Nikos A. Gavalas

Reversible cold inactivation of C4-phosphoenolpyruvate carboxylase: factors affecting reactivation and stability

Summary Phosphoenolpyruvate carboxylase (EC 4.1.1.31), in dilute leaf extracts from Cynodon dactylon (L.) Pers., is inactivated during incubation at 0°–25 °C. The rate and extent of inactivation are increased at lower temperatures. The inactivation at 0 °C is partially reversible upon transfer to 25 °C and more than fully reversible when glucose-6-phosphate (10 mM) is also added. The activating effect of glucose-6phosphate becomes faster and more pronounced as the temperature is increased. Inactivation is favored by high pH, dilution and high ionic strength. Dissociation of the active tetrameric form into smaller subunits is most probably the cause of inactivation.

Artifacts in the assay of maize leaf phosphoenolpyruvate carboxylase activity due to its instability.

When the assay of maize leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity is started with phosphoenolpyruvate, much lower reaction rates are obtained as compared to the enzyme-initiated reaction. The difference is due to the lability of the dilute enzyme in the absence of its substrate and is increased with incubation time in the absence of substrate or stabilizers. The activation of the enzyme by glucose-6-phosphate is overestimated with the substrate-initiated assay since a part of the apparent activation is due to stabilization of the enzymic activity by this effector during the minus-substrate preincubation. In contrast, the inhibitory effect of malate is underestimated when the reaction is started with the substrate. The enzyme-initiated assay is recommended provided that the necessary corrections for apparent activity in the absence of substrate and for inactivation during the assay at low substrate levels are made.

Calcium Inhibition of Phosphoenolpyruvate Carboxylase: Possible Physiological Consequences for C4-Photosynthesis

Summary Calcium is a strong inhibitor of phosphoenolpyruvate carboxylase at concentrations low enough (0.5-1.0 mM) to be of physiological significance. The inhibition is competitive in respect to magnesium and affects both C 3 - and C 4 -phosphoenolpyruvate carboxylases. The results are consistent with the hypothesis that low soluble calcium is a prerequisite for C 4 -photosynthetic carbon fixation.

Assaying for pyruvate, orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.

Modulation of Phosphoenolpyruvate Carboxylase by 3-Phosphoglycerate: Probable Physiological Significance for C4-photosynthesis

Summary The effect of 3-phosphoglycerate on the activity of phosphoenolpyruvate carboxylase from C 4 plants depends on pH and substrate (PEP) concentration. At low pH (7.2) a strong activation is observed due to the elimination of the positive cooperativity; therefore, it is most evident at low substrate levels. This activation may have a physiological significance for C 4 -photosynthesis, since 3-PGA is a major photosynthetic metabolite which can also serve as a stabilizer of PEP concentration during steady — state photosynthesis.

Effects of glycerol on the in vitro stability and regulatory activation/inactivation of pyruvate, orthophosphate dikinase of Zea mays L.

Glycerol stabilizes the activity of pyruvate, orthophosphate dikinase extracted from darkened or illuminated maize leaves. It serves as a better protectant of activity than dithiothreitol for the active day-form and the glycerol concentration needed for full protection is inversely related to the level of protein. The night-form of the enzyme is also protected by glycerol not only against inactivation, but also against partial reactivation in storage. Glycerol does not prevent the Pi-dependent activation nor the ADP-dependent inactivation of pyruvate, orthophosphate dikinase, but the rates of both processes are substantially decreased. The ability of the inactive night-form for Pi-dependent activation is also sustained by glycerol for at least 2 h at 20°C, apparently through stabilization of the labile regulatory protein.

Effects of anions in ion exchange chromatography on the specific activity of C4 phosphoenolpyruvate carboxylase

Summary The specific activity and yield of phosphoenolpyruvate carboxylase (EC 4.1.1.31), partially purified from Cynodon dactylon (L.) Pers. (Bermuda grass) leaves, is affected by the anions of the elution gradient in the ion exchange chromatography step of purification. Specific activities around 60 µmol min -1 · mg -1 protein were obtained at peak fractions with sulphate and phosphate. Chloride decreases the specific activity to 27 µmol min -1 · mg -1 protein and should be avoided in elution gradients. It is concluded that the activation state of phosphoenolpyruvate carboxylase can be modified during extraction and purification, and that the specific activity of the fully activated enzyme is much higher than realized up to the present.