LAUSR

(1,3)-β-D-Glucan synthase (GS) catalyzes formation of the linear (1,3)-β-D-glucan in the fungal cell wall, and is a tar-get of clinically approved antifungal antibiotics. The catalytic subunit of GS, FKS protein, does not exhibit significant sequence homologies to other glycosyltransferases, and thus a significant ambiguity remains about its catalytic mechanism. One of the major technical barriers in studying GS is the absence of activity assay methods that allow characterization of the lengths and amounts of (1,3)-β-D-glucan due to its poor solubility in water and organic solvents. Here, we report a successful development of a novel GS activity assay based on size-exclusion chromatography coupled with pulsed amperometric detection and radiation counting (SEC-PAD-RC), which allows for the simultaneous characterization of the amount and length of the polymer product. The assay revealed that the purified yeast GS produces glucan with a length of ~2,000 - 7,000 mer, consistent with the reported degree of polymerization of (1,3)-β-D-glucan isolated from intact cells. Pre-steady state kinetics analysis revealed a highly efficient but rate-determining chain elongation rate of ~50 sec-1, which represents the first observation of chain elongation by a nucleotide-sugar dependent polysaccharide synthase. Coupling the SEC-PAD-RC method with substrate analog mechanistic probes provided the first unambiguous evidence that GS catalyzes non-reducing end polymerization. Based on these observations, we propose a detailed model for the catalytic mechanism of GS. The approaches described here can be used to determine the mechanism of catalysis of other polysaccharide synthases.