Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: relevance of arginine 70 for catalysis.
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 CO2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site 1 decrease the enzyme catalytic efficiency and the affinity of the protein for PEP, evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg70, whose guanidinium group is close to the PEP carboxyl group. Arg70 mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg70Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn2+ complex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg70 and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis.
Cristina Ravanal
M
M
Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago 33, Chile.
Flores
Michel
M
Pérez
Estela
E
Aroca
Francisco
F
Cardemil
Emilio
E
D000244:Adenosine Diphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D001120:Arginine; D019764:Phosphoenolpyruvate Carboxykinase (ATP)