Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-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 0.8kcalmol(-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 2.3kcalmol(-1) and 3.2kcalmol(-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.
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