Mutational and Fluorescence Evidence For a PEP Activation Site in Phosphoenolpyruvate Carboxylase

The interaction of phosphoenolpyruvate carboxylase (PEPc) with its substrate PEP is unusual because PEP serves as both substrate (as Mg-PEP complex) and allosteric activator (as free, uncomplexed PEP). Based on results from mutational analysis and the location of these residues within the same pocket on the surface of maize phosphoenolpyruvate carboxylase, we propose that the residues R753, N755, N942, and I956 contribute to the substrate activation site. Although they are not located in the enzyme’s active site, modification of any of these residues via site-directed mutagenesis significantly reduces the enzyme’s affinity for the substrate PEP. Using the program Computed Atlas of Surface Topography of proteins, it was determined that pocket 217 houses these four residues in addition to R232 which, based on previous mutational analysis, is also believed to be involved in substrate activation. Mutations R753E and N755A displayed a 9-fold increase and a 3-fold increase, respectively, in the Km. R753E displayed a 3 fold reduction in the Vmax, while N755A only showed a one third reduction. N942D showed a two-fold increase in Km and roughly a 1/3 reduction in Vmax. Preliminary studies of I956F showed a 52-fold loss of affinity for substrate. These results suggest that these five residues within pocket 217 are critical to the activation of PEPc by its substrate. The effects of these mutations are currently being evaluated by means of fluorescence assays of PEP binding.

This work was supported by the NIH MBRS-RISE grant GM61331.