Abstract
A series of enzymes for Kemp elimination of 5-nitrobenzisoxazole has been recently designed and tested. In conjunction with the design process, extensive computational analyses were carried out to evaluate the potential performance of four of the designs, as presented here. The enzyme-catalyzed reactions were modeled using mixed quantum and molecular mechanics (QM/MM) calculations in the context of Monte Carlo (MC) statistical mechanics simulations. Free-energy perturbation (FEP) calculations were used to characterize the free-energy surfaces for the catalyzed reactions as well as for reference processes in water. The simulations yielded detailed information about the catalytic mechanisms, activation barriers, and structural evolution of the active sites over the course of the reactions. The catalytic mechanism for the designed enzymes KE07, KE10(V131N), and KE15 was found to be concerted with proton transfer, generally more advanced in the transition state than breaking of the isoxazolyl N-O bond. On the basis of the free-energy results, all three enzymes were anticipated to be active. Ideas for further improvement of the enzyme designs also emerged. On the technical side, the synergy of parallel QM/MM and experimental efforts in the design of artificial enzymes is well illustrated.