Oligomerization and Regulation of Higher Plant Phosphoenolpyruvate Carboxylase

Abstract

The specific activity of phosphoenolpyruvate (PEP) measured at a saturating level of substrate diminishes as the enzyme is diluted at about the same rate that specific light scattering by the diluted enzyme decreases. The presence of PEP in the assay causes an increase in activity with increasing dilution. This is accompanied by an increase in light scattering of the diluted enzyme. The reverse situation obtains with the addition of malate to assays: the activity decreases with increasing dilution but light scattering is not substantially changed, indicating that the enzyme is already brought to a smaller aggregate by the dilution itself. In this case, the inhibition by malate in the assay probably is the noncompetitive type not involved in regulatory control by malate. Glucose-6-phosphate in the range from 1 to 6 millimolar causes an increase in activity of the enzyme run at a substrate level less than K,, and an associated increase in light scattering is found, indicating an increase in the mean size of the enzyme. When PEP is added to a 1/80 diluted enzyme, light scattering increases and is associated with a more rapid activity of the enzyme. When malate is added to the same cuvette, the activity decreases and the light scattering diminishes, thus showing that the ligand response is immediately reversible. When malate is added first, followed by PEP, the reverse sequence of activity and light scattering change is observed.

PHOSPHOENOLPYRUVATE CARBOXYLASE: A Ubiquitous, Highly Regulated Enzyme in Plants

“…PEP, G6P, Mg 2+ , or a higher [PEPC] favors conversion of α 2 to α 4 , whereas l-malate or a lower [PEPC] shifts the equilibrium toward the dimeric form (79,128,129). Similar in vitro association/dissociation properties have been reported for the active C 4 homotetramer from maize (123,127). There is no evidence, however, to support the involvement of these aggregation-state changes in the diel regulation of the CAM and C 4 isoforms in vivo.…”

Evolution of the Enzymatic Characteristics of C4Phosphoenol Pyruvate Carboxylase

“…The last step also made it possible to distinguish between the oligomeric forms of the enzyme. There are several reports that the C, P-pyruvate carboxylase can rapidly loose or reduce its activity during preparation and storage (Uedan and Sugiyama, 1976;Gavalas et al, 1982;Angelopoulos et al, 1988;Stamatakis et al, 1988;Fujikura and Sun, 1991;Wedding et al, 1994) and that these changes may be due to alterations in the oligomeric structure (Walker et al, 1986;Podesti and Andreo, 1989;Willeford and Wedding, 1992). The dimer may also be active but differs in properties compared to the tetramer.…”

Inactivation of Maize Phosphoenolpyruvate Carboxylase by Urea

“…Because urea does not bind to the enzyme, it is unlikely that these effects are due to displacement of urea from the enzyme. Because both PEP and glc-6-P have been shown in other studies to increase the size of the PEPC aggregate (15,17), it is reasonable at this point to speculate that the "protective" effect of these ligands is due to their independent effect in inducing aggregation, perhaps by reforming hydrogen bonds broken by urea. Light-scattering measurements tend to confirm such an assumption, because an increase in emission was observed when either PEP or glc-6-P was added to PEPC in 1.5 M urea.…”

“…This effect of urea, which disaggregates proteins by breaking hydrogen bonds (12), offers a different way of probing the factors involved in equilibria of enzymic oligomers. Our interest in this question arises from the aggregation and disaggregation of PEPC,2 which is a major factor in regulating the activity of that enzyme (15,17).We have studied the effect of moderate concentrations of urea on the inactivation of PEPC and have investigated the effect on the response to urea of ligands known to regulate the enzyme. We have also shown, using light scattering, that the effect of urea is to dissociate the enzyme.…”

“…This effect of urea, which disaggregates proteins by breaking hydrogen bonds (12), offers a different way of probing the factors involved in equilibria of enzymic oligomers. Our interest in this question arises from the aggregation and disaggregation of PEPC,2 which is a major factor in regulating the activity of that enzyme (15,17).…”