Measuring the effects of reaction coordinate and electronic treatments in the QM/MM reaction dynamics of Trypanosoma cruzi trans-sialidase

The free energy of activation, as defined in transition state theory, is central to calculating reaction rates, distinguishing between mechanistic paths and elucidating the catalytic process. Computational free energies are accessible through the reaction space that is comprised of the conformational and electronic degrees of freedom orthogonal to the reaction coordinate. The overarching aim of this thesis was to address theoretical and methodological challenges facing current methods for calculating reaction free energies in glycoenzyme systems. Tractable calculations balance chemical accuracy and sampling efficiency that necessitates simplification of these complex reaction spaces through quantum mechanics/molecular mechanics partitioning and use of a semi-empirical electronic method to sample an approximated reaction coordinate. Here I directly and indirectly interrogate both the appropriate levels of sampling as well as the accuracy of the semi-empirical method required for reliable analysis of glycoenzyme reaction pathways. Free Energies from Adaptive Reaction Coordinates Forces, a method that builds the potential of mean force from multiple iterations of reactive trajectories, was used to construct reaction surfaces and volumes for the glycosylation and deglycosylation reactions comprising the T. cruzi trans-sialidase catalytic itinerary. This enzyme was chosen for the wealth of experimental data available for it built from its significance as a potential drug target against Chagas disease. Of equal importance is the identification of an elimination reaction competing with the primary transferase activity. The identification of this side reaction, that is observable only in the absence of the trans-sialidase or sialic acid acceptor, presented the opportunity to study the means by which enzymes selectivity bias in favor of a single reaction path. I therefore set out to explore the molecular details of how T. cruzi transsialidase asserts a precision and selectivity synonymous with enzyme catalysis. The chemical nature of the transition sate, formally defined as a dividing hypersurface separating the reactant and product regions of phase space, was characterized for the deglycosylation reaction. More than 40 transition state configurations were isolated from reactive trajectories, and the sialic acid substrate conformations were analyzed as well as the substrate interactions with the nucleophile and catalytic acid/base. A successful barrier crossing requires that the substrate pass through a family of E₅, ⁴H₅ and ⁶H₅ puckered conformations, all of which interact slightly differently with the enzyme. This work brings new evidence to the prevailing premise that there are several pathways from reactant to product passing through the saddle and successful product formation is not restricted to the minimum energy path. Increasing the reaction space with use of a multi-dimensional (3-D) reaction coordinate allowed simultaneous monitoring of the hitherto unexplored competition between a minor elimination reaction and the dominant displacement reaction present in both steps of the catalytic cycle. The dominant displacement reactions display lower barriers in the free energy profiles, greater sampling of favorable reactant stereoelectronic alignments and a greater number of possible transition paths leading to successful crossing reaction trajectories. The effects on the electronic degrees of freedom in reaction space were then investigated by running density functional theory reactive trajectories on the semi-empirical free energy. In order to carry out these simulations Free Energies from Adaptive Reaction Coordinates Forces was ported as a Fortran 90 library that interfaces with the NWChem molecular dynamics package. The resulting B3LYP/6-31G/CHARMM crossing trajectory provides a molecular orbital description of the glycosylation reaction. Direct investigation of the underlying potential energy functions for B3LYP/6-31G(d), B3LYP/6-31G and SCC-DFTB/MIO point to the minimal basis set as the primary limitation in using self-consistent charge density functional tight binding as the quantum mechanical model for modeling of enzymatic reactions transforming sialic acid substrates

Design and SAR Analysis of Covalent Inhibitors Driven by Hybrid QM/MM Simulations

Quantum mechanics/molecular mechanics (QM/MM) hybrid technique is emerging as a reliable computational method to investigate and characterize chemical reactions occurring in enzymes. From a drug discovery perspective, a thorough understanding of enzyme catalysis appears pivotal to assist the design of inhibitors able to covalently bind one of the residues belonging to the enzyme catalytic machinery. Thanks to the current advances in computer power, and the availability of more efficient algorithms for QM-based simulations, the use of QM/MM methodology is becoming a viable option in the field of covalent inhibitor design. In the present review, we summarized our experience in the field of QM/MM simulations applied to drug design problems which involved the optimization of agents working on two well-known drug targets, namely fatty acid amide hydrolase (FAAH) and epidermal growth factor receptor (EGFR). In this context, QM/MM simulations gave valuable information in terms of geometry (i.e., of transition states and metastable intermediates) and reaction energetics that allowed to correctly predict inhibitor binding orientation and substituent effect on enzyme inhibition. What is more, enzyme reaction modelling with QM/MM provided insights that were translated into the synthesis of new covalent inhibitor featured by a unique combination of intrinsic reactivity, on-target activity, and selectivity

