AFFINITY CLEAVAGE AT THE METAL-BINDING SITE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly in activated by micromolar concentrations of ferrous sulfate in the prese nce of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ w ith Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, of Co2+ at 100-fol d molar excess over Fe2+ offered complete protection from Fe2+/ascorba te-induced inactivation. The substrates PEP and GTP, bur not OAA, GDP, or CO2, offered full protection from inactivation. The addition of 5 mM EDTA stopped further inactivation of the enzyme. Thermodynamic stud ies indicate that the inactive enzyme no longer binds Mn2+ but still h ad high affinity for GTP indicating that the inactivation process was specific for the metal sire, A decrease in cysteine content was observ ed over time following PEPCK treatment with. Fe2+ and ascorbate, The a pparent first-order rate constant for free sulfhydryl loss (0.085 +/- 0.005 min(-1)) is similar to the apparent first-order rate constant fo r inactivation (0.067 +/- 0.005 min(-1)). Amino acid composition analy sis revealed that cysteic acid was generated upon Fe2+/ascorbate addit ion to PEPCK. Native chicken liver PEPCK has an M-r, of 67 kDa. SDS-PA GE of the inactivated enzyme showed the presence of two new bands at 3 1.7 and 35.3 kDa indicating that PEPCK was specifically cleaved, al a single site. The rate of cleavage was slower than the rate of inactiva tion and fully inactivated enzyme was only 50% cleaved, The Fe2+/ascor bate-catalyzed inactivation was not solely due to protein cleavage, Th e protein fragments generated by cleavage were separated by C4 reverse phase HPLC, The cleavage exposed a new N-terminus which was identifie d to be the 35.3 kDa C-terminal half of PEPCK. Sequencing of the fragm ents indicated that the site of cleavage was between Asp296 and Ile297 . These results indicate that Asp296 is involved in metal chelation. T his agrees with previous studies [Hlavaty, J. J., & Nowak, T. (1997) B iochemistry 36, 3389-3403] that suggested that Asp295 and Asp296 are i nvolved in metal binding.