Kinetics of phosphoenolpyruvate carboxylase from Zea mays leaves at high concentration of substrates.
At low concentrations of phosphoenolpyruvate and magnesium, the substrate of phosphoenolpyruvate carboxylase (PEPC) from Zea mays leaves is the MgPEP complex and free phosphoenolpyruvate (fPEP) is an allosteric activator [A. Tovar-Méndez, R. Rodríguez-Sotres, D.M. López-Valentín, R.A. Muñoz-Clares, Biochem. J. 332 (1998) 633-642]. To further the understanding of this photosynthetic enzyme, we have re-investigated its kinetics covering a 500-fold range in fPEP and free Mg(2+) (fMg(2+)) concentrations. Apparent V(max) values were dependent on the concentration of the fixed free species, suggesting that these species are substrates of the PEPC-catalyzed reaction. However, when substrate inhibition was taken into account, similar V(max) values were obtained in all saturation curves for a given varied free species, indicating that MgPEP is indeed the reaction substrate. As substrate inhibition may be the result of the rise in ionic strength of the assay medium, we studied its effects on the kinetics of the enzyme. Mixed inhibition against MgPEP was found, with apparent K(ic) and K(iu) values of 36 and 1370 mM, respectively. Initial velocity patterns determined at constant ionic strength, 600 mM, were consistent with MgPEP being the true PEPC substrate, fPEP an allosteric activator, and fMg(2+) a weak, non-competitive inhibitor, thus confirming the kinetic mechanism determined previously at low concentrations of PEP and Mg(2+), and indicating that apparent substrate inhibition by MgPEP in maize leaf PEPC is caused by inhibition by high magnesium and ionic strength.
Tovar-Méndez
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Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, México D.F. 04510, Mexico.
Muñoz-Clares
R A
RA
D007700:Kinetics; D008956:Models, Chemical; D009994:Osmolar Concentration; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D010730:Phosphoenolpyruvate Carboxylase