Information from experiments is necessary, but often insufficient to characterize the mechanisms and energetics of chemical reactions in enzymes and DNA. The recently developed pseudobond quantum mechanical/molecular mechanical approach will be applied to simulate the reactions in enzymes and DNA; the systems are chosen because of their intrinsic biomedical interests, the availability of experimental structural data as starting point, and the collaborations established with investigators whose laboratories have investigated the systems and continue to do so. The long- term goal of this project is to develop and establish the DFT-based QM/MM simulation as a partner equal to experiments for the study of structure and chemical reactions in enzymes and DNA and to provide insight into the chemical reaction mechanisms in biological systems. The following are the specific tasks of this project: (1) 4-Oxalocrontonate tautomerase (4OT) belongs to a family of enzymes which use their amino-terminal proline as a general base in catalysis. Experimental studies have only provided a primitive picture of the reaction mechanism of 4OT. The pseudobond QM/MM free energy approach will be employed to explore the mechanistic details of 4OT and provide understanding of the experimental observations. (2) Cyclic nucleotide phosphodiesterases(PDE) are enzymes forming a diverse super family and play fundamental roles in cell signal transfer by their common activity of hydrolyzing the most common second messengers 3',5'-cyclic adenosine monophosphate(cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Selective inhibitors of PDEs are of potential therapeutic values. We propose to study the mechanism by which PDE4 hydrolyze cAMP, to address whether the mechanism is associative or dissociative, and to provide insight and guide to inhibitor design. (3) Nitrile hydratase (NHase), a bacterial metalloenzyme catalyzing the hydration of nitriles, is well-known as one of the most industrially successful enzymes. Important structural and mechanistic details concerning this active center is still not clear. The pseudobond QM/MM method will be employed to determine the structure of its active site, study the structure basis for this unique spin-preference of Fe (III) center and explore the catalytic reaction mechanisms. (4) The chemical reactivity of nucleic acids, in particular, nucleobase oxidation reactions, is of intrinsic interest in understanding the mechanisms by which DNA and RNA is naturally damaged, leading to aging and cancer. The pesudobond QM/MM methods will be applied to investigate the guanine oxidation reactions in DNA, leading to a quantitative description of the mechanism and electronic structure. (5) Further methodology development will focus on the design of the effective core potential for the carbon boundary atom in the C-N single bond for DNA calculations.
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