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

Abstract

Studies of the response of phosphoenolpyruvate carboxylase from C3 (wheat [Tritium aestivum Lj), C4 (maize [Zea mays LI), and Crassulacean acid metabolism (CAM) (Crassula) leaves to the activator glucose-6-phosphate as a function of pH showed that the binding of the activator and the response path to activation were essentially identical for all three enzymes. The level of affinity for the activator differed, with the CAM enzyme having the highest affinity and the maize enzyme the lowest. The observed pK values suggest that histidine and cysteine groups may be involved in activation by glucose-6-phosphate. The presence of glucose-6-phosphate protected the enzyme against inactivation of the activation response by p-chloromercuribenzoate. The maximal activation response to glucose-6-phosphate showed differences among the three enzymes including different pH optima and different pH profiles. Here the maize leaf enzyme showed a potential response about twice as great as that of the C3 and CAM enzymes.lope. The PEPCs of C3 plants are mostly responsible to these same effectors, and Latzko and Kelly (10) have listed 11 possible functions of PEPC in C3 plants, although these seem less likely to need tight regulation.Many studies (8, 9, 14-16, 22, 28) have reported the activation of a variety of PEPCs by Glc-6-P, and a number of possibilities have been postulated for the way in which the activity of the enzyme is stimulated by this sugar phosphate. It seemed potentially beneficial to understanding the mechanism of this activation to study the effect of pH on the activation of PEPC by Glc-6-P as a means of providing clues to the groups involved in binding of Glc-6-P and in the expression of its activating effect. The study was extended to include examples of three different types of plants, C3, C4, and CAM, both because a direct comparison of Glc-6-P activation of the PEPC of these various types of plants has not been available, and because differences in the activating mechanism might be revealing of the underlying characteristics of the process which results in activation and of the metabolic patterns of the plants themselves.Regulation of PEP2 carboxylase in CAM and C4 plants is often attributed to an interaction of two effectors-activation by Glc-6-P and inhibition by malate (1, 8, 12, 14-16, 24, 27, 28). 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) 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. MATERIALS AND METHODS EnzymesThree different forms of PEPC (EC 4.1.1.31) were compared in these studies. The first was the enzyme prepared in our laboratory from field-grown Crassula argentea by the procedure previously described (19). The specific activity of this p...

Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

“…At high pH values, the enzyme showed a low affinity for its substrate, indicated by the increased Km and low value of the Vmax/Km ratio. These experiments reinforced our previous studies suggesting strong changes in the binding properties of PEPC at different pHs accompanied by a change in the metal requirement (12,17,21,22 When Glc-6-P was added after malate ( Fig. 2A, trace c), no detectable change in the fluorescence of ANS could be measured.…”

Inhibition of Phosphoenolpyruvate Carboxylase by Malate

“…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).…”

“…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).…”

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.…”