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Most QM-cluster models of enzymes are constructed based on X-ray crystal structures, which limits comparison toin vivostructure and mechanism. This work introduces an MD to QM-cluster model workflow. 

New computational and experimental studies have been carried out for the MgCCH radical in its X2Σ+ state. Coupled cluster theory with single, double, and perturbative triples, CCSD(T), was used in conjunction with post-CCSD(T) and scalar relativistic additive corrections to compute vibrational quartic force fields for this molecule. From the quartic force fields, higher-order spectroscopic properties, including rotational constants, were obtained. In tandem, the five lowest energy rotational transitions for MgCCH, N = 1→0 through N = 5→4, were measured for the first time using Fourier transform microwave/millimeter wave methods in the frequency range 9 -50 GHz. The radical was created in the Discharge Assisted Laser Ablation Source (DALAS) developed in the Ziurys group. A combined fit of these data with previous millimeter direct absorption measurements have yielded the most accurate rotational constants for MgCCH to date. The computed principle rotational constant lies within 1.51-1.65 MHz of the experimental one, validating the computational approach. High-level theory was then applied to produce accurate rovibrational spectroscopic constants for MgCCH+, including a rotational constant of B0 = 5354.5–5359.5 MHz.. These new predictions will further the experimental study of MgCCH+, and aid in the low-temperature characterization of MgCCH, detected towards the circumstellar shell of IRC+10216, a carbon-rich star. 

Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism of Glycoside Hydrolase Family 7 (GH7) was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol −1 for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction; and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol −1 for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme. 

Designing realistic quantum mechanical (QM) models of enzymes is dependent on reliably discerning and modeling residues, solvents, and cofactors important in crafting the active site microenvironment. Interatomic van der Waals contacts have previously demonstrated usefulness toward designing QM-models, but their measured values (and subsequent residue importance rankings) are expected to be influenceable by subtle changes in protein structure. Using chorismate mutase as a case study, this work examines the differences in ligand-residue interatomic contacts between an x-ray crystal structure and structures from a molecular dynamics simulation. Select structures are further analyzed using symmetry adapted perturbation theory to compute ab initio ligand-residue interaction energies. The findings of this study show that ligand-residue interatomic contacts measured for an x-ray crystal structure are not predictive of active site contacts from a sampling of molecular dynamics frames. In addition, the variability in interatomic contacts among structures is not correlated with variability in interaction energies. However, the results spotlight using interaction energies to characterize and rank residue importance in future computational enzymology workflows.