In:
FEBS Letters, Wiley, Vol. 185, No. 1 ( 1985-06-03), p. 83-88
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The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide‐directed, site‐specific mutagenesis of the lac Y gene.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 (HgCl 2 , p ‐(chloro‐mercuri)benzenesulfonate and N ‐ethylmaleimide) than in the wild type, and β‐D‐galactosyl 1‐thio‐β‐D‐galactoside does not protect the mutant carrier against slow inactivation by N ‐ethylmaleimide. It is concluded that the Cys 148 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 Cys 148 → Gly 148 replacement.
Type of Medium:
Online Resource
ISSN:
0014-5793
,
1873-3468
DOI:
10.1016/0014-5793(85)80745-6
Language:
English
Publisher:
Wiley
Publication Date:
1985
detail.hit.zdb_id:
1460391-3
SSG:
12
Bookmarklink