Partial purification and characterization of the N-acetylglucosaminyl-phosphatidylinositol De-N-acetylase of glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes

N-Acetylglucosaminylphosphatidylinositol (GlcNAc-PI) de-JV-acetylase was solubilized from the bloodstream form of African trypanosomes using Zwittergent 3-14. The solubilized GlcNAc-PI de-JV-acetylase was assayed using radiolabeled GlcNAc-PI substrates. The enzyme was partially purified about 140-fold from washed trypanosome membranes using conventional liquid chromatography. The enzyme has a Km of 1.5 UM. Replacement of the di-O-substituted D-myo-inositol of the natural GlcNAc-PI substrate by the L-myo-inositol isomer did not significantly alter the ability of the compound to act as a substrate for the de-N-acetylase, suggesting that the C-2 to C-5 hydroxyl groups of the myoinositol ring do not play a critical role in substrate recognition. A substrate analogue lacking fatty acids was a relatively poor substrate for the enzyme, indicating that the lipid component plays an important role in substrate recognition and/or presentation of the substrate to the enzyme in detergent micelles. Substrate analogues lacking the glycerophosphate component were not recognized by the enzyme, suggesting that this component is important in the substrate recognition process

Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein

Acyl-CoA binding protein (ACBP) maintains a pool of fatty acyl-CoA molecules in the cell and plays a role in fatty acid metabolism. The biochemical properties of Plasmodium falciparum ACBP are described together with the 2.0 AÊ resolution crystal structures of a P. falciparum ACBP-acyl-CoA complex and of bovine ACBP in two crystal forms. Overall, the bovine ACBP crystal structures are similar to the NMR structures published previously; however, the bovine and parasite ACBP structures are less similar. The parasite ACBP is shown to have a different ligand-binding pocket, leading to an acyl-CoA binding speci®city different from that of bovine ACBP. Several non-conservative differences in residues that interact with the ligand were identi®ed between the mammalian and parasite ACBPs. These, together with measured bindingspeci®city differences, suggest that there is a potential for the design of molecules that might selectively block the acyl-CoA binding site. # 2001 Academic Pres