Plasmodium falciparum enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. This enzyme is thus a potential target for anti‐malarial treatments. A unique feature of Pfeno is the presence of a pentapeptide insert ¹⁰⁴EWGWS¹⁰⁸. The functional role of tryptophan residues in this insert was investigated using site‐directed mutagenesis. Replacement of these two Trp residues with alanines (or lysines) resulted in a near complete loss of enolase activity and dissociation of the normal dimeric form into monomers. Molecular modeling indicated that ³⁴⁰R forms π‐cation bonds with the aromatic rings of ¹⁰⁵W and ⁴⁶Y. Mutation induced changes in the interactions among these three residues were presumably relayed to the inter‐subunit interface via a coil formed by ⁴⁶Y : ⁵⁹Y, resulting in the disruption of a salt bridge between ¹¹R : ⁴²⁵E and a π‐cation interaction between ¹¹R : ⁵⁹Y. This led to a drop of ~ 4 kcal·mole⁻¹ in the inter‐subunit docking energy in the mutant, causing a ~ 10³ fold decrease in affinity. Partial restoration of the inter‐subunit interactions led to reformation of dimers and also restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme's active site. Since Plasmodium enolase is a moonlighting protein with multiple parasite‐specific functions, it is likely that these functions may map on to the highly conserved unique insert region of this protein.

Enolase(EC4.2.1.11).