Kinetics and equilibrium binding of phosphoenolpyruvate to phosphoenolpyruvate carboxylase from Zea mays

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Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C4 plants shows different affinities for the substrate phosphoenolpyruvate (PEP) at pH 7.0 and pH 8.0. This has been demonstrated by determination of the enzymatic activity, applying fluorescence titrations and fast reaction techniques such as iodine laser temperature jump (ILTJ) and stopped flow (SF). The binding reaction of PEP to PEPC from Zeamays was measured using the fluorescence probe 2-p-toluidinonaphthalene-6-sulfonate (TNS). The kinetics are described by an allosteric mechanism with a fast reversible bimolecular binding reaction of PEP to a high affinity (tensed) form of PEPC which is in equilibrium with its low affinity (relaxed) form. The association and dissociation rate constants k+A and k-A for the fast binding reaction to the high affinity form were determined to be 1.4±0.15×104 M-1 s-1 and 17±6 s-1 at pH 8.0. The corresponding dissociation constants Kd=1.2±0.5 mM for PEP calculated from the kinetic constants, measured by ILTJ and SF, are in good agreement with Kd values achieved in our equilibrium titration experiments or from the data of Michaelis–Menten-type kinetic experiments. PEP preferentially binds to the high affinity binding site of PEPC, shifting the isomerisation equilibrium strongly towards the tensed form, with the consequence that PEPC is activated. Rate constants for the isomerisation process were obtained as kB+(0)=4.95±0.35 s-1 and kB-(0)=1.25±0.1 s-1 at pH 8. Our kinetic data are consistent with the concerted sequential allosteric mechanism introduced by Monod, Wyman and Changeux. In summary, in this study we present, for the first time, data on the kinetics of PEP binding and on the rate of the isomerisation reaction between the two allosteric forms of PEPC.