Enzyme design has tremendous applications in industry as well as medicinal chemistry.1 The advancement in the field of computational tools coupled with experiments has to led to an increased understanding of enzymatic reactions.2 Current trends indicate that computational strategies are now being widely employed to design new enzymes.3 However, there is still a huge gap between the natural and artificially designed enzymes in terms of catalytic abilities. The knowledge of the contribution of individual residues to catalysis is of prime importance in enzyme design. It is known that mutation of active site residues drastically changes the catalytic properties of an enzyme. However, mutation of residues farther away from the active site is not known. In the current study we intend to identify the electrostatic contribution of such residues to the activation energy of an SN2 reaction between dichloroethane and an aspartate residue catalyzed by haloalkane dehalogenase (Dh1A). The enzyme does not require any metal ion or cofactors and thus is an attractive alternative for the degradation of toxic halogenated compounds. Empirical valence bond (EVB) approach which is a semiempirical QM/MM type is used to simulate catalysis in the enzyme and reproduce the observed activation barrier.4 To do so, first the reaction in water is simulated to generate a set of EVB parameters which are consequently used for the simulation in protein. The observed activation free energies are found to be in accordance with experimental values. Next, we studied the electrostatic contribution of each residue on moving from the reactant state (RS) to the transition state (TS). After identifying the hot spots, the residues are mutated and the effect of such mutation will be studied using the EVB approach. The current method provides a fast screening of the residues and provides useful hints for mutations. Further studies involving validation of these results using experiments will be carried out. References (1) Toscano, M. D.; Woycechowsky, K. J.; Hilvert, D. Angew. Chem. Int. Ed. 2007, 46, 3212. (2) Roca, M.; Vardi-Kilshtain A.; Warshel, A. Biochemistry 2009, 48, 3046. (3) Rothlisberger, D.; Khersonsky, O.; Wollacott, A. M.; Jiang, L.; DeChancie, J.; Betker, J.; Gallaher, J. L.; Althoff, E. A.; Zanghellini, A.; Dym, O.; Albeck, S.; Houk, K. N.; Tawfik, D. S.; Baker, D. Nature 2008, 453, 190. (4) Frushicheva, M. P.; Cao, J.; Chu, Z. T.; Warshel, A. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 16869.

Identifying hotspot residues for rational enzyme design in haloalkane dehalogenase

251st National Meeting (2016)

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