Photoaffinity labeling of phosphoenolpyruvate carboxykinase (GTP) by 8-azido-GTP
Mammalian phosphoenolpyruvate carboxykinase (PEPCK) exhibits the unusual nucleotide specificity of utilizing GTP or ITP as substrates, but not ATP. The photoaffinity probe 8-azido-GTP (8-N/sub 3/-GTP) fulfills the criteria of an affinity label for PEPCK from rat liver cytosol. Inactivation of homogeneous PEPCK by the probe exhibits saturation kinetics with a K/sub i/ of 2.0 +/- 0.4 ..mu..M. Photolabeling in the presence of 5 ..mu..M 8-N/sub 3/-GTP results in a maximal 50% inactivation of the PEPCK-catalyzed carboxylation of PEP or decarboxylation of OAA. At a concentration of approximately twice its K/sub m/, GTP provides 40% protection against inactivation by the probe, with up to 80% protection afforded at higher concentrations of GTP; AMP, which is not a substrate, provides insignificant protection against inactivation. Although 2 mM Mg/sup 2 +/ or Mn/sup 2 +/ also protect substantially against inactivation, the substrates OAA, PEP and HCO/sub 3//sup -/ are ineffective. 8-N/sub 3/-GTP exhibits catalytic competency in that it can serve as a substrate with a K/sub m/ and V/sub max/ very similar to those of GTP. Thus, 8-N/sub 3/-GTP behaves as an affinity label of PEPCK and can cause a high level of inactivation at low concentrations; comparison of a peptide labeled with (..gamma..-/sup 32/P)8-N/sub 3/-GTP against the known sequence of the protein will allow identification of the GTP binding site.
- Research Organization:
- Univ. of Tennessee, Memphis
- OSTI ID:
- 6028589
- Report Number(s):
- CONF-870644-; TRN: 87-037192
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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