Computational Studies on Functionalized ZnO Surfaces and Nanostructures

In this work, we have used computer simulations to investigate the effect of organic functionalization on ZnO surfaces and nanostructures. Density Functional Theory has been employed to study the interactions of ZnO surfaces with different organic groups, identifying stabilization mechanisms involved in each case and the most promising anchoring groups for ZnO functionalization. Additionally, a semi-empirical model for ZnO large scale simulations has been developed and validated by comparison against DFT calculations. The was successful in simulating Zn-containing bulk solids and molecular complexes, ZnO surfaces and nanostructures, and the adsorption of organic acids on (1010)-ZnO surfaces. We have also employed this model to characterize native defects in ZnO nanowires. Finally, we have demonstrated that the interaction of surface oxygen vacancies with organic acids may explain the suppression of photoluminescence anomalies observed for polymer coated ZnO nanowires

Developing methods to construct ring pucker free energy hypersurfaces applied to the analysis of glycosidase enzyme catalytic mechanisms

Includes bibliographical references.Carbohydrates consist of one or more sub-units usually various 5- and 6-membered cycles (furanoses and pyranoses) which can twist, bend or flip into a variety of conformers that differ in strain - this is ring puckering. These puckers notably the strained puckering conformers are observed during enzymatically assisted bond formation or cleavage of the glycosidic bonds of carbohydrate substrates. In this thesis, the free energy of ring puckering is calculated by implementing the Hill-Reilly reduced coordinate pucker description into the sampling enhancing Free Energies from Adaptive Reaction Coordinate Forces (FEARCF) method. FEARCF non-Boltzmann simulations of prototypical sugars β-Dribose and β-D-glucose converged to yield free energy pucker surfaces and volumes when using several semi-empirical QM methods - AM1, PM3, PM3CARB-1 and SCC-DFTB. From this, the accessible puckering conformations and minimum free energy paths of puckering were reasoned An analysis of the furanose and pyranose free energy pucker surfaces and volumes compared with both Density Functional Theory RB3LYP/6-311++G** optimised structures and a Hartree-Fock free energy surface revealed that SCC-DFTB provides the best semi-empirical description of 5- and 6- membered carbohydrate ring deformation. This illustrates that necessary high energy ring conformations observed in enzymatic binding sites requires the enzyme to induce and preserve high energy conformations required for successful hydrolyses and synthesis of the glycosidic bond. To further test this hypothesis, a 5- and 6-membered cycle were studied within enzymatic environments. The polysaccharide cellulose contains β 1-4 linked glucose subunit and is degraded by cellulase, a glycosidase. Specifically, the retaining cellobiohydrolase I (CBHI) of Trichoderma Reesei which cleaves cellobiose units from crystalline cellulose.The free energy volumes of puckering for the glucose sub-unit (in the catalytic position of an 8 unit cellulosic fragment - cellooctaose) were calculated and explored in vacuum, water and in the active site of CBHI. It was observed that the binding pocket of enzymes limits the ring pucker and that the active site amino acids preferentially stabilise certain puckering conformations. For CBHI, the first part of the glycosidase reaction is the glycosylation step. This was driven to completion during SCC-DFTB QM/MD FEARCF calculations where GLU212, ASP214 and GLU217 and part of the substrate were treated quantum mechanically. The general hybrid orbital method was used to connect the QM and MM regions. The free energy barriers of glycosylation were computed and the puckering statistics during the conversion of cellooctaose to products were correlated with this. Guanosine, a 5-membered ribose derivative is phosphorylated by Purine Nucleoside Phosphorylase (PNP) in order to salvage the guanine base. The effect of the PNP protein environment on ring pucker was studied by using FEARCF SCC-DFTB QM/MD non Boltzmann free energy calculations to quantify the pucker change induced in guanosine when changing environment from vacuum, to water and to the protein. In vacuo, the E4 and E1 pucker conformers were observed as minima. Upon solvation, the puckering phase space became less restricted with the 3T4 and 2T3 pucker conformers as minima. In the PNP active site pucker became restricted with only the 4E conformer observed

Implementation and benchmark of a long-range corrected functional in the density functional based tight-binding method

