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Identification of reactive lysines in phospho enol pyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae
Author(s) -
Bazaes Sergio,
Goldie Hughes,
Cardemil Emilio,
Jabalquinto Ana María
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00107-k
Subject(s) - saccharomyces cerevisiae , escherichia coli , biochemistry , chemistry , identification (biology) , microbiology and biotechnology , biology , yeast , gene , botany
Escherichia coli and Saccharomyces cerevisiae phospho enol pyruvate 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 Mn 2+ . 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. Lys 286 of bacterial PEPCK and Lys 289 of the yeast enzyme were identified as the reactive amino acid residues. The modified lysine residues are conserved in all ATP‐dependent phospho enol pyruvate carboxykinases described so far.