(653g) Using Hybrid Quantum Mechanics and Force-Field Methods for Improving Enzymatic Activity

In this talk we discuss ground and transition state computations to explore how specific mutations in engineered variants of dihydrofolate reductase (DHFR) and cytochrome P450 BM3 confer improved activity on dihydrofolate and new activity on ethane, respectively. We first focus on the rate limiting step reported in the literature in each of the reaction mechanisms, and use quantum mechanical (QM) calculations to converge on the equilibrium ground and transition state structures. Density functional theory (DFT) calculations in Gaussian 03 at the UB3LYP/Lanl2DZ level of theory were used for all QM calculations. All transition states were optimized and confirmed using vibrational frequency analysis. Subsequently, we parameterize bonds, angles, dihedral angles, and Mulliken charges of the ground and transition state structures into the molecular mechanics force field CHARMM, introducing constraints to preserve the geometries. We assess the effects of different experimentally isolated mutations using a computational saturation mutagenesis procedure and interaction energy calculations at the ground and transition states of the rate limiting steps. We systematically select design positions drawing upon sequence, structure, and energetic factors, and proceed to iteratively redesign using our previously presented Iterative Protein Redesign and Optimization algorithm. We finally assess top designs using hybrid quantum mechanical/molecular mechanics (QM/MM) calculations to determine their effect on binding energy.