UDP-N-acetylglucosamine pyrophosphorylase, a key enzyme in encysting Giardia, is allosterically regulated

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzy me in any system studied to date, kinetic data including Hill plots demonst rate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purif ied from encysting Giardia, is allosterically activated anabolically by phy siological levels of glucosamine 6-phosphate (3 mu M). Capillary electropho resis demonstrates that within 24 h after trophozoites are induced to encys t, the level of glucosamine g-phosphate increases 3-fold over that of non-e ncysting cells and that by 48 h into encystment the level of glucosamine 6- phosphate has decreased to non-encysting levels or below. UDP-N-acetylgluco samine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immu noaffinity purified from encysting and non-encysting cells exhibited the sa me molecular weight, amino acid composition, and circular dichroism spectra , Moreover, regardless of whether the enzyme came from encysting or nonency sting cells, the change in its circular dichroism spectra and up to a 6-fol d increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-a cetylglucosamine pyrophosphorylase is a major regulatory point in amino sug ar synthesis in encysting Giardia and that its allosteric anabolic activati on may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.