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Toxins TcdA and TcdB from Clostridioides difficile glucosylate human colon Rho GTPases. TcdA and TcdB glucosylation of RhoGTPases results in cytoskeletal changes, causing cell rounding and loss of intestinal integrity. Clostridial toxins TcdA and TcdB are proposed to catalyze glucosylation of Rho GTPases with retention of stereochemistry from UDP-glucose. We used kinetic isotope effects to analyze the mechanisms and transition-state structures of the glucohydrolase and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase glucosylation with retention of stereochemistry, while hydrolysis of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic analysis revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an S N i mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mechanical calculations revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an S N i mechanism with the formation of a distinct oxocarbenium phosphate ion pair transition state where the glycosidic bond to the UDP leaving group breaks prior to attack of the threonine nucleophile from Rho GTPase.