, His322, and Glu325 may be components of a H+ relay system that plays an important role in the coupled translocation of lactose and H+.

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