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Biochemistry. 1999 Jan 12;38(2):813-9.

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli.

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1
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles 90095-1662, USA.

Abstract

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.

PMID:
9888822
DOI:
10.1021/bi982200h
[Indexed for MEDLINE]
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