Oligomerization and the Sensitivity of Phosphoenolpyruvate Carboxylase to Inactivation by Proteinases

Self Cite

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).

Section: Ph Profile Of Maximal Inhibition By Malatementioning

confidence: 62%

“…This difference was found between enzyme prepared from CAM leaves gathered just before daylight or late in the afternoon (26), and the convertibility of the dimer to the tetramer and the resistance of the tetramer to malate inhibition and the susceptibility of the dimer to malate has been demonstrated (19,20,27,29).…”

Section: Discussionmentioning

confidence: 97%

Inhibition of Phosphoenolpyruvate Carboxylase by Malate

Wedding¹,

Black²,

Meyer³

1990

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“…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).…”

mentioning

confidence: 95%

“…It has been suggested that producing a highly purified PEPC requires such a long preparation time that it may result in the loss by action of proteinases of the N-terminal segment that contains the seryl group that is phosphorylated (3), thus eliminating the possibility of control by phosphorylation. An opposing sug-gestion is that incubation of the CAM PEPC with trypsin causes a decrease in activity (13), but the residual activity is still inhibited by malate and activated by Glc-6-P, making it unlikely that some crucial regulatory site has been removed.…”

mentioning

confidence: 99%

Oligomerization and Regulation of Higher Plant Phosphoenolpyruvate Carboxylase

Willeford¹,

Wedding²

1992

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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.

“…The fluorescence of the extrinsic fluorophore ANS was measured with a Perkin Elmer LS-5 spectrofluorimeter in a 3-mL cuvette thermostated at 25°C and magnetically stirred (19). ANS was excited at 350 nm (slit = 5 nm) and emission was measured at 490 nm (slit = 15 nm).…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

Rustin

Meyer²,

Wedding³

1991

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The chemical modification of phosphoenolpyruvate carboxylase purified from Crassula argentea leaves was studied using the fluorescence of the extrinsic probe 8-anilino-1-napthalenesulfonate. The effects of ligands on kinefic parameters of phosphoenolpyruvate carboxylase activity, and its response to pH and metal cations, were associated with the binding of the ligands to the enzyme as measured by fluorescence. Binding of the ligands phosphoenolpyruvate, malate, and glucose-6-phosphate revealed by fluorescence measurements corresponds to competitive phenomena observed in kinetic studies. The fluorescence measurements also suggest the involvement of specific amino acids in the binding of a given ligand. Arginyl residues modified by 2,3-butanedione appear to be directly involved in the binding of phosphoenolpyruvate and malate to the active and the inhibition sites, respectively. A histidyl residue was involved in the binding of malate, accounting for the lack of inhibition by malate in kinetic studies of the enzyme treated with diethylpyrocarbonate. Although activity was lost, there was no decrease in the ability of the treated enzyme to bind phosphoenolpyruvate, suggesting that additional histidyl residues are essential for activity although not directly involved in the binding of phosphoenolpyruvate. The lysine reagent trinitrobenzenesulfonate caused a loss of activity and a reduction in malate inhibition and glucose-6-phosphate activation, but these modifications were not related to changes in the ability of the enzyme to bind any of the three ligands. This suggests that lysine residues were not directly involved in the binding of these ligands.