title: On the challenge of exploring the evolutionary trajectory from phosphotriesterase to arylesterase using computer simulations.
abstract: The ability to design effective enzymes presents a fundamental challenge in biotechnology and also in biochemistry. Unfortunately, most of the progress on this field has been accomplished by bringing the reactants to a reasonable orientation relative to each other, rather than by rational optimization of the polar preorganization of the environment, which is the most important catalytic factor. True computer based enzyme design would require the ability to evaluate the catalytic power of designed active sites. This work considers the evolution from a phosphotriesterase (with the paraoxon substrate) to arylesterase (with the 2-naphthylhexanoate (2NH) substrate) catalysis. Both the original and the evolved enzymes involve two zinc ions and their ligands, making it hard to obtain a reliable quantum mechanical description and then to obtain an effective free energy sampling. Furthermore, the options for the reaction path are quite complicated. To progress in this direction we started with DFT calculations of the energetics of different mechanistic options of cluster models and then used the results to calibrate empirical valence bond (EVB) models and to generate properly sampled free energy surfaces for different mechanisms in the enzyme. Interestingly, it is found that the catalytic effect depends on the Zn-Zn distance making the mechanistic analysis somewhat complicated. Comparing the activation barriers of paraoxon and the 2NH ester at the beginning and end of the evolutionary path reproduced the observed evolutionary trend. However, although our findings provide an advance in exploring the nature of promiscuous enzymes, they also indicate that modeling the reaction mechanism in the case of enzymes with a binuclear zinc center is far from trivial and presents a challenge for computer-aided enzyme design.
authors.affiliation: Department of Chemistry, University of Southern California , 3620 McClintock Avenue, Los Angeles, California 90089, United States.
authors.forename: Ram Prasad
authors.initials: RP
authors.lastname: Bora
authors.forename: Matthew J L
authors.initials: MJ
authors.lastname: Mills
authors.forename: Maria P
authors.initials: MP
authors.lastname: Frushicheva
authors.forename: Arieh
authors.initials: A
authors.lastname: Warshel
mesh_terms: D002208:Caproates / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D002384:Catalysis; D003198:Computer Simulation; D006868:Hydrolysis; D007477:Ions / Q000737:chemistry; D008956:Models, Chemical; D010261:Paraoxon / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry*; D011422:Propionates / Q000737:chemistry; D011789:Quantum Theory; D015032:Zinc / Q000737:chemistry
chemical_list: D002208:Caproates; D007477:Ions; D010710:Phosphates; D011422:Propionates; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D044345:Phosphoric Triester Hydrolases; D015032:Zinc; D010261:Paraoxon