Formation of an intramolecular cystine disulfide during the reaction of 8-azidoguanosine 5'-triphosphate with cytosolic phosphoenolpyruvate carboxykinase (GTP) causes inactivation without photolabeling.
Phosphoenolpyruvate carboxykinase (GTP) (PEPCK) specifically utilizes a guanosine or inosine nucleotide as a substrate, yet it does not share extended sequence homology with other GTP-binding proteins, and the molecular basis for its nucleotide specificity is not understood. In an effort to locate the enzyme's nucleotide-binding site, we have studied the interaction of cytosolic PEPCK from rat liver with the photoprobe 8-azidoGTP, which fulfills the criteria of a specific photoaffinity label for PEPCK. The photoprobe binds reversibly to the enzyme prior to modification and at low concentrations causes greater than 60% inactivation (Ki = 1.2 microM). GTP provides nearly complete protection against inactivation by 8-azidoGTP, whereas phosphoenolpyruvate and metal ions provide partial protection. In addition, the photoprobe is a substrate for the enzyme and has a Km similar to that for GTP. However, the extent of covalent modification by [32P]8-azidoGTP as measured by three independent techniques is significantly lower than the extent of enzyme inactivation. Further investigation of this anomaly has revealed that the loss in enzymatic activity is caused by modification of a critical cysteine residue in a reaction that does not terminate with covalent attachment of the photolabel. Quantitation of the total free thiols of modified PEPCK shows that 2 mol of cysteine is lost per mole of inactivated enzyme. These results indicate that the photoinactivation of PEPCK by 8-azidoGTP is caused by the formation of an intramolecular cystine disulfide bridge, thus providing evidence for the existence of a pair of proximal cysteine residues within the GTP-binding site. The interaction of cysteine residues with the reactive photogenerated derivatives of 8-azidopurines is discussed.
Lewis
C T
CT
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163.
Haley
B E
BE
Carlson
G M
GM
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D003553:Cystine / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D004220:Disulfides; D013438:Sulfhydryl Compounds; D003553:Cystine; D006160:Guanosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)