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

27 February 1995

journal article
research article
Published by Wiley in FEBS Letters

Vol. 360 (2) , 207-210
https://doi.org/10.1016/0014-5793(95)00107-k

Abstract

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.

Keywords

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