Mitochondrial phosphoenolpyruvate carboxykinase from the chicken. Comparison of the cDNA and protein sequences with the cytosolic isozyme.

Weldon, Sharon L.; Rando, A.; Matathias, Amy S.; Hod, Yaacov; Kalonick, Patricia A.; Savon, Summer; Cook, Jonathan S.; Hanson, Richard W.

doi:10.1016/s0021-9258(19)39115-x

Cited by 64 publications

(29 citation statements)

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“…This demonstrates that the labeling process is reversible and that no deleterious irreversible damage by oxidation of PEPCK has occurred during the modification procedure. Native PEPCK contains 13 cysteine residues, none of which are in disulfide bonds (Makinen et al, 1983;Weldon et al, 1990). Co 3+ -PEPCK has 13 free cysteine residues, the same number as apoPEPCK.…”

Section: Discussionmentioning

confidence: 99%

“…The amino acid sequences of PEPCK from chicken liver mitochondria (Chicken mito.) (Weldon et al, 1990), chicken liver cytosol (Chicken cyto.) (Cook et al, 1986), Ascaris suum (A. suum) (Geary et al, 1993), Haemonchus contortus (H.…”

Section: Discussionmentioning

confidence: 99%

“…Sequences were aligned to maximize overall identity between all of the sequences shown. The numbering system is based on the sequence of chicken mitochondrial PEPCK (Weldon et al, 1990). The preceding amino acid in chicken mitochondrial PEPCK is 275 Lys.…”

Section: Discussionmentioning

confidence: 99%

“…Mass, amino acid composition, and sequence analyses identified the peptide as the segment Thr276-Lys301. This highly conserved region is located near the central portion of PEPCK and is near the putative nucleotide binding region (Weldon et al, 1990). Amino acids Asp295 and Asp296 are the most feasible metal ligands in this peptide, discounting backbone carbonyl groups as ligands.…”

Section: Discussionmentioning

confidence: 99%

“…Nucleotide and derived protein sequences of the mature form of the mitochondrial isoenzyme of PEPCK from chicken liver have been derived from cDNA clones (Weldon et al, 1990). Crystallographic analysis of PEPCK from animal sources has not been possible due to the current inability to crystallize the enzyme.…”

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confidence: 99%

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Formation and Characterization of an Active Phosphoenolpyruvate Carboxykinase−Cobalt(III) Complex

Hlavaty

Nowak

1997

Biochemistry

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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