Kinetics of phosphoenolpyruvate carboxylase from Zea mays leaves at high concentration of substrates

At low concentrations of phosphoenolpyruvate and magnesium, the substrate o f phosphoenolpyruvate carboxylase (PEPC) from Zea mays leaves is the MgPEP complex and free phosphoenolpyruvate (fPEP) is an allosteric activator [A. Tovar-Mendez, R. Rodriguez-Sotres, D.M. Lopez-Valentin, R.A. Munoz-Clares, Biochem. J. 332 (1998) 633-642]. To further the understanding of this photo synthetic enzyme, we have re-investigated its kinetics covering a 500-fold range in fPEP and free Mg2+ (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, indicat ing that MgPEP is indeed the reaction substrate. As substrate inhibition ma y be the result of the rise in ionic strength of the assay medium, we studi ed its effects on the kinetics of the enzyme. Mixed inhibition against MgPE P was found, with apparent K-ic and K-iu values of 36 and 1370 mM, respecti vely. Initial velocity patterns determined at constant ionic strength, 600 mM, were consistent with MgPEP being the true PEPC substrate, FPEP an allos teric activator, and fMg(2+) a weak, non-competitive inhibitor, thus confir ming the kinetic mechanism determined previously at low concentrations of P EP and Mg2+, and indicating that apparent substrate inhibition by MgPEP in maize leaf PEPC is caused by inhibition by high magnesium and ionic strengt h. (C) 2001 Elsevier Science B.V. All rights reserved.