Regulation of Phosphoenolpyruvate Carboxylase from Crassula argentea

Abstract

Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at -70°C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage-which is composed of an equilibrium mixture of dimer and tetramer-with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. (Crassula argentea Thunb.) were grown in a greenhouse as described previously (17).Chemicals. The chemicals used in this study were identical with those described earlier (17, 18) and were of the highest purity commercially available.Enzyme Assay. The activity of PEP carboxylase was measured at 25°C in 1.0 ml of 50 mm Mops (pH 7.2) containing 0.15 mm NADH, 5 mM MgSO4, 10 mM NaHCO3, 5 mM PEP, and 1 IU of malate dehydrogenase (from Sigma). The reaction was started by the addition of enzyme and the oxidation of NADH was followed at 340 nm in the cell compartment of a Beckman DU-50 spectrophotometer controlled at 25°C. Kinetic parameters were determined by fitting assays at varying concentrations of PEP to the Michaelis-Menten equation modified to provide estimates of the Hill number as well as Vm and Km (17).Enzyme Purification. The enzyme was purified from day leaves collected after 4 to 5 h of light. The purification procedure was a modification ofthe method previously described (18). All steps were performed at 0 to 4°C. Leaves were homogenized in 2 volumes of 0.05 M Hepes buffer (pH 7.2), with 1 mM EDTA, 1 mM DTT and 20% (v/v) glycerol. A Polytron homogenizer was run in an ice bath at full speed for two 15 s bursts with a 15 s cooling interval. After filtering through two layers of fine nylon mesh, the homogenate was centrifuged at 5OOg for 10 min and the supernatant liquid discarded. The pellet was extracted with the extraction buffer containing 2% Triton X-1 14 but omitting glycerol, following the method of Pryde (10) for solubilizing membrane-associated proteins. After centrifugation, the material soluble in Triton X-1 14 was fractionated by ammonium sulfate precipitation, with the fraction precipitating between 35 and 60% ammonium sulfate saturation collected by centrifugation at 16,000g for 15 min and suspended in the extraction buffer without either Triton or glycerol. The preparation was then desalted using a 2 x 20 cm Sephadex G-25 column and applied to either a DEAE-cellulose

Oligomerization and Regulation of Higher Plant Phosphoenolpyruvate Carboxylase

“…The other line shows a similar response in light scattering. The greater response at high dilutions may represent the formation of aggregates from the monomeric enzyme that we have previously seen ( 18,19) to be without activity. Figure 2B shows that the presence of malate causes a decrease in the velocity of the reaction that reaches a minimal level at maximal dilution.…”

“…Uncovering the fact that small oligomers of PEPC, e.g. monomer and dimer, have little or no activity, whereas tetramer and larger molecules possess considerable activity (7,9,(15)(16)(17)(18)(19), has suggested oligomerization as a means of controlling the activity of the enzyme.…”

Posttranslational Regulation of Phosphoenolpyruvate Carboxylase in C4 and Crassulacean Acid Metabolism Plants

“…the night/ day activity ratio is about 8 [15,29]) which are paralleled by the classical physiological changes in the activity ofCAM (e.g. external C02 fixation, titratable acidity [21] (20,29 (30,31) further showed that the substrate PEP and Mg2+, a bivalent cation required for catalysis, favor the conversion of a2 to a4, whereas L-malate, a feedback inhibitor, shifts the dimer-tetramer equilibrium toward the dimeric enzyme-form. These in vitro findings suggested that interconversion between the malatesensitive dimer and malate-insensitive tetramer of PEPC might be the molecular mechanism for the diel regulation of PEPC activity in CAM plants.…”

Activation of Higher Plant Phosphoenolpyruvate Carboxylases by Glucose-6-Phosphate

“…Although other factors such as enzyme phosphorylation (3,7,11), aggregation/disaggregation of the enzyme (29,30,32), temperature (2,21,28,31), and other allosteric effectors (8, 9, 14-17, 19,22,28) seem to be involved in at least some cases, it is clear that these two effectors have powerful influences on the post-translational activity of PEPC regardless of whether other factors may also be involved. The 2Abbreviations: PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; Aces, N(2-acetamido)-2-aminoethanesulfonic acid; Ches, 2(N-cyclohexyl-amino)ethane sulfonic acid; PCMB, pchloromercuribenzoate; Glc-6-P, glucose-6-phosphate.…”