期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 99, 期 10, 页码 6613-6618出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.102178299
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资金
- NIDDK NIH HHS [DK51131:06, R56 DK051131, R01 DK051131] Funding Source: Medline
N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [C-14]NEM. Remarkably, however, D-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [C-14]galactose transport by lactose, melibiose, or beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. in addition, although the conservative mutation Ala-122 --> Ser causes only mild inhibition of lactose transport, the mutations Ala-122 --> Phe and Ala-122 --> Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the nongalactosyl moiety Of D-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of D-galactopyranosides.
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