Chorismate mutase (CM) is an enzyme that catalyzes the Claisen rearrangement of chorismate to prephenate. In a recent effort to understand the basis for catalysis by CM, Kienhöfer and co-workers (J. Am. Chem. Soc. 2003, 125, 3206-3207) reported results on the mutation of Arg90 in Bacillus subtilis CM (BsCM) to citrulline (Cit), an isosteric but neutral arginine analogue. An ca. 10(4)-fold decrease in kcat or 5.9 kcal/mol increase in the free-energy barrier (ΔG(‡)) for the overall catalysis was observed upon mutation. In this work, attention is turned to determining the key factors that contribute to the reduced catalytic efficiency of Arg90Cit BsCM. Using a combined QM/MM Monte Carlo/Free-Energy Perturbation method, a ΔΔG(‡) value of 3.3 kcal/mol is obtained. The higher free-energy barrier for the mutant is exclusively related to inferior stabilization of the TS, particularly one of its carboxylate groups, by neutral Cit. In addition, the reaction becomes 2.0 kcal/mol more exergonic. As BsCM is limited by product release, this step contributes to the remainder of the 10(4)-fold decrease in the rate constant in going from Arg90 to Cit.