Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 126.96.36.199] is completely inactivated by the 2′,3′-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 ± 17 μM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 ° C is 0.200 ± 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.
ASJC Scopus subject areas
- Molecular Biology