Projector-Based Embedding Eliminates Density Functional Dependence for QM/MM Calculations of Reactions in Enzymes and Solution

Combined quantum mechanics/molecular mechanics (QM/MM) methods are increasingly widely utilized in studies of reactions in enzymes and other large systems. Here, we apply a range of QM/MM methods to investigate the Claisen rearrangement of chorismate to prephenate, in solution, and in the enzyme chorismate mutase. Using projector-based embedding in a QM/MM framework, we apply treatments up to the CCSD­(T) level. We test a range of density functional QM/MM methods and QM region sizes. The results show that the calculated reaction energetics are significantly more sensitive to the choice of density functional than they are to the size of the QM region in these systems. Projector-based embedding of a wave function method in DFT reduced the 13 kcal/mol spread in barrier heights calculated at the DFT/MM level to a spread of just 0.3 kcal/mol, essentially eliminating dependence on the functional. Projector-based embedding of correlated ab initio methods provides a practical method for achieving high accuracy for energy profiles derived from DFT and DFT/MM calculations for reactions in condensed phases

Conformational Change and Ligand Binding in the Aristolochene Synthase Catalytic Cycle

Terpene synthases are potentially useful biocatalysts for the synthesis of valuable compounds, such as anticancer drugs and antibiotics. The design of altered activities requires better knowledge of their mechanisms, for example, an understanding of the complex conformational changes that are part of their catalytic cycle, how they are coordinated, and what drives them. Crystallographic studies of the sesquiterpene synthase artistolochene synthase have led to a proposed sequence of ligand binding and conformational change but have provided only indirect insight. Here, we have performed extensive molecular dynamics simulations of multiple enzyme–ligand complexes (over 2 μs in total). The simulations provide clear evidence of what drives the conformational changes required for reaction. They support a picture in which the substrate farnesyl diphosphate binds first, followed by three magnesium ions in sequence, and, after reaction, the release of aristolochene and two magnesium ions followed by the final magnesium ion and diphosphate. Binding of farnesyl diphosphate leads to an increased level of sampling of open conformations, allowing the first two magnesium ions to bind. The closed enzyme conformation is maintained with a diphosphate moiety and two magnesium ions bound. The open-to-closed transition reduces flexibility around the active site entrance, partly through a lid closing over it. The simulations with all three magnesium ions and farnesyl diphosphate bound provide, for the first time, a realistic model of the Michaelis complex involved in reaction, which is inaccessible to experimental structural studies. These insights could help with the design of altered activities in a range of terpene synthases

Conformational effects in enzyme catalysis: reaction via a high energy conformation in fatty acid amide hydrolase.

Quantum mechanics/molecular mechanics and molecular dynamics simulations of fatty acid amide hydrolase show that reaction (amide hydrolysis) occurs via a distinct, high energy conformation. This unusual finding has important implications for fatty acid amide hydrolase, a key enzyme in the endocannabinoid system. These results demonstrate the importance of structural fluctuations and the need to include them in the modeling of enzyme reactions. They also show that approaches based simply on studying enzyme-substrate complexes can be misleading for understanding biochemical reactivity

Quantum Mechanics/Molecular Mechanics Modeling of Regioselectivity of Drug Metabolism in Cytochrome P450 2C9

[Image: see text] Cytochrome P450 enzymes (P450s) are important in drug metabolism and have been linked to adverse drug reactions. P450s display broad substrate reactivity, and prediction of metabolites is complex. QM/MM studies of P450 reactivity have provided insight into important details of the reaction mechanisms and have the potential to make predictions of metabolite formation. Here we present a comprehensive study of the oxidation of three widely used pharmaceutical compounds (S-ibuprofen, diclofenac, and S-warfarin) by one of the major drug-metabolizing P450 isoforms, CYP2C9. The reaction barriers to substrate oxidation by the iron-oxo species (Compound I) have been calculated at the B3LYP-D/CHARMM27 level for different possible metabolism sites for each drug, on multiple pathways. In the cases of ibuprofen and warfarin, the process with the lowest activation energy is consistent with the experimentally preferred metabolite. For diclofenac, the pathway leading to the experimentally observed metabolite is not the one with the lowest activation energy. This apparent inconsistency with experiment might be explained by the two very different binding modes involved in oxidation at the two competing positions. The carboxylate of diclofenac interacts strongly with the CYP2C9 Arg108 side chain in the transition state for formation of the observed metabolite—but not in that for the competing pathway. We compare reaction barriers calculated both in the presence and in the absence of the protein and observe a marked improvement in selectivity prediction ability upon inclusion of the protein for all of the substrates studied. The barriers calculated with the protein are generally higher than those calculated in the gas phase. This suggests that active-site residues surrounding the substrate play an important role in controlling selectivity in CYP2C9. The results show that inclusion of sampling (particularly) and dispersion effects is important in making accurate predictions of drug metabolism selectivity of P450s using QM/MM methods

Conformational Effects in Enzyme Catalysis: Reaction via a High Energy Conformation in Fatty Acid Amide Hydrolase

Quantum mechanics/molecular mechanics and molecular dynamics simulations of fatty acid amide hydrolase show that reaction (amide hydrolysis) occurs via a distinct, high energy conformation. This unusual finding has important implications for fatty acid amide hydrolase, a key enzyme in the endocannabinoid system. These results demonstrate the importance of structural fluctuations and the need to include them in the modeling of enzyme reactions. They also show that approaches based simply on studying enzyme-substrate complexes can be misleading for understanding biochemical reactivity