PMID:3922794

Citation	Neuhaus, JM, Soppa, J, Wright, JK, Riede, I, Blöcker, H, Frank, R and Overath, P (1985) Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution. FEBS Lett. 185:83-8
Abstract	The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.
Links	PubMed
Keywords	Amino Acid Sequence; Biological Transport, Active; Cloning, Molecular; Codon; Cysteine; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Galactosides/metabolism; Genes, Bacterial; Membrane Transport Modulators; Membrane Transport Proteins/antagonists & inhibitors; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Monosaccharide Transport Proteins; Mutation; Serine; Structure-Activity Relationship; Sulfhydryl Reagents/pharmacology; Symporters
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:LACY		GO:0015528: lactose:hydrogen symporter activity	ECO:0000315:		F		Table 1: Shows kinetic constants for mutant compared to wild-type for lactose uptake.	complete CACAO 4140
ECOLI:LACY	enables	GO:0015528: lactose:proton symporter activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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