Influence of allosteric effectors on the kinetics and equilibrium binding of phosphoenolpyruvate (PEP) to phosphoenolpyruvate carboxylase (PEPC) fromZea mays

Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C-4 plants, shows the features of an allosteric enzyme. Allosteric acti vators such as D-glucose-6-phosphate and glycine increase the affinity of P EPC for its substrate PEP at pH 8.0 and pH 7.0. Allosteric inhibitors like L-malate and L-aspartate predominantly decrease the affinity of the carboxy lase for PEP at pH 7.0. This was demonstrated by determination of the enzym atic activity and stopped flow (SF) fluorimetry. The binding reaction of PE P to PEPC from Zea mays was measured using the fluorescence probe 2-p-tolui dinonaphthalene-6-sulfonate (TNS). The kinetics are described by an alloste ric mechanism with a fast reversible bimolecular binding step of PEP to a h igh affinity (tensed) form of PEPC, which is in equilibrium with its low af finity (relaxed) form. The influence of allosteric effectors on the conform ational transition step is demonstrated in support of the description of th e kinetics of PEPC by applying a concerted allosteric mechanism as introduc ed by Monod, Wyman and Changeux. In summary, we present data for the influe nce of allosteric activators on the kinetics of PEP binding to PEPC and on the concentration dependence of the isomerisation reaction between two allo steric forms of PEPC. (C) 2001 Elsevier Science B.V. All rights reserved.