The N-Acetyl-D-glucosaminylphosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis Is a Zinc Metalloenzyme

The de- N -acetylation of N -acetyl- d -glucosaminylphosphatidylinositol (GlcNAc-PI) is the second step of mammalian and trypanosomal glycosylphosphatidylinositol biosynthesis. Glycosylphosphatidylinositol biosynthesis is essential for Trypanosoma brucei , the causative agent of African sleeping sickness, and GlcNAc-PI de- N -acetylase has previously been validated as a drug target. Inhibition of the trypanosome cell-free system and recombinant rat GlcNAc-PI de- N -acetylase by divalent metal cation chelators demonstrates that a tightly bound divalent metal cation is essential for activity. Reconstitution of metal-free GlcNAc-PI de- N -acetylase with divalent metal cations restores activity in the order Zn 2+ > Cu 2+ > Ni 2+ > Co 2+ > Mg 2+ . Site-directed mutagenesis and homology modeling were used to identify active site residues and postulate a mechanism of action. The characterization of GlcNAc-PI de- N -acetylase as a zinc metalloenzyme will facilitate the rational design of anti-protozoan parasite drugs.