Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
 4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Avian mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) was incubated with Co2+ and H2O2 to form a stable Co3+-PEPCK complex. PEPCK, similarly incubated with H2O2 and either Mg2+ or Mn2+, resulted in no significant loss in activity over 30 min. PEPCK, incubated with Co2+ and H2O2 at pH 7.4, showed rapid inhibition as observed by a 40% decrease in activity after 5 min. The loss of activity is linear with the incorporation of cobalt into PEPCK, resulting in 15-25% activity for the stoichiometric Co3+-PEPCK complex. The incorporation of and inhibition by Co3+ is protected by PEP and GTP (ITP). Treatment of the Co3+-PEPCK complex with beta-mercaptoethanol results in a loss of cobalt and full recovery of activity. The reduction and reactivation are protected by PEP and GTP (ITP). EPR, PRR, circular dichroism, and fluorescence studies all indicate that Co3+ has been selectively incorporated into the cation site of PEPCK, resulting in a catalytically active enzyme-cation species. The substrates form Michaelis complexes with Co3+-PEPCK, and the catalytic reaction occurs as a second sphere complex as previously suggested [Lee & Nowak (1984) Biochemistry 23, 6506); Duffy & Nowak (1985) Biochemistry 24, 1152]. Proteolytic digestion of the Co3+-PEPCK complex and isolation of the cobalt-containing peptide by reverse phase HPLC were performed to identify the location of the cation binding site. From mass, amino acid composition, and sequence analyses of the isolated cobalt-peptide, the region Thr276-Lys301 is responsible for metal chelation. This very homologous region, located in the central portion of PEPCK, contains two highly conserved aspartic acids, Asp295 and Asp296, that are the only feasible metal binding ligands.
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PMID:Formation and characterization of an active phosphoenolpyruvate carboxykinase-cobalt(III) complex. 911 19

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) requires two divalent cations for activity. One cation activates the enzyme through a direct interaction with the protein at site n(1). The second cation, at site n(2), acts in the cation-nucleotide complex that serves as a substrate. The Co(3+)(n(1))-PEPCK and Cr(3+)(n(1))-PEPCK complexes were used to examine the kinetic, mechanistic, and binding properties of the n(2) metal. EPR studies performed on the Co(3+)(n(1))-PEPCK-GTP complex yielded a stoichiometry of 1 mol of Mn(2+) bound per mole of Co(3+)(n(1))-PEPCK-GTP with a K(D) of 5 microM. PRR studies show a significant enhancement for the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complex. A change in enhancement in the presence of PEP suggests that PEP interacts with the second metal ion. The distance between Mn(2+) at site n(2) on PEPCK and the cis and trans protons and the (31)P of PEP are 7.0, 7.5, and 4.8 A, respectively, as measured by high-resolution NMR. PRR studies of the Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GTP and Co(3+)(n(1))-PEPCK-Mn(2+)(n(2))-GDP complexes as a function of frequency (omega(I)) were used to estimate the hydration number of the n(2) metal to be between 0.5 and 0.7. The metal-metal distance for the M(n(1))-PEPCK-M(n(2))-GTP complex is approximately 8.3 A, and the distance for the M(n(1))-PEPCK-M(n(2))-GDP complex is 9.2 A. The change in the metal-metal distance suggests a conformational change at the active site of PEPCK occurs during catalysis. The Co(3+)(n(1))-PEPCK complex was incubated with Co(2+), GTP, and H(2)O(2) to create a doubly labeled and inactive Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex. The Co(3+)(n(1))-PEPCK-Co(3+)(n(2))-GTP complex was digested by LysC, and two cobalt-containing peptides were purified using RP-HPLC. Amino acid sequencing of the second cobalt-containing peptide points to the region of Tyr57-Lys76 of PEPCK. Asp66, Asp69, and Glu74 are all feasible ligands to the site n(2) metal.
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PMID:Characterization of the second metal site on avian phosphoenolpyruvate carboxykinase. 1068 18