Understanding the role of active-site residues in enzymatic catalysis is of fundamental importance for a microscopic description of the catalytic mechanism, but also for the design of effective enzyme mimics or improvement of existing enzymes (or abzymes) for catalyzing chemical reactions. Site-directed mutagenesis and kinetic studies are able to identify key residues involved in the catalysis, but determination of their exact role from experimental information alone can be difficult. This is the case for the chorismate mutase of Bacillus subtilis (BsCM), which catalyzes the Claisen rearrangement of chorismate to prephenate (Figure 1 a and b) in the biosynthetic pathway that forms Tyr and Phe. [1] Mutation experiments [1][2][3][4][5] have identified several residues that are important for the catalysis. For instance, it was shown that replacement of Arg 90 by Gly or Ala leads to a significant reduction in k cat by a factor of more than 10 5 , while k cat /K m is decreased by a factor of 10 5 -10 7 . Interestingly, the double mutants Cys 88 Lys/Arg 90 Ser and Cys 88 Ser/ Arg 90 Lys restore a factor of more than 10 3 in k cat . [5] Arg 90 interacts with the ether oxygen atom of the transition-state analogue (TSA) in the X-ray structure of the wildtype BsCM complex, [6][7] and this observation led to the suggestion that Arg 90 stabilizes developing negative charge on the enolic oxygen atom in the transition state [1][2][3]6] as well as the transition-state conformation. [2,6] Nevertheless, the exact role of Arg 90 and other active-site residues is not clear.
Herein we apply quantum-mechanical/molecularmechanical (QM/MM) molecular-dynamics simulations to study the role of Arg 90 in the BsCM catalysis. It is demonstrated that the existence of this residue is essential for stabilizing the reactive-substrate conformation (CHAIR) in the active site, as both the Arg 90!Ala and Arg 90!Gly mutations destroy the ability of the wildtype enzyme to stabilize CHAIR. [8] Free-energy simulations suggest that the loss of this ability may contribute significantly to the reduction of k cat for Arg 90 Ala and Arg 90 Gly observed [*] Prof.