We present a theoretical study of the reaction mechanism of monoethanolamine (MEA) with CO₂ in an aqueous solution. We have used molecular orbital reaction pathway calculations to compute reaction free energy landscapes for the reaction steps involved in the formation of carbamic acids and carbamates. We have used the conductor-like polarizable continuum model to calculate reactant, product, and transition state geometries and vibrational frequencies within density functional theory (DFT). We have also computed single point energies for all stationary structures using a coupled cluster approach with singles, doubles, and perturbational triple excitations using the DFT geometries. Our calculations indicate that a two-step reaction mechanism that proceeds via a zwitterion intermediate to form carbamate is the most favorable reaction channel. The first step, leading to formation of the zwitterion, is found to be rate-determining, and the activation free energies are 12.0 (10.2) and 11.3 (9.6) kcal/mol using Pauling (Bondi) radii within the CPCM model at the CCSD(T)/6-311++G(d,p) and CCSD(T)/6-311++G(2df,2p) levels of theory, respectively, using geometries and vibrational frequencies obtained at the B3LYP/6-311++G(d,p) level of theory. These results are in reasonable agreement with the experimental value of about 12 kcal/mol. The second step is an acid-base reaction between a zwitterion and MEA. We have developed a microkinetic model to estimate the effective reaction order at intermediate concentrations. Our model predicts an equilibrium concentration for the zwitterion on the order of 10⁻¹¹ mol/L, which explains why the existence of the zwitterion intermediate has never been detected experimentally. The effective reaction order from our model is close to unity, also in agreement with experiments. Complementary ab initio QM/MM molecular dynamics simulations with umbrella sampling have been carried out to determine the free energy profiles of zwitterion formation and proton transfer in solution; the results confirm that the formation of the zwitterion is rate-determining.
T he global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) has become an urgent public health concern (1,2). In 2016, the World Health Organization included CRE in a list of antimicrobial-resistant priority pathogens on which to concentrate future drug development strategies. Of note, carbapenem-resistant Klebsiella pneumoniae (CRKP) account for 60%-90% of clinical CRE infections in the United States, Europe, and China (1-3), resulting in an increased mortality rate of up to 40%-50% in nosocomial settings (4). The dissemination of CRKP is mostly clonal, and the population structure is geographically specific. Since its emergence during the early to mid-2000s, sequence type (ST) 258 has become the most prevalent CRKP clone in North America, Latin America, and Europe (5). However, in Asia, especially China, ST11 is the predominant clone, accounting for up to 60% of CRKP (3). ST11 is a single-locus (tonB) variant of ST258, and both types belong to the clonal group 258. A recombination event is thought to have occurred between a recipient ST11 and a donor ST442like strain, giving rise to ST258 during 1985-1997 (6,7). A phylogenomic study revealed that the ST258 population consists of >2 clades, resulting from an ≈215-kb recombination event that includes the capsule polysaccharide (cps) synthesis locus (6). The genetic differences generated by the resulting capsular switch are supposed to be primarily responsible for the ST258 diversification (8). Likewise, a segregation was identified in the ST11 population, resulting in >3 clades with different capsular loci (KL) (9-11). These studies consistently indicate that cps is a recombination hotspot in K. pneumoniae. However, the K-type distribution within ST11 in clinical settings is unclear. More important, the biological, epidemiologic, and
Acetylcholinesterase (AChE) is a remarkably efficient serine hydrolase responsible for the termination of impulse signaling at cholinergic synapses. By employing Born-Oppenheimer molecular dynamics simulations with B3LYP/6-31G(d) QM/MM potential and the umbrella sampling method, we have characterized its complete catalytic reaction mechanism for hydrolyzing neurotransmitter acetylcholine (ACh) and determined its multi-step free energy reaction profiles for the first time. In both acylation and deacylation reaction stages, the first step involves the nucleophilic attack to the carbonyl carbon with the triad His447 serving as the general base, and leads to a tetrahedral covalent intermediate stabilized by the oxyanion hole. From the intermediate to the product, the orientation of His447 ring needs to be adjusted very slightly, and then the proton transfers from His447 to the product and the break of the scissile bond happen spontaneously. For the three-pronged oxyanion hole, it only makes two hydrogen bonds with the carbonyl oxygen at either the initial reactant or the final product state, but the third hydrogen bond is formed and stable at all transition and intermediate states during the catalytic process. At the intermediate state of the acylation reaction, a short and low-barrier hydrogen bond (LBHB) is found to be formed between two catalytic triad residues His447 and Glu334, and the spontaneous proton transfer between two residues has been observed. However, it is only about 1 ~ 2 kcal/mol stronger than the normal hydrogen bond. In comparison with previous theoretical investigations of the AChE catalytic mechanism, our current study clearly demonstrates the power and advantages of employing Born-Oppenheimer ab initio QM/MM MD simulations in characterizing enzyme reaction mechanisms.
The active-site dynamics of apo CphA beta-lactamase from Aeromonas hydropila and its complex with a beta-lactam antibiotic molecule (biapenem) are simulated using a quantum mechanical/molecular mechanical (QM/MM) method and density functional theory (DFT). The quantum region in the QM/MM simulations, which includes the Zn(II) ion and its ligands, the antibiotic molecule, the catalytic water, and an active-site histidine residue, was treated using the self-consistent charge density functional tight binding (SCC-DFTB) model. Biapenem is docked at the active site unambiguously, based on a recent X-ray structure of an enzyme-intermediate complex. The substrate is found to form the fourth ligand of the zinc ion with its 3-carboxylate oxygen and to hydrogen bond with several active-site residues. The stability of the metal-ligand bonds and the hydrogen-bond network is confirmed by 500 ps molecular dynamics simulations of both the apo enzyme and the substrate-enzyme complex. The structure and dynamics of the substrate-enzyme complex provide valuable insights into the mode of catalysis in such enzymes that is central to the bacterial resistance to beta-lactam antibiotics.
