Characterization of Phosphoenolpyruvate Carboxykinase from Panicum maximum

Abstract

PEPCK has been characterized from several microorganisms and animals (1, 5-7, 9). However, only limited information is available on the characteristics of this enzyme in higher plants and most of this has been obtained using crude extracts (2, 10-14, 16, 18). In addition, the data on plant PEPCK are on either the exchange reaction or the ADP-dependent carboxylation reaction. Using either of these reactions to study PEPCK in relation to C4 photosynthesis is open to criticism since the proposed role of PEPCK is as a decarboxylase. The exchange reaction has been found to occur at rates much faster (3-to 30-fold) than either the carboxylation or decarboxylation reaction in animal tissue (7) Even though PEPCK is proposed as the decarboxylase in bundle sheath cells of certain C4 plants, no information is available on the characteristics of the ATP-dependent decarboxylation of OAA by the enzyme (12)(13)(14). This report presents data on the partial purification of PEPCK to remove PEP carboxylase using the C4 grass Panicum maxinum. The ATP-dependent decarboxylation reaction has been characterized and compared to the exchange reaction and the ADP-dependent carboxylation reaction.Phosphoenolpyruvate carboxykinase catalyzes the nucleotidedependent carboxylation of PEP2 to produce OAA in a reversible fashion as shown in equation 1. CO2 + ADP + PEP+&I% +OAA + ATP (1) In addition, PEPCK catalyzes a nucleotide-dependent exchange of CO2 into OAA (16,20).PEPCK has been proposed to act as a decarboxylase during photosynthesis in bundle sheath cells of specific plants which carry out C4 photosynthesis such as Panicum maximum. Through the action of PEPCK in these C4 plants, CO2 is released from OAA in the bundle sheath cells where the CO2 is refixed by the C3 cycle (3,12,14). In other C4 plants either a NADP+-or NAD+-dependent malic enzyme acts as the decarboxylase in bundle sheath cells. In all C4 plants the 4-carbon organic acids 1

The Central Role of Phosphoenolpyruvate in Plant Metabolism

“…Utter & Kurahashi (171) first noted that PEP carboxykinase from chicken liver catalyzes the formation of pyruvate from oxaloacetate in the presence of IDP. A similar reaction has been demonstrated in yeast (21), and ADP-stimulated oxaloacetate decarboxylation has been reported with partially purified preparations of PEP carboxykinase from Panicum maximum (135) (32). However, the absence of ADP-stimulated oxaloacetate decarboxyla tion in preparations of PEP carboxykinase from Chloris gayana has been reported (62).…”

Improved analysis of C4and C3photosynthesis via refinedin vitroassays of their carbon fixation biochemistry

“…2A ). In contrast to the alkali pH 8 preference of PEPC ( Greenway et al , 1978 ), the activity of PEPCK is optimal at pH 7 and 80% lower at pH 8.0 ( Ray and Black, 1976 ; Pierre et al , 2004 ). To minimize, possibly preclude, PEPC activity, the extraction and measurement of PEPCK carboxylase activity was undertaken at pH 7.0 with Mg 2+ (required for PEPC activity) omitted and replaced with 2mM Mn 2+ , a PEPCK cofactor ( Fig.…”

Phosphoenolpyruvate carboxykinase from higher plants: Purification from cucumber and evidence of rapid proteolytic cleavage in extracts from a range of plant tissues

“…270 kDa) suggesting that, in common with PEPCK from C 4 grasses (hexamer; Burnell 1986), yeast (tetramer; Tortora et al 1985) and Trypanosoma cruzi (dimer; Urbina 1987), the cucumber enzyme was a multimer. Whether or not these The activity of P E P C K was stable for several months when the purified preparation was stored at -20 ~ C. The presence of D T T was essential at all stages of the purification to prevent loss of activity (see also Ray and Black 1976;Burnell 1986). Kinetic studies using purified enzyme showed that it possessed similar kinetic properties to those of P E P C K s isolated from other plant tissues (Daley et al 1977;Leegood and ap Rees 1978;Burnell 1986).…”