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 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. 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 phosphoenolpyruvate carboxykinases described so far.