Sensitivity of efflux-driven carrier turnover to external pH in mutants of the Escherichia coli lactose carrier that have tyrosine or phenylalanine substituted for histidine-322. A comparison of lactose and melibiose.
Two Escherichia coli lactose carrier mutants (tyrosine or phenylalanine substituted for histidine 322) were studied under conditions of net efflux or equilibrium exchange. Net lactose efflux by either mutant was 10-20-fold slower than by the parent and was sensitive to extracellular pH (5.6-8.0). The presence of extracellular lactose (equilibrium exchange) failed to accelerate loss of [14C]lactose, indicating that the step(s) rate limiting for exchange were also rate limiting for net lactose efflux. Net melibiose efflux by the Phe-322 mutant was comparable to the normal carrier, while that by the Tyr-322 mutant was 5-fold faster (pH 7.0). Melibiose efflux by either mutant was sensitive to pH (5.6-8.0). Melibiose in the extracellular medium significantly accelerated loss of [3H]melibiose from either mutant, showing that slow exchange is a sugar-specific phenomenon and not an intrinsic property of these mutants. The sugar-specific effect of these mutations could mean that the defect in these mutants is not on the path of the proton, although alternative explanations cannot as yet be eliminated. The modest effect of these mutations on the transport rate indicates that His-322 contributes a far smaller free energy increment to catalyzing of H+/galactoside cotransport than active site histidines contribute to catalyzing peptide bond hydrolysis in serine proteases. We interpret this to mean that in chemical terms the function of these catalytic histidine residues differ considerably.
King
S C
SC
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Wilson
T H
TH
D004187:Disaccharides / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D008553:Melibiose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008962:Models, Theoretical; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010649:Phenylalanine; D027981:Symporters; D014443:Tyrosine
D004187:Disaccharides; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; C021499:lactose permease; D008553:Melibiose; D004492:Edetic Acid; D007785:Lactose