ABSTRACT
Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated cytosolic enzyme situated at a crucial branch point of central plant metabolism. The structure of AtPPC3, a C3 PEPC isozyme of the model plant Arabidopsis thaliana, in complex with the inhibitors aspartate and citrate was solved at 2.2-Å resolution. This represents the first PEPC structure with citrate bound. Aspartate and citrate binding sites are in close proximity (5.1-5.3â¯Å) and interactions between citrate and specific residues were identified. Citrate functions as a mixed (allosteric) inhibitor as it reduced AtPPC3's Vmax while increasing Km(PEP) values. The PEP saturation data gave an excellent fit to the mixed inhibition model, yielding Ki and Ki' (citrate) values of 9.3 and 42.5â¯mM, respectively. Citrate and aspartate inhibition of AtPPC3 was non-additive, likely due to their closely positioned binding sites, their similar negative charge, and type of binding residues. Fewer interactions and lower affinity for citrate support its observed weaker inhibition of AtPPC3 relative to aspartate. Citrate does not appear to induce further conformational change beyond aspartate owing to the similar structural mechanism of inhibition. AtPPC3 largely exhibits root-specific expression in Arabidopsis, where it is markedly upregulated during stresses such as excessive salinity or nutritional Pi deprivation that necessitate large increases in anaplerotic PEP carboxylation. The cytosolic citrate concentration of potato tubers suggests that AtPPC3's inhibition by citrate may be physiologically relevant. Our results provide novel insights into the structural basis of allosteric PEPC control and the kinetic effects brought about upon inhibitor binding.
