REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM THE GREEN-ALGA SELENASTRUM-MINUTUM - PROPERTIES ASSOCIATED WITH REPLENISHMENT OF TRICARBOXYLIC-ACID CYCLE INTERMEDIATES DURING AMMONIUM ASSIMILATION

Two isoforms of phosphoenolpyruvate carboxylase (PEPC) with very different regulatory properties were partially purified from the green alga Selenastrum minutum. They were designated PEPC1 and PEPC2. PEPC1 showed sigmoidal kinetics with respect to phosphoenolpyruvate (PEP) whereas PEPC2 exhibited a typical Michaelis-Menten response. The S0.5(PEP) of PEPC1 was 2.23 millimolar. This was fourfold greater than the S0.5(PEP) of PEPC2, which was 0.57 millimolar. PEPC1 was activated more than four-fold by 2.0 millimolar glutamine and sixfold by 2.0 millimolar dihydroxyacetone phosphate (OHAP) at a subsaturating PEP concentration of 0.625 millimolar. In contrast, PEPC2 showed only 8% and 52% activation by glutamine and DHAP, respectively. The effects of glutamine and DHAP were additive. PEPC1 was more sensitive to inhibition by glutamate, 2-oxoglutarate, and aspartate than PEPC2. Both isoforms were equally inhibited by malate. All of these metabolites affected only the S0.5(PEP) not the Vmax. The regulatory properties of S. minutum PEPC in vitro are discussed in terms of (a) increased rates of dark carbon fixation (shown to be catalyzed predominantly by PEPC) and (b) changes in metabolite levels in vivo during enhanced NH4+ assimilation. Finally, a model is proposed for the regulation of PEPC in vivo in relation to its role in replenishing tricarboxylic acid cycle intermediates consumed in NH4+ assimilation.