Identification of vicinal thiols of phosphoenolpyruvate carboxykinase (GTP).
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.
Lewis
C T
CT
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163.
Seyer
J M
JM
Cassell
R G
RG
Carlson
G M
GM
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis / Q000737:chemistry; D003553:Cystine / Q000032:analysis / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D004220:Disulfides / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000032:analysis* / Q000737:chemistry / Q000528:radiation effects; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
C021961:8-azidoguanosine triphosphate; D001386:Azides; D004220:Disulfides; D007461:Iodoacetates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D003553:Cystine; D006160:Guanosine Triphosphate; D004228:Dithionitrobenzoic Acid; D014357:Trypsin; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003545:Cysteine; D019807:Iodoacetic Acid