Inhibition of Phosphoenolpyruvate Carboxylase by Malate

Abstract

Malate has been noted to be a 'mixed' inhibitor of phosphoenolpyruvate (PEP) carboxylase. The competitive portion of this inhibition appears to be fairly constant regardless of the condition of the enzyme being measured, but the noncompetitive (V-type) inhibition is subject to variation depending on the source of the enzyme, its storage condition, the presence or absence of various ligands, and differences in pH. In the case of the maize (Zea mays L.) phosphoenolpyruvate carboxylase (PEPC), the V-type inhibition by malate is much less pronounced at pH 8 than at pH 7. Examinafton of the response of the maize PEPC to PEP concentration reveals a pronounced cooperativity at pH 8 which is not present at pH 7, and which results in the disappearance of the V-type inhibition at pH 8. The ability of high concentrations of PEP to convert PEPC from a form readily inhibited by malate to one resistant to malate inhibition has been previously demonstrated and we attribute the cooperativity shown at pH 8 to this response to high levels of PEP. Support for this proposal is provided by studies of the enzyme at pH 7 and pH 8 run in 20% glycerol. In this case there was no V-type inhibition of PEPC at either pH. Treatment with 20% glycerol has been shown to result in the aggregation of maize PEPC.enzyme from C4 plants, 0.006 to 6.2 mm (19,24,25,27) for the CAM enzyme while the C3 enzyme is much less sensitive to malate (8). This variability stems in part from the changing sensitivity ofthe enzyme to malate, which occurs as a function of time and other factors in the intact cell (1 1, 23, 24) and during storage after extraction (28). It is also due in part to the fact that the kinetic mechanism of inhibition changes from the purely competitive one found at the time when the enzyme is evaluated as resistant to malate inhibition to a mixed type of inhibition, displaying both a K and a V effect (1, 5, 13). The V effect produces the inhibition usually observed in physiological studies of inhibition by malate, while the competitive inhibition often goes unnoticed in experimental studies unless for some reason low levels of substrate are present.The present study has been undertaken as a means of providing a broader base of information concerning the response of PEPC to malate and in the hope that some of the specific characteristics of the enzyme relating to malate inhibition may be revealed. Both a C4 and a CAM enzyme were used in these studies because of earlier indications that the PEPC from these sources differ in some significant ways (23,28).

Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean‐acid‐metabolism plant

“…The K, values are significantly higher than these described by other authors for the PyrPC of maize and Bryophyllurn, i.e. C4 and constitutive CAM plant [5, 7. 8, 10, 25, 261; however, some authors described similarly high values for the K, in vitro for C4 and CAM PyrPC in the presence of 15% glycerol [29,301. The authors argue that glycerol mimics the environment of the cytosol in vitro [30].…”

Oligomerization and Regulation of Higher Plant Phosphoenolpyruvate Carboxylase

“…The small effect of malate on light scattering when added first shows that the 1/80-diluted PEPC is near the dimeric equilibrium. The somewhat larger effect on activity is due to the noncompetitive inhibition of malate (6,12,14) and is not related to a change in aggregation state. It is of interest that the addition of either PEP or malate directly to the 1/80-diluted enzyme causes a smaller change than the subsequent addition ofthe other ligand.…”

“…Some workers do not find changes in the size of the molecule under conditions that are thought to result in different activities. We and others using HPLC and light scattering have found that PEPC has a diurnal change in size (1,17,18) and aggregates or disaggregates under the influence of ligands and other treatments that result in differing activities (10,(12)(13)(14).…”

“…This is potentially useful in vivo but does not provide for shutting down PEPC during the period when decarboxylation is the primary function of a CAM system, for example. Malate inhibition is so pervasive (1,4,12,14) that it must have some role in turning off PEPC. Under some circumstances, the inhibition can be large (12,14), but using 13C as a tracer to follow the in vivo activity of PEPC (8) has shown that the CAM enzyme is completely shut down in daylight.…”

Kinetic Analysis of the Non-Phosphorylated, in Vitro Phosphorylated, and Phosphorylation-Site-Mutant (Asp8) Forms of Intact Recombinant C4 Phosphoenolpyruvate Carboxylase from Sorghum

“…Indeed, although many detailed kinetic studies have been performed on C 4 and CAM Ppyruvate carboxylase (e.g. [16][17][18][19][20][21]), the documented effect of proteolysis during enzyme preparation [11,[13][14][15] and the relatively low malate sensitivity of P-pyruvate carboxylase often observed in these previous studies [16][17][18][19] suggest that the enzyme used in these prior investigations was partially or completely truncated at its N-terminus.…”