PURIFICATION AND CHARACTERIZATION OF HIGH-MOLECULAR-MASS AND LOW-MOLECULAR-MASS ISOFORMS OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM CHLAMYDOMONAS-REINHARDTII - KINETIC, STRUCTURAL AND IMMUNOLOGICAL EVIDENCE THAT THE GREEN ALGAL ENZYME IS DISTINCT FROM THE PROKARYOTIC AND HIGHER-PLANT ENZYMES

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the supply o f carbon skeletons for the assimilation of nitrogen by green algae. Tw o PEPC isoforms with respective native molecular masses of 400 (PEPC1) and 650 (PEPC2) kDa have been purified from Chlamydomonas reinhardtii CW-15 cc1883 (Chlorophyceae). SDS/PAGE, immunoblot and CNBr peptide-m apping analyses indicate the presence of the same 100 kDa PEPC catalyt ic subunit in both isoforms. PEPC1 is a homotetramer, whereas PEPC2 se ems to be a complex between the PEPC catalytic subunit and other immun ologically unrelated polypeptides of 50-70 kDa. Kinetic analyses indic ate that these PEPC isoforms are (1) differentially regulated by pH, ( 2) activated by glutamine and dihydroxyacetone phosphate and (3) inhib ited by glutamate, aspartate, 2-oxoglutarate and malate. These results are consistent with the current model for the regulation of anaplerot ic carbon fixation in green algae, and demonstrate that green algal PE PCs are uniquely regulated by glutamine. Several techniques were used to assess the structural relationships between C. reinhardtii PEPC and the higher plant or prokaryotic enzyme. Inmunoblot studies using anti -(green algal or higher plant PEPC) IgGs suggested that green algal (C . reinhardtii, Selenastrum minutum), higher plant (maize, banana fruit , tobacco) and prokaryotic (Synechococcus leopoliensis, Escherichia co li) PEPCs have little or no immunological relatedness. Moreover, the N -terminal amino acid sequence of the C. reinhardtii PEPC subunit did n ot have significant similarity to the highly conserved corresponding r egion in enzymes from higher plants, and CNBr cleavage patterns of gre en algal PEPCs were distinct from those of higher plant and cyanobacte rial PEPCs. These results point to significant evolutionary divergence between green algal, higher plant and prokaryotic PEPCs.