Influence of allosteric effectors on the kinetics and equilibrium binding of phosphoenolpyruvate (PEP) to phosphoenolpyruvate carboxylase (PEPC) from Zea mays.
Phosphoenolpyruvate carboxylase (PEPC) the carbon dioxide processing enzyme of C(4) plants, shows the features of an allosteric enzyme. Allosteric activators such as D-glucose-6-phosphate and glycine increase the affinity of PEPC 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 carboxylase for PEP at pH 7.0. This was demonstrated by determination of the enzymatic activity and stopped flow (SF) fluorimetry. The binding reaction of PEP to PEPC from Zea mays 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 step of PEP to a high affinity (tensed) form of PEPC, which is in equilibrium with its low affinity (relaxed) form. The influence of allosteric effectors on the conformational transition step is demonstrated in support of the description of the kinetics of PEPC by applying a concerted allosteric mechanism as introduced by Monod, Wyman and Changeux. In summary, we present data for the influence of allosteric activators on the kinetics of PEP binding to PEPC and on the concentration dependence of the isomerisation reaction between two allosteric forms of PEPC.
Frank
J
J
Fritz-Haber-Institut, Max-Planck-Society, Faradayweg 4-6, D-14195 Berlin, Germany. frank_j@fhi-berlin.mpg.de
Clarke
R J
RJ
Vater
J
J
Holzwarth
J F
JF
D000465:Algorithms; D001224:Aspartic Acid / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D005470:Fluorometry; D019298:Glucose-6-Phosphate / Q000737:chemistry; D007700:Kinetics; D008293:Malates / Q000494:pharmacology*; D008968:Molecular Conformation; D010728:Phosphoenolpyruvate / Q000737:chemistry*; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D003313:Zea mays / Q000201:enzymology / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008293:Malates; D001224:Aspartic Acid; D019298:Glucose-6-Phosphate; D010728:Phosphoenolpyruvate; C030298:malic acid; D010730:Phosphoenolpyruvate Carboxylase