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Enzyme II/sup Mtl/ of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier

Journal Article · · Biochemistry; (United States)
OSTI ID:7188288
;

The cysteine of the membrane-bound mannitol-specific enzyme II (EII/sup Mtl/) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with (/sup 14/C)iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing.

Research Organization:
Univ. of Groningen (Netherlands)
OSTI ID:
7188288
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:15; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETAMIDE
AMIDES
AMINO ACID SEQUENCE
AMINO ACIDS
AZINES
BACTERIA
BIOCHEMISTRY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
CYSTEINE
DECOMPOSITION
ENZYMES
ESCHERICHIA COLI
HETEROCYCLIC COMPOUNDS
LABELLED COMPOUNDS
LABELLING
LIQUID COLUMN CHROMATOGRAPHY
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEOLYSIS
PYRIDINES
SEPARATION PROCESSES
THIOLS
TRANSFERASES