Single Hydrogen Bond Research Articles

Diaminomethylenemalononitrile as a Chiral Single Hydrogen Bond Catalyst: Application to Enantioselective Conjugate Addition of α‐Branched Aldehydes

An improved diaminomethylenemalononitrile organocatalyst, bearing a N,N-disubstituted structure, promoted enantioselective conjugate addition reaction of α-branched aldehydes with vinyl sulfone, affording adducts with excellent enantioselectivities (up to 96% ee). Mechanistic studies revealed that the diaminomethylenemalononitrile motif holds the vinyl sulfone substrate using a single hydrogen bond accompanied by multiple weak interactions, including electrostatic C-H⋅⋅⋅O interactions.

A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.

Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure–activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N–H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.

Molecular Tailoring Approach for the Estimation of Intramolecular Hydrogen Bond Energy.

Hydrogen bonds (HBs) play a crucial role in many physicochemical and biological processes. Theoretical methods can reliably estimate the intermolecular HB energies. However, the methods for the quantification of intramolecular HB (IHB) energy available in the literature are mostly empirical or indirect and limited only to evaluating the energy of a single HB. During the past decade, the authors have developed a direct procedure for the IHB energy estimation based on the molecular tailoring approach (MTA), a fragmentation method. This MTA-based method can yield a reliable estimate of individual IHB energy in a system containing multiple H-bonds. After explaining and illustrating the methodology of MTA, we present its use for the IHB energy estimation in molecules and clusters. We also discuss the use of this method by other researchers as a standard, state-of-the-art method for estimating IHB energy as well as those of other noncovalent interactions.

Structure and intermolecular interactions of the fully negative solvatochromic merocyanine in the crystal phase

Herein we report an X-ray crystallographic characterization of tetramethine merocyanine based on benzimidazole and thiobarbituric acid, which shows negative solvatochromism in the entire polarity range of organic solvents. In the crystal, there are three types of non-equivalent dye molecules (A, B, C) and one water molecule in the independent part of the unit cell. The former constitute the AACBBC π-stacked columns, while the latter serves as a hydrogen-bonded bridge between the B and C molecules of the adjacent stacks. The negative bond-length alternation (BLA) values for the polymethine chain of all merocyanine species indicate a greater contribution of the zwitterionic limiting structure in their electronic structure in the crystal. The dipolarity is somewhat lesser for the molecule A, which does not form H-bonds with water. The DFT-B3LYP calculations of the studied merocyanine, its monohydrate, and the π-stacked dimer in vacuo and in a model environment (IEFPCM) have shown that a single hydrogen bond should have lesser effect on its electronic structure in the crystal than π-stacking and other multiple close-range interactions.

Advances in mechanisms of water photodissociationon TiO<sub>2</sub> surfaces

<p indent=0mm>Solar H₂O splitting to H₂ and O₂ is one of the most interesting ways to achieve clean and renewable energy and remains one of holy grails in catalysis. Since the pioneer work about photoelectrochemical H₂O splitting into H₂ and O₂ on Pt and TiO₂ electrodes was reported by Fujishima and Honda in 1972, the photochemistry of H₂O on TiO₂ surfaces has been extensively investigated by various techniques during the past decades because of its potential applications in energy chemistry. However, due to its complexity, an unambiguous understanding of elementary processes that underlie the H₂O splitting reaction on TiO₂ surfaces is still lacking. Thus, it is of great value to get an insightful understanding of the mechanisms of H₂O photochemistry on TiO₂ surfaces at a molecular level. In this review, we highlight the recent progress that provides fundamental insight into H₂O photochemistry on the TiO₂ model surfaces using surface science techniques. First of all, the structures of TiO₂ single crystal surfaces and the adsorption behaviors of H₂O on these surfaces are discussed, which will affect the photochemistry of H₂O on the surfaces. Nearly no H₂O dissociation occurs at the regular Ti⁴⁺ sites of the TiO₂ surfaces. Next, the details of H₂O photochemistry on the TiO₂ surfaces are presented, and the roles of surface structures, intermolecular hydrogen bond and photon energy in H₂O photochemistry are discussed. The mechanisms of H₂O photochemistry on different TiO₂ surfaces are similar, which occurs via the ejection of OH radical into vacuum, leaving behind the remaining H atom on the adjacent O_b site. Hydrogen bond is believed to play a double-edged sword role in H₂O dissociation: The single hydrogen bond between H₂O molecules can promote H₂O dissociation efficiently, but one-dimensional hydrogen bonding chain hinders the dissociation of H₂O significantly. Moreover, the arrangement of H₂O changes dramatically on different TiO₂ surfaces, which can further affect the dissociation probability of H₂O. In addition, the dissociation probability of H₂O on rutile TiO₂(110) is found to be highly dependent on photon energy, which raises doubt about the widely accepted photocatalysis model in which the excess energy of charge carriers is nearly useless for photocatalysis. Therefore, a sophisticated photocatalysis model that can perfectly incorporate the effect of charge carrier energy and the interaction between adsorbates (or intermediates) and TiO₂ surfaces should be developed. Afterwards, an open discussion on a new photocatalysis model is presented, which highlights the energy of charge carriers and the interaction between charge carriers and adsorbates in H₂O photochemistry on TiO₂. This will deepen our understandings of fundamental processes in photocatalysis and provide clues for the development of more efficient photocatalysis. In the end, the challenges and opportunities of the mechanistic studies of TiO₂ photocatalysis at a molecular level are discussed briefly, which may guide the development of TiO₂ photocatalysis in the future.

