Protein phosphatase (type 2A)
Less sensitive towards malate Sensitive towards glucose-6-P active
(CAM: nocturnal; C4: during the day)
In the reaction, the energy-rich phosphate group of PEP is transferred to the bicarbonate ion forming carboxyphosphate as an intermediate.
The final product of the reaction, oxaloacetate, is an unstable //-keto acid and is reduced by NAD malate dehydrogenase to the stable malate. The NADH originates from glycolysis.
Since PEPC does not react with C02 but with HCO3, the carboxylation of PEP is insensitive to 02, in contrast to the carboxylation of RuBP by Rubisco.
PEPC is an extensively analysed plant enzyme because of its function as the pacemaker enzyme of CAM. Differences in the reported kinetic parameters (Michaelis constant, vmax) are not because of the different sources of the enzyme but are due, most of all, to differences in the isolated protein; often a protein was isolated which had been partially truncated by proteolysis. Furthermore, the enzyme also reversibly forms dimers or tetramers, which originally was regarded as allosteric regulation of the mechanism. The activity of the enzyme is allosterically influenced by effectors. Glucose-6-phosphate and triose-phosphate stimulate activity, whilst malate and aspartate have a strong inhibitory effect, particularly at a slightly alkaline (cytosolic) pH. The regulation of the CAM-PEPC has been shown to take place via the reversible phosphorylation of a serine residue of the 110-kDa subunit (e.g. Ser 8 for millet, Ser 15 for maize) near the N-terminus. Phosphorylation at this single site renders the enzyme insensitive to malate; even in the presence of this allosteric inhibitor it remains in the active form, in particular as it now reacts more sensitively to the positive effectors mentioned above. The phosphorylation of CAM-PEPC occurs through a strongly regulated protein kinase, dephosphorylation (and inactiva-tion) by a protein phosphatase of the type 2A. This phosphatase does not show any fluctuations in activity, in contrast to the kinase. The activity of the protein kinase is controlled diurnally, which, in contrast to the C4-PEPC, does not react to a change from dark to light (Fig. 1.5.16).
The question why CAM-PEPC does not compete during the day for C02 released by malic enzyme (in the chloroplast) can be answered by the consideration that the dephosphorylated enzyme is inhibited by malate. In addition to the post-translational regulation of activity by phosphorylation and dephosphorylation, there is another regulation through gene expression, which was observed during the induction of CAM in C3/CAM intermediates. The genome of Mesem-bryanthemum crystallinum, for example, contains two genes for PEPC, Ppcl and Ppc2. Regulation of expression is induced by drought, ABA and high salinity, where certain transcription factors recognise AT-rich regions in the promoter.
The problems of transitory malic acid storage in the vacuole and the diurnal oscillations of CAM-PEPC activity have been discussed here in more detail, but CAM also shows other peculiarities which are only partly understood, e.g. the question of what happens to the pyruvate formed during the release of C02 in the chloro-plast. Some scientists assume it is used in regeneration of PEP catalysed by pyruvate P; dikinase, analogous to the C4 pathway of photosynthesis, whilst others believe that respiration in the mitochondria is more probable. A further unsolved question is the opening of the stomates during the dark period, as stomata usually close in the dark (blue light is the effective component of the white light for the opening of stomata, see Fig. 2.2.15).
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