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Identification of vicinal thiols of phosphoenolpyruvate carboxykinase (GTP).
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1993 Jan 25; Vol. 268 (3), pp. 1628-36. - Publication Year :
- 1993
-
Abstract
- Phosphoenolpyruvate carboxykinase (PEPCK) from the cytosol of rat liver has 13 cysteines, at least one of which (Cys288) is known to be very reactive and critical for catalytic activity (Lewis, C. T., Seyer, J. M., and Carlson, G. M. (1989) J. Biol. Chem. 264, 27-33). Previous results provided evidence for the existence of at least 1 pair of vicinal cysteines within or near the active site of PEPCK (Lewis, C. T., Haley, B. E., and Carlson, G. M. (1989) Biochemistry 28, 9248-9255). An intramolecular cystine disulfide is induced to form upon treatment of PEPCK with equimolar 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) or upon irradiation of the enzyme in the presence of the photoaffinity probe 8-azidoGTP. In each case, modification is accompanied by a substantial loss in catalytic activity, and substrates protect against inactivation and modification. We now report the identification of these modified thiols by differential alkylation of cysteines and half-cystines with radioactive iodoacetate, followed by isolation and sequencing of the modified tryptic peptides. The results indicate that the disulfide formed by equimolar Nbs2 lies within a 15-residue region of the PEPCK sequence that includes Cys399, Cys407, and Cys413. In addition, Cys407 and/or Cys413 also appear to participate in formation of the disulfide induced by 8-azidoGTP. These thiols lie very near a consensus sequence that has been suggested to represent the binding site for the guanine ring of GTP.
- Subjects :
- Alkylation
Amino Acid Sequence
Animals
Azides pharmacology
Chromatography, High Pressure Liquid
Cysteine analysis
Cysteine chemistry
Cystine analysis
Cystine chemistry
Cytosol enzymology
Disulfides chemistry
Dithionitrobenzoic Acid pharmacology
Guanosine Triphosphate analogs & derivatives
Guanosine Triphosphate pharmacology
Iodoacetates
Iodoacetic Acid
Molecular Sequence Data
Peptide Fragments chemistry
Peptide Fragments isolation & purification
Phosphoenolpyruvate Carboxykinase (GTP) chemistry
Phosphoenolpyruvate Carboxykinase (GTP) radiation effects
Rats
Trypsin metabolism
Liver enzymology
Phosphoenolpyruvate Carboxykinase (GTP) analysis
Sulfhydryl Compounds analysis
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 268
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8420937