Vibrational Signature of Dynamic Coupling of a Strong Hydrogen Bond.

Elucidating the dynamic couplings of hydrogen bonds remains an important and challenging goal for spectroscopic studies of bulk systems, because their vibrational signatures are masked by the collective effects of the fluctuation of many hydrogen bonds. Here we utilize size-selected infrared spectroscopy based on a tunable vacuum ultraviolet free electron laser to unmask the vibrational signatures for the dynamic couplings in neutral trimethylamine-water and trimethylamine-methanol complexes, as microscopic models with only one single hydrogen bond holding two molecules. Surprisingly broad progression of OH stretching peaks with distinct intensity modulation over ∼700 cm-1 is observed for trimethylamine-water, while the dramatic reduction of this progression in the trimethylamine-methanol spectrum offers direct experimental evidence for the dynamic couplings. State-of-the-art quantum mechanical calculations reveal that such dynamic couplings are originated from strong Fermi resonance between the stretches of hydrogen-bonded OH and several motions of the solvent water/methanol, such as translation, rocking, and bending, which are significant in various solvated complexes commonly found in atmospheric and biological systems.

Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single A\u03b242 oligomers

Significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aβ42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer’s disease. Our results demonstrate that the carboxyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

Methanol degradation mechanisms and permeability phenomena in novolac epoxy and polyurethane coatings

On a global scale, methanol is one of the most important feedstocks and is used widely as solvent and co-solvent. However, due to the polar nature and associated ability to conduct current, the small molecule can take part in galvanic corrosion of metal storage tanks and degrade the barrier properties of protective coatings. In the present work, we investigated the degradation of two novolac epoxy coatings and a polyurethane (PU) coating exposed to methanol with the aim of quantifying the various degradation paths. Absorption and desorption rates were measured and the thermomechanical properties followed by dynamic mechanical analysis. For evaluation of the coating barrier properties (i.e., breakthrough time and steady state permeation rates of methanol), permeation cells were applied. During methanol absorption, simultaneous leaching of certain coating ingredients and bonding of methanol to the binder matrix via hydrogen bonds was evidenced. In terms of classification, the bonding of methanol took place by two types of mechanisms. In Type I, the methanol molecule forms a single hydrogen bond to the coating network, thereby acting as a plasticizer, which decreases the coating storage modulus and glass transition temperature. For Type II bonding of methanol, on the other hand, two hydrogen bonds to the coating network form per molecule, resulting in so-called physical crosslinking. The Type I mechanism boosted segmental mobility and contributed to the leaching of the plasticizer benzyl alcohol from the novolac epoxy coatings and residual solvents (i.e., naphtha and xylene) from the PU coating. Following the methanol desorption, and attributed to an increased effective crosslinking density from Type II bound methanol, the novolac epoxy and PU coatings exhibited significant increases in the glass transition temperatures. In addition, for the three coatings, a gradual decline in the permeability rate of methanol was observed over time. These enhanced (and unexpected) barrier properties result from a combination of effects ascribed to Type II bound methanol and the leaching process.