SUMMARY Rhomboid proteases regulate key cellular pathways, but their biochemical mechanism including how water is made available to the membrane-immersed active site remains ambiguous. We performed four prolonged molecular dynamics simulations initiated from both gate-open and gate-closed states of Escherichia coli rhomboid GlpG in a phospholipid bilayer. GlpG was notably stable in both gating states, experiencing similar tilt and local membrane thinning, with no observable gating transitions, highlighting that gating is rate-limiting. Analysis of dynamics revealed rapid loss of crystallographic waters from the active site, but retention of a water cluster within a site formed by His141, Ser181, Ser185 and/or Gln189. Experimental interrogation of 14 engineered mutants revealed an essential role for at least Gln189 and Ser185 in catalysis with no effect on structural stability. Our studies indicate that spontaneous water supply to the intra-membrane active site of rhomboid proteases is rare, but its availability is ensured by an unanticipated active site element, the water-retention site.
Protein Arginine Deiminase 4 (PAD4) catalyzes the citrullination of the peptidylarginine, and plays a critical role in rheumatoid arthritis (RA) and gene regulation. Understanding its catalytic mechanism is not only of fundamental importance, but also of significant medical interest for the rational design of new inhibitors. By employing on-the-fly Born-Oppenheimer ab initio QM/MM molecular dynamics simulations, we have demonstrated that it is unlikely for the active site cysteine and histidine to exist as a thiolate-imidazolium ion-pair in the PAD4 Michaelis reactant complex. Instead, a substrate-assisted proton transfer mechanism for the deimination reaction step has been characterized: both Cys645 and His471 in the PAD4 active site are neutral prior to the reaction; the deprotonation of Cys645 by the substrate arginine occurs in concert with the nucleophilic addition of the Cys thiolate to C ζ of the substrate, and leads to a covalent tetrahedral intermediate; then the C ζ -N η1 bond cleaves and the resulted ammonia is displaced by a solvent water molecule. The initial deprotonation and nucleophilic attack step is found to be rate-determining. The computed free energy barrier with B3LYP(6-31G*) QM/MM MD simulations and umbrella sampling is 20.9 kcal·mol −1 , consistent with the experimental kinetic data. During the deimination, His471 plays an important role in stabilizing transition state through the formation of the hydrogen bond with the guanidinium group. Our current studies further demonstrated the viability and strength of the ab initio QM/MM molecular dynamics approach in simulating enzyme reactions.
Aspirin, one of the oldest and most common anti-inflammatory agents, has recently been shown to reduce cancer risks. The principal pharmacological effects of aspirin are known to arise from its covalent modification of cyclooxygenase-2 (COX-2) through acetylation of Ser530, but the detailed mechanism of its biochemical action and specificity remains to be elucidated. In this work, we have filled this gap by employing a state-of-the-art computational approach, Born–Oppenheimer molecular dynamics simulations with ab initio quantum mechanical/molecular mechanical potential and umbrella sampling. Our studies have characterized a substrate-assisted inhibition mechanism for aspirin acetylating COX: it proceeds in two successive stages with a metastable tetrahedral intermediate, in which the carboxyl group of aspirin serves as the general base. The computational results confirmed that aspirin would be 10–100 times more potent against COX-1 than against COX-2, and revealed that this inhibition specificity between the two COX isoforms can be attributed mainly to the difference in kinetics rate of the covalent inhibition reaction, not the aspirin-binding step. The structural origin of this differential inhibition of the COX enzymes by aspirin has also been elucidated.
The authors present a new five-dimensional potential energy surface for H2-CO2 including the Q3 normal mode for the nu3 antisymmetric stretching vibration of the CO2 molecule. The potential energies were calculated using the supermolecular approach with the full counterpoise correction at the CCSD(T) level with an aug-cc-pVTZ basis set supplemented with bond functions. The global minimum is at two equivalent T-shaped coplanar configurations with a well depth of 219.68 cm-1. The rovibrational energy levels for four species of H2-CO2 (paraH2-, orthoH2-, paraD2-, and orthoD2-CO2) were calculated employing the discrete variable representation (DVR) for radial variables and finite basis representation (FBR) for angular variables and the Lanczos algorithm. Our calculations showed that the off-diagonal intra- and intermolecular vibrational coupling could be neglected, and separation of the intramolecular vibration by averaging the total Hamiltonian with the wave function of a specific vibrational state of CO2 should be a good approximation with high accuracy. The calculated band origin shift in the infrared spectra in the nu3 region of CO2 is -0.113 cm-1 for paraH2-CO2 and -0.099 cm-1 for orthoH2-CO2, which agrees well with the observed values of -0.198 and -0.096 cm-1. The calculated rovibrational spectra for H2-CO2 are consistent with the available experimental spectra. For D2-CO2, it is predicted that only a-type transitions occur for paraD2-CO2, while both a-type and b-type transitions are significant for orthoD2-CO2.
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