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The intramembrane active site of GlpG, an E. coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates.
- Source :
-
Molecular microbiology [Mol Microbiol] 2007 Apr; Vol. 64 (2), pp. 435-47. - Publication Year :
- 2007
-
Abstract
- Escherichia coli GlpG is an orthologue of the rhomboid proteases that catalyse intramembrane proteolysis of specific membrane proteins. We previously showed that it can cleave a type I model membrane protein, Bla-LY2-MBP, having the second transmembrane region of lactose permease (LY2) in vivo and in vitro at the predicted periplasm-membrane boundary region of LY2. Here we investigated the environment of the active site regions of GlpG in the membrane-integrated state by examining the modifiability of Cys residues introduced into the regions around the catalytic residues with membrane-permeable and -impermeable alkylating reagents. The results indicate that the enzyme active site is fully open to the external aqueous phase. GlpG also cleaved a similar fusion protein, Bla-GknTM-MBP, having the transmembrane region of Gurken (GknTM), a physiological substrate of Drosophila rhomboids. Engineered Cys residues in the cleavage site regions of the LY2 and GknTM sequences were efficiently modified with a membrane-impermeable alkylating reagent, showing that these regions are exposed to the periplasm. These results suggest that GlpG cleaves an extramembrane region of substrates, unlike the currently prevailing view that this class of membrane proteases acts against a membrane-embedded polypeptide segment after its lateral entrance into the enzyme's active site.
- Subjects :
- Alkylating Agents pharmacology
Amino Acid Sequence
Binding Sites drug effects
Carrier Proteins metabolism
Cell Membrane drug effects
Drosophila Proteins metabolism
Escherichia coli drug effects
Escherichia coli Proteins metabolism
Ethylmaleimide pharmacology
Hydrolysis drug effects
Maltose-Binding Proteins
Molecular Sequence Data
Molecular Weight
Monosaccharide Transport Proteins metabolism
Periplasm drug effects
Polyethylene Glycols pharmacology
Protein Structure, Tertiary drug effects
Substrate Specificity drug effects
Sulfhydryl Compounds metabolism
Symporters metabolism
Transforming Growth Factor alpha metabolism
beta-Lactamases metabolism
Cell Membrane metabolism
DNA-Binding Proteins chemistry
Endopeptidases chemistry
Escherichia coli enzymology
Escherichia coli Proteins chemistry
Membrane Proteins chemistry
Peptides metabolism
Water metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0950-382X
- Volume :
- 64
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Molecular microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 17493126
- Full Text :
- https://doi.org/10.1111/j.1365-2958.2007.05679.x