Hydrogen bonds dominated frictional stick-slip of cellulose nanocrystals

Crystalline cellulose, the most abundant natural polymer on earth, features exceptional physical and mechanical properties. Using atomistic simulation, this study reports the mechanical behavior of cellulose-cellulose nanocrystal hydrophilic interface and systematically examines the impact of loading direction, interfacial moisture, misalignment and surface types. The density, orientation or distribution of interfacial hydrogen bonds are shown to explain the series of findings presented here, including stick-slip behavior, stiffness recovery after an irreversible slip, direction-dependent behavior and weakening induced by hydration or misalignment. Correlation analysis shows that, regardless of the various loading conditions, the interfacial stress, shear velocity and interaction energy are strongly correlated with the density of interfacial hydrogen bonds, which quantitatively supports the central role of hydrogen bonding. Based on this correlation, the friction force rendered by a single hydrogen bond is inferred to be fHB ∼1.3 E-10 N under a shearing speed of 1 m s−1.

Histidine-Gated Proton-Coupled Electron Transfer to the CuA Site of Nitrous Oxide Reductase.

Copper-containing nitrous oxide reductase (N2OR) is the only known enzyme to catalyze the conversion of the environmentally critical greenhouse gas nitrous oxide (N2O) to dinitrogen (N2) as the final step of bacterial denitrification. Other than its unique tetranuclear active site CuZ, the binuclear electron entry point CuA is also utilized in other enzymes, including cytochrome c oxidase. In the CuA site of Pseudomonas stutzeri N2OR, a histidine ligand was found to undergo a conformational flip upon binding of the substrate N2O between the two copper centers. Here we report on the systematic mutagenesis and spectroscopic and structural characterization of this histidine and surrounding H-bonding residues, based on an established functional expression system for PsN2OR in E. coli. A single hydrogen bond from Ser550 is sufficient to stabilize an unbound conformation of His583, as shown in a Asp576Ala variant, while the additional removal of the hydrogen bond in a Asp576Ala/Ser550Ala double variant compelled His583 to stay in a bound conformation as a ligand to CuA. Systematic mutagenesis of His583 to Ala, Asp, Asn, Glu, Gln, Lys, Phe, Tyr, and Trp showed that although both the CuZ and CuA sites were present in all the variants, only the ones with a protonable side chain, i.e., His, Asp, and Glu, were able to mediate electron transfer at physiological pH. This observation is in line with a proton-coupled electron transfer mechanism at the CuA site of N2OR.

Protein secondary structure motifs: A kinematic construction.

The kinematic geometry of protein backbone structures, constrained by either single or multiple hydrogen bonds (H-bonds), possibly in a periodic array, is discussed. These structures include regular secondary structure elements α-helices and β-sheets but also include other short H-bond stabilized irregular structural elements like β-turns. The work here shows that the variations observed in such structures have simple geometrical correlations consistent with constrained motion kinematics. A new classification of the ideal helices is given, in terms of the parameter α, the angle at a Cα atom to its two neighboring Cα 's along the helix, and shown how it can be generalized to include nonideal helices. Specifically, we derive an analytical expression of the backbone dihedrals, (ϕ, ψ), in terms of the parameter α subject to the constraint that the peptide planes are parallel to the helical axis. Helices constructed in this way exhibit near-vertical alignment of the C = O and N - H units and are the canonical objects of this study. These expressions are easily modifiable to include perturbations of parameters relevant to nonplanar peptide units and noncanonical angles. The addition of a second parameter, ε0 , inclination of successive peptide planes along a helix with respect to the helical axis leads to a generalization of the previous expression and provides an efficient parametrization of such structures in terms of coordinates consistent with H-bond parameters. An analogs parametrization of β-turns, using inverse kinematic methods, is also given. Besides offering a unifying viewpoint, our results may find useful applications to protein and peptide design.

Local structure of NH4Cl solution and its correlation with osmotic coefficient by molecular dynamics simulation and Raman spectroscopy

The NH4Cl aqueous solution ranging from 1.00 wt.% to 22.00 wt.% have been studied by molecular dynamic simulation, Raman spectroscopy and polarized Raman spectroscopy. Radial distribution function (RDF), diffusion property, structure of hydrogen bond network, excess Raman spectroscopy and non-coincidence effect (NCE) were obtained. The contact ion pairs of cation and anion appeared in NH4Cl solution of 15.00 wt.%, leading to fundamental changes in hydrogen bonds and the hydrated shell. The evolution of hydrogen bond with mass fraction in solution was described. The osmotic coefficient of NH4Cl aqueous solution was obtained by thermodynamic calculation, and its linear relationship with double donor single acceptor hydrogen bond (DDA-OH) was preliminarily summarized. Moreover, the experimental results are in good agreement with the simulation results.

Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF-DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Rising trend on the halogen and non-halogen derivatives (Br, Cl, CF3, F, CH3 and NH2) in ruminal β-d-Xylopyranose – a quantum chemical perspective

The utmost aim of the current study is to find significance of the binding affinity in the halogen and non-halogen derivatives: Br, Cl, CF3, F, CH3 and NH2 of β-d-Xylopyranose with the hinge region amino acids of ruminant-β-glycosidase. The interaction energy analysis was carried out in detail through various density functional studies as M062X/def2-QZVP, M062X/LANL2DZ, B3LYP/LANL2DZ and M06HF/LANL2DZ level of theories. The total interaction energy of halogen derivatives: Br, Cl, F and CF3 are −618.21, −599.00, −720.45 and −553.08 kcal/mol respectively, and non-halogen derivative: amine group (NH2) is −87.96 kcal/mol at M062X/def2-QZVP level of theory, which exist with strong binding affinity. Ligand properties: dipole moment, polarizability, volume, molecular mass, electrostatic potential map was evaluated to understand its electrostatic and structural behavior. The nature of the bonds was inferred from the electrostatic potential map for all the halogen and non-halogen derivatives ligand. The stabilization energy from NBO analysis reveals the stability of single hydrogen and halogen bonds (N–H…Br, C–Br…O, N–H…Cl, C–Cl…O, O–H…F, C–H…F, N–H…F, C–F…O, N–H…O, O–H…O, N–H…N, O–H…N) in β-d-Xylopyranose and its derivatives. Overall, this study paves way for scientist and medicinal chemist in modelling new drugs. Further, it suggests mutations that increase the binding and may enhance the catalytic action and strengthen the complex diet in animals and hence recommended for experimental synthesis. Communicated by Ramaswamy H. Sarma

Effects of intramolecular hydrogen bond and electron delocalization on the basicity of proton sponges and superbases with benzene, pyridine, pyrazine and pyrimidine scaffolds

There are several classes of organic superbases whose basicity increases because of the formation of intramolecular hydrogen bond or/and of enhancement of electron delocalization in their protonated structures. In this work, effects of these two factors on the basicity of π-π and n-π conjugated nitrogen bases (mainly imines) are studied theoretically using the B3LYP/6-311++G(d,p) method. Comparison of proton affinities (PA) and gas phase basicities (GB) of investigated bases shows that change of electron delocalization after protonation in one ring enhances PA even by 30–40 kcal mol−1. Comparison of PAs of some proton sponges and “proton sponge-like” structures reveals that formation of a single hydrogen bond enhances PA by about 10 kcal mol−1. During this work, a large number of new superbases were designed and their basicities were assessed in the gas phase (with PA between 270 and 295 kcal mol−1) and acetonitrile (with pKa between 30 and 40).

The therapeutic potential of phytol towards Trypanosoma congolense infection and the inhibitory effects against trypanosomal sialidase

The search for novel therapeutic candidates against animal trypanosomiasis is an ongoing scientific endevour because of the negative impacts of the disease to the African livestock industry. In this study, the in vivo therapeutic potentials of phytol toward Trypanosoma congolense infection and the inhibitory effects on trypanosomal sialidase were investigated. Rats were infected with T. congolense and administered daily oral treatment of 50 and 100 mg/kg BW of phytol. Within the first 10 days of the treatment, no antitrypanosomal activity was recorded but a moderate trypanostatic activity was observed from day 17-day 21 pi. However, at 100 mg/kg BW, phytol demonstrated a significant (p < 0.05) ameliorative potentials toward T. congolense-induced host-associated pathological damages such as anaemia, hepatic and renal damages; and the data was comparable to diminazine aceturate. Moreover, the T. congolense caused a significant (p < 0.05) increase in free serum sialic acid level which was significantly (p < 0.05) prevented in the presence of phytol (100 mg/kg BW). In an in vitro analysis, phytol inhibited partially purified T. congolense sialidase using an uncompetitive inhibition pattern with inhibition binding constant of 261.24 μmol/mL. Subsequently, molecular docking revealed that the compound binds to homology modelled trypanosomal sialidase with a binding free energy of −6.7 kcal/mol which was mediated via a single hydrogen bond while Trp324 and Pro274 were the critical binding residues. We concluded that phytol has moderate trypanostatic activity but with a great potential in mitigating the host-associated cellular damages while the anaemia amelioration was mediated, in part, through the inhibition of sialidase.

