Abstract
PEPC catalyzes the primary CO2-fixation reaction in C4 photosynthesis. The maize PEPC is feedback inhibited by L-malate (MA) or L-aspartate (Asp) in an allosteric manner. Here we report successful desensitization to feedback inhibitors by site-directed mutagenesis. Previously we reported the 3-dimensional structures of PEPC of E. coli complexed with Asp and maize. The structural data indicated the involvement of 4 conserved residues for the binding of inhibitor. These residues are Arg647, Lys835, Arg894 and Asn968 in numbering of Zea mays PEPC. Recombinant mutant enzymes of K835G, R894G and K835G/R894G were prepared by site-directed mutagenesis. Each of K835G and R894G showed a marked desensitization to the inhibitors retaining almost the same catalytic activity and sensitivities to activators as wild-type enzyme. Thus specific participation of these residues in the binding with allosteric inhibitor was demonstrated. The mutant PEPCs (K773G, R832G) of E. coli were prepared and its properties will also be presented.
