Gaining quantitative description of enzymatic reactions is one of the challenges of molecular biology. Thus we propose a continuation of our research aimed at the development, validation and implementation of computational tools for simulations of enzymatic reactions. We hope that our progress will help in the advances in key medical and biotechnological frontiers. During the previous grant periods we developed methods for simulating reactions in enzymes and examined their performance. The results were encouraging and showed the general applicability our EVB approach. In recent years, we have started to examine more rigorous ab initio approaches that exploit the increasing availability of computer power. The QM(ai)/MM method, which uses the EVB as a reference potential, allows us to start evaluating ab initio free energies of enzymatic reactions. In addition to the development projects we made very significant progress in studying different classes of enzymatic reactions and in exploring the feasibility of different catalytic mechanisms.ln order to exploit our advances we propose the following projects: (i) Developing ab initio-FEP approaches to a level where they can be used routinely in studies of enzymatic reactions. This will include: (a) improving the use of EVB reference potentials for QM(ai)/MM -FEP simulations, (b) Improving the search of transition states on QM(ai)/LD surfaces of solution reactions, (c) Refining the use of the CDFT method in studies of metalloenzymes. (ii) Quantifying the key contributions to enzyme catalysis, including the elucidation of the relationship between folding and catalysis. These studies will involve the following subprojects; (a) Exploring the relationship between the preorganization of active sites and the local stability of the protein, (b) Exploring the relationship between thermostability and catalysis (c) Exploring the catalysis in molten globules and (d) Computer aided enzyme design, (iii) Continuing to elucidate the mechanism of different classes of biologically important enzymes and exploring the validity of different catalytic proposals in well-defined test cases, (iv) Contributing to the field of computer-aided enzyme design by exploring more quantitative design concepts that will use the calculated contributions of different residues to transition state stabilization. This study will also explore the reason for the difference between the activities of catalytic antibodies and enzymes that catalyze similar reactions. ? ? ?
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