Clostridium difficile is a Gram-positive bacterial pathogen that causes serious and potentially fatal inflammatory disease of the colon. During infection, C. difficile produces two virulence factors, TcdA and TcdB which are large multi-domain containing protein toxins and are responsible for human intestinal pathogenesis. TcdA and TcdB mediate host cytotoxicity by glucosylating and inactivating host GTPases Rac1, RhoA and Cdc42 using UDP-glucose as a glucosyl donor. We use kinetic isotope effects (KIEs) and transition state theory to solve the transition state structure of the glucosyltransferase domain of TcdB (TcdB-GTD). Knowledge of the transition state structure provides a blueprint for the design of transition state analogues which are extremely potent enzyme inhibitors. Experimental analysis of the transition state requires KIEs which compare the chemical steps of enzymatic reaction rates with isotopically labeled substrates (heavy) and unlabeled substrates (light). KIEs provide critical information on the catalytic mechanism and also bond order, lengths and geometry of the anomeric carbon at the transition state. 1-3H Glucose, 1-14C Glucose, 1-18O Glucose and 2-3H Glucose were used to synthesize ‘heavy’ UDP-glucose substrates and, 6-3H-glucose and 6-14C glucose were used to synthesize ‘light’ UDP-glucose substrates. KIEs will be measured on the glucohydrolase reaction of TcdB-GTD, where water is used as a nucleophile to hydrolyse the glycosidic bond between the anomeric carbon of the glucose ring and the uridine diphosphate (UDP) moiety of UDP-glucose. In this assay, labeled glucose product is separated from unreacted labeled UDP-glucose. The resulting product 3H/14C ratios are compared to the starting 3H/14C ratio to calculate the experimental KIE. Experimental KIEs will be matched to theoretical KIEs generated from computationally derived transition state structures of TcdB-GTD, permitting the design of stable analogues which have the potential to be developed into novel anti-virulence agents targeting C. difficile TcdB toxin.