Photochemical cross-linking of guanosine 5'-triphosphate to phosphoenolpyruvate carboxykinase (GTP).
Mammalian phosphoenolpyruvate carboxykinase (PEPCK) specifically requires a guanosine or inosine nucleotide as a substrate; however, the structural basis for this nucleotide specificity is not yet known. Because affinity labels derived from guanosine have not yielded a stable, modified peptide in quantities sufficient for sequence analysis, we have investigated the utility of direct photochemical cross-linking of GTP to PEPCK in order to identify the nucleotide binding site. UV irradiation at a distance of 2 cm by a Mineralight lamp (330 microW/cm2) results in the attachment of [alpha-32P]GTP to PEPCK via a stable, covalent linkage in a reaction that is dependent upon GTP concentration and duration of irradiation. After 10 min of irradiation, more than 0.2 mol of [alpha-32P] GTP is incorporated per mole of PEPCK; under these conditions the GTP concentration required for half-maximal labeling is 69 microM. The substrates phosphoenolpyruvate, ITP, and GDP provide protection against photolabeling, as do Mn2+ and Mg2+. One major and one minor radioactive peptide derived from proteolytic digests of photolabeled PEPCK have been isolated and identified. The major modified peptide has been provisionally assigned to an acidic region near the C-terminus, and the minor peptide has been identified as Ser462-Lys471.
Lewis
C T
CT
Department of Biochemistry, University of Tennessee, College of Medicine, Memphis 38163.
Seyer
J M
JM
Carlson
G M
GM
D000249:Adenosine Monophosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry*; D007293:Inosine Triphosphate / Q000737:chemistry; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000378:metabolism
D003432:Cross-Linking Reagents; D007293:Inosine Triphosphate; D006153:Guanosine Diphosphate; D000249:Adenosine Monophosphate; D006160:Guanosine Triphosphate; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)