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 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.
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
Claudia
C
Bueno
Fernando D
FD
González-Nilo
María
M
Victoria Encinas
Emilio
E
Cardemil
D019943:Amino Acid Substitution; D009154:Mutation; D010728:Phosphoenolpyruvate / Q000378:metabolism*; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D010728:Phosphoenolpyruvate; D019764:Phosphoenolpyruvate Carboxykinase (ATP)