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

Glu126 and Arg144 in the lactose permease are indispensable for substrate b inding 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-->A la or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, eff lux, 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 accu mulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Gl u126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ i nflux to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->A sp permease exhibits about a 6-fold increase in the K-m for active transpor t relative to wild-type permease with a comparable V-max. Direct binding as says using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitro phenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substra te protection against [C-14]-N-ethylmaleimide labeling of single-Cys148 per mease reveal an apparent K-d of 3-5 mM for lactose and 15-20 mu M for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of G lu126-->Asp/Cys148 permease for lactose is markedly decreased (K-d > 80 mM) , while TDG affinity is altered to a much lesser extent (K-d ca. 80 mu M). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding a nd support the idea that Arg144 plays a major role in substrate specificity .