Crystal structure and ligand-induced folding of the SAM/SAH riboswitch.

While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem–loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen–Watson–Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

Binding studies of crocin to β-Lactoglobulin and its impacts on both components

Light susceptibility of crocin and high degree of unsaturation limit its application in food systems. This study aimed to investigate the impact of molecular binding of crocin to β-lactoglobulin (β-LG) on crocin stability as well as the conformational stability of β-LG. The overall binding affinity was estimated about 1.74 × 103 M−1 through following UV spectral changes of β-LG at different concentrations of crocin. A 1:1 binding stoichiometry for the complex of crocin and β-LG was recognized using Job's method. The results of molecular docking and molecular dynamic simulation indicated that in addition to a single hydrogen bond with an oxygen linker attached to polyene chain, hydrophobic interactions play the major role in binding of crocin's polyene chain to β-LG surface. Three-dimensional emission and CD spectra showed conformational alterations in both tertiary and secondary structure of β-LG varying with crocin concentration. Depending on the temperature, crocin showed a dual effect on the thermodynamic stability of β-LG. Crocin destabilized β-LG up to room temperature, but stabilized at higher temperatures. The CD spectra of crocin in the Soret region and simulation results proved the bending of polyene chain and changes in the angle between sugar groups and polyene chain in combination with the protein. β-LG complexation enhanced the chemical stability of crocin by 20% during a 30-day storage, while its degradation was accelerated under UV irradiation. According to the results, it is predicted crocin functionalities to be affected by β-LG association and vice versa.

Sterically controlled and pH-dependent self-aggregation of synthetic zinc 3-(alkylamino)methylated chlorophyll-a derivatives in aqueous micellar solution

Zinc methyl 3-(methyl/ethylamino)methyl-pyropheophorbides-[Formula: see text] were prepared as models of photosynthetically active bacteriochlorophyll-[Formula: see text] possessing the 3[Formula: see text]-hydroxy group. The synthetic methylamino analog predominantly dimerized via two Zn[Formula: see text]N coordination bonds in aqueous Triton X-100 micelles, while the more sterically crowding and less coordinating ethylamino derivative self-aggregated to form large oligomers through single coordination and hydrogen bonds, Zn[Formula: see text]N–H[Formula: see text]O=C-13, similarly as in the natural self-aggregates of bacteriochlorophyll-[Formula: see text]. The artificial self-aggregates were dependent on the aqueous pH, but the dimer was less pH-sensitive and also more tolerant to the additional Triton X-100.

Molecular docking and molecular dynamics simulations of a mutant Acinetobacter haemolyticus alkaline-stable lipase against tributyrin

We previously reported on a mutant lipase KV1 (Mut-LipKV1) from Acinetobacter haemolyticus which optimal pH was raised from 8.0 to 11.0 after triple substitutions of surface aspartic acid (Asp) with lysine (Lys). Herein, this study further examined the Mut-LipKV1 by molecular docking, molecular dynamics (MD) simulations and molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) calculations to explore the structural requirements that participated in the effective binding of tributyrin and its catalytic triad (Ser165, Asp259 and His289) and identify detailed changes that occurred post mutation. Mut-LipKV1 bound favorably with tributyrin (−4.1 kcal/mol) and formed a single hydrogen bond with His289, at pH 9.0. Despite the incongruent docking analysis data, results of MD simulations showed configurations of both the tributyrin-Mut-LipKV1 (RMSD 0.3 nm; RMSF 0.05 − 0.3 nm) and the tributyrin-wildtype lipase KV1 (tributyrin-LipKV1) complexes (RMSD 0.35 nm; RMSF 0.05 − 0.4 nm) being comparably stable at pH 8.0. MM-PBSA analysis indicated that van der Waals interactions made the most contribution during the molecular binding process, with the Mut-LipKV1-tributyrin complex (−44.04 kcal/mol) showing relatively lower binding energy than LipKV1-tributyrin (−43.83 kcal/mol), at pH 12.0. All tributyrin-Mut-LipKV1 complexes displayed improved binding free energies over a broader pH range from 8.0 − 12.0, as compared to LipKV1-tributyrin. Future empirical works are thus, important to validate the improved alkaline-stability of Mut-LipKV1. In a nutshell, our research offered a considerable insight for further improving