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

Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C-4 plants shows different affinities for the substrate phosphoenolpyru vate (PEP) at pH 7.0 and pH 8.0. This has been demonstrated by determinatio n of the enzymatic activity, applying fluorescence titrations and fast reac tion techniques such as iodine laser temperature jump (ILTJ) and stopped fl ow (SF). The binding reaction of PEP to PEPC from Zea mays was measured usi ng the fluorescence probe 2-p-toluidinonaphthalene-6-sulfonate (TNS). The k inetics are described by an allosteric mechanism with a fast reversible bim olecular binding reaction of PEP to a high affinity (tensed) form of PEPC w hich is in equilibrium with its low affinity (relaxed) form. The associatio n and dissociation rate constants k(+A) and k(-A) for the fast binding reac tion to the high affinity form were determined to be 1.4 +/- 0.15 x 10(4) M -1 s(-1) and 17 +/- 6 s(-1) at pH 8.0. The corresponding dissociation const ants K-d = 1.2 +/- 0.5 mM for PEP calculated from the kinetic constants, me asured by ILTJ and SF, are in good agreement with K-d values achieved in ou r equilibrium titration experiments or from the data of Michaelis-Menten-ty pe kinetic experiments. PEP preferentially binds to the high affinity bindi ng site of PEPC, shifting the isomerisation equilibrium strongly towards th e tensed form, with the consequence that PEPC is activated. Rate constants for the isomerisation process were obtained as k(B+(0)) = 4.95 +/- 0.35 s(- 1) and k(B-(0)) = 1.25 +/- 0.1 s(-1) at pH 8. Our kinetic data are consiste nt with the concerted sequential allosteric mechanism introduced by Monod, Wyman and Changeux. In summary, in this study we present, for the first tim e, data on the kinetics of PEP binding and on the rate of the isomerisation reaction between the two allosteric forms of PEPC.