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 enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 mu M). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine g-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra, Moreover, regardless of whether the enzyme came from encysting or nonencysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.