Mutational evidence for a substrate activating site in phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPc) is a key enzyme in algae, fungi, and plants. It has been established as a crucial regulatory enzyme in photosynthetic metabolism where it is responsible for the irreversible beta-carboxylation of PEP in the mesophyll cells of C4 plants. Several studies have proposed the existence of a binding site, in which PEP will bind without forming a complex with Mg2+. Through a combination of site directed mutagenesis and bioinformatics, this study identified several key residues which may be part of this binding site. Replacement of R753, N755, E952, I956 or K960 showed anywhere from a 4-fold to a 25-fold decrease in the enzyme's affinity for PEP. Mutating I956 to Trp or Phe caused a complete loss of enzyme activation. In addition, because the mutations had a global effect on the activation of the enzyme by other activators, it is speculated that all activation sites reside within this area