Formation of an intramolecular cystine disulfide during the reaction of 8-azidoguanosine 5 prime -triphosphate with cytosolic phosphoenolpyruvate carboxykinase (GTP) causes inactivation without photolabeling

Journal Article · Mon Nov 27 23:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00450a003· OSTI ID:7138178

Lewis, C T; Carlson, G M ^[1]; Haley, B E ^[2]

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1. Univ. of Tennessee, Memphis (USA)
2. Univ. of Kentucky, Lexington (USA)

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, the authors 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-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 K{sub m} similar to that for GTP. However, the extent of covalent modification by ({sup 32}P)8-azido-GTP 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.

• OSTI ID: 7138178
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:24; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AROMATICS
AZAARENES
AZIDO COMPOUNDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLIC ACIDS
CHEMICAL BONDS
CYSTINE
DAYS LIVING RADIOISOTOPES
DISULFIDES
DOSE-RESPONSE RELATIONSHIPS
ENZYME ACTIVITY
ENZYMES
GUANOSINE
HETEROCYCLIC COMPOUNDS
INACTIVATION
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
LYASES
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PURINES
RADIOISOTOPES
REACTION KINETICS
RIBOSIDES
UPTAKE