Substrate binding to fluorescent labeled wild type, Lys213Arg, and His233Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the γ-phosphoryl group of ATP to yield PEP, ADP, and CO 2 in the presence of a divalent metal ion. Previous experiments have shown that mutation of amino acid residues at metal site 1 decrease the steady-state affinity of the enzyme for PEP, suggesting interaction of PEP with the metal ion [Biochemistry 41 (2002) 12763]. To more completely understand this enzyme interactions with substrate ligands, we have prepared the phosphopyridoxyl (P-pyridoxyl)-derivatives of wild type, Lys213Arg, and His233Gln S. cerevisiae PEP carboxykinase and used the changes in the fluorescence probe to determine the dissociation equilibrium constants of PEP, ATPMn 2−, and ADPMn 1− from the corresponding derivatized enzyme–Mn 2+ complexes. Homology modeling of P-pyridoxyl-PEP carboxykinase and P-pyridoxyl-PEP carboxykinase–substrate complexes agree with experimental evidence indicating that the P-pyridoxyl group does not interfere with substrate binding. ATPMn 2− binding is 0.8 kcal mol −1 more favorable than ADPMn 1− binding to wild type P-pyridoxyl–enzyme. The thermodynamic data obtained in this work indicate that PEP binding is 2.3 kcal mol −1 and 3.2 kcal mol −1 less favorable for the Lys213Arg and His233Gln mutant P-pyridoxyl-PEP carboxykinases than for the wild type P-pyridoxyl–enzyme, respectively. The possible relevance of N and O ligands for Mn 2+ in relation to PEP binding and catalysis is discussed.