Identification of reactive lysines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae

Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases (PEPCKs), were inactivated by pyridoxal 5′‐phosphate followed by reduction with sodium borohydride. Concomitantly with the inactivation, one pyridoxyl group was incorporated in each enzyme monomer. The modification and...

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Bibliographic Details
Published in:FEBS letters 1995-02, Vol.360 (2), p.207-210
Main Authors: Bazaes, Sergio, Goldie, Hughes, Cardemil, Emilio, Jabalquinto, Ana María
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
Chemical modification
Escherichia coli
Escherichia coli - enzymology
HEPES
high-performance liquid chromatography, TFA, trifluoracetic acid
HPLC
Lysine - chemistry
Lysyl residues
Molecular Sequence Data
N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
OAA
oxalacetic acid
PEP
PEPCK
Peptides - chemistry
phenylthiohydantoin
phosphoenolpyruvate
Phosphoenolpyruvate carboxykinase
Phosphoenolpyruvate Carboxykinase (GTP) - antagonists & inhibitors
Phosphoenolpyruvate Carboxykinase (GTP) - chemistry
PLP
PTH
pyridoxal 5′-phosphate
Pyridoxal Phosphate - chemistry
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Sequence Alignment
Sequence Homology, Amino Acid
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Description
Summary:Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases (PEPCKs), were inactivated by pyridoxal 5′‐phosphate followed by reduction with sodium borohydride. Concomitantly with the inactivation, one pyridoxyl group was incorporated in each enzyme monomer. The modification and loss of activity was prevented in the presence of ADP plus Mn2+. After digestion of the modified protein with trypsin plus protease V‐8, the labeled peptides were isolated by reverse‐phase high‐performance liquid chromatography and sequenced by gas‐phase automatic Edman degradation. Lys286 of bacterial PEPCK and Lys289 of the yeast enzyme were identified as the reactive amino acid residues. The modified lysine residues are conserved in all ATP‐dependent phosphoenolpyruvate carboxykinases described so far.
ISSN:0014-5793
1873-3468
DOI:10.1016/0014-5793(95)00107-K