N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase from Entamoeba histolytica

Abstract

PIG-L/GPI12 proteins are endoplasmic reticulum-resident membrane proteins involved in the second step of glycosylphosphatidylinositol anchor biosynthesis in eukaryotes. We show that the Entamoeba histolytica PIG-L protein is optimally active in the acidic pH range. The enzyme has an intrinsic low level of de-N-acetylase activity in the absence of metal and is significantly stimulated by divalent cations. Metal binding induces a large conformational change in the protein that appears to improve catalytic rates while not altering the affinity of the enzyme for its substrate.

First Step of Glycosylphosphatidylinositol (GPI) Biosynthesis Cross-talks with Ergosterol Biosynthesis and Ras Signaling in Candida albicans

“…GPI-GnT assay was done using microsomes from each strain with 1000 g of total protein, as described previously (11).…”

Catalysis by N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase (PIG-L) from Entamoeba histolytica

“…In a previous report we showed that unlike rat PIG-L and other known de-Nacetylases, EhPIG-L is actually capable of low activity in the absence of metal, and the catalysis is stimulated by divalent cations (9). We also showed that, unlike other known PIG-L enzymes, EhPIG-L preferred divalent cations such a Mg 2ϩ , Mn 2ϩ , or Co 2ϩ rather than Zn 2ϩ…”

“…Briefly, protein expression was induced with 0.25 mM isopropyl 1-thio-␤-D-galactopyranoside, and the cells were grown at 16°C for another 16 h. The proteins were affinity-purified from amylose beads and used without removal of the MBP tag for all the enzyme assays. We have previously shown that the MBP tag does not significantly alter the activity of the enzyme (9).…”

“…Protein expression and purification were carried out essentially as described previously (9). Briefly, protein expression was induced with 0.25 mM isopropyl 1-thio-␤-D-galactopyranoside, and the cells were grown at 16°C for another 16 h. The proteins were affinity-purified from amylose beads and used without removal of the MBP tag for all the enzyme assays.…”

“…We also showed that the role of the metal appeared to be in altering catalytic rates by inducing a catalytically efficient conformation of the enzyme rather than in altering the affinity of the enzyme for its substrate. Thus, metal altered V max rather than the K m of the enzyme for its substrate (9).…”