Bridging the gap between first principles methods and empirical schemes, the density functional based tight-binding method (DFTB) has become a versatile tool in predictive atomistic simulations over the past years. One of the major restrictions of this method is the limitation to local or gradient corrected exchange-correlation functionals. This excludes the important class of hybrid or long-range corrected functionals, which are advantageous in thermochemistry, as well as in the computation of vibrational, photoelectron and optical spectra. The present work provides a detailed account of the implementation of DFTB for a long-range corrected functional in generalized Kohn-Sham theory. We apply the method to a set of organic molecules and compare ionization potentials and electron affinities with the original DFTB method and higher level theory. The new scheme cures the significant overpolarization in electric fields found for local DFTB, which parallels the functional dependence in first principles density functional theory (DFT). At the same time the computational savings with respect to full DFT calculations are not compromised as evidenced by numerical benchmark data

Structure and stability of molecular crystals with many body dispersion inclusive density functional tight binding

Accurate prediction of structure and stability of molecular crystals is crucial in materials science and requires reliable modeling of long-range dispersion interactions. Semi-empirical electronic structure methods are computationally more efficient than their ab initio counterparts, allowing structure sampling with significant speedups. Here, we combine the Tkatchenko-Scheffler van-der-Waals method (TS) and the many body dispersion method (MBD) with third-order density functional tight-binding (DFTB3) via a charge population-based method. We find an overall good performance for the X23 benchmark database of molecular crystals, despite an underestimation of crystal volume that can be traced to the DFTB parametrization. We achieve accurate lattice energy predictions with DFT+MBD energetics on top of vdW-inclusive DFTB3 structures, resulting in a speedup of up to 3000 times compared to a full DFT treatment. This suggests that vdW-inclusive DFTB3 can serve as a viable structural prescreening tool in crystal structure prediction

Quantum Mechanical/Molecular Mechanical Molecular Dynamics Simulations on Enzymes

The dynamic nature of proteins in solution is often an indispensable factor in biological function such as enzymatic catalysis. Complementary to the conventional structural analysis, computational simulations have the advantage to reflect the dynamic nature of proteins or enzymes. One of the computational simulation methods, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations, has been widely applied to the research in structural analysis, ligand-receptor binding and enzymatic catalysis. In this dissertation, QM/MM MD simulations were applied to the studies on cytidine deaminase (CDA), yeast cytosine deaminase (yCD), and kumamolisin-As, as well as two protein lysine methyltransferases (PKMTs), DIM-5 and SET7/9. In the simulations of the transition state analogue (TSA) binding of zebularine 3, 4-hydrate to CDA and of 4-[R]-hydroxyl-3,4-dihydropyrimidine (DHP) to yCD, proton transfers were observed between the TSA and a catalytic Glu residue in both cases. Such general acidbase mechanism was also observed in the stabilization of the tetrahedral intermediate by a critical Asp residue during the acylation of kumamolisin-As. Moreover, dynamic substrate-assisted catalysis (DSAC) involving the His of the substrate at P1 site was proposed. It was suggested that DSAC may contribute to the transition state stabilizations and substrate specificity of kumamolisin-As. The origin of the product specificities of PKMTs was studied by comparison of QM/MM MD simulations on the first, second and third methyl transfers in the trimethylase DIM5 and the monomethylase SET7/9. The product specificities of the enzymes can be well explained by population distributions of well-aligned reactive structures and the relative free energy barriers for the methyl transfers. The structural and energetic reasons for the product specificities were discussed and a triplet code based on the relative free energy barriers for the three methyl transfers was proposed in the determination of product specificities of PKMTs

DFTB+, a software package for efficient approximate density functional theory based atomistic simulations

DFTB+ is a versatile community developed open source software package offering fast and efficient methods for carrying out atomistic quantum mechanical simulations. By implementing various methods approximating density functional theory (DFT), such as the density functional based tight binding (DFTB) and the extended tight binding method, it enables simulations of large systems and long timescales with reasonable accuracy while being considerably faster for typical simulations than the respective ab initio methods. Based on the DFTB framework, it additionally offers approximated versions of various DFT extensions including hybrid functionals, time dependent formalism for treating excited systems, electron transport using non-equilibrium Green's functions, and many more. DFTB+ can be used as a user-friendly standalone application in addition to being embedded into other software packages as a library or acting as a calculation-server accessed by socket communication. We give an overview of the recently developed capabilities of the DFTB+ code, demonstrating with a few use case examples, discuss the strengths and weaknesses of the various features, and also discuss on-going developments and possible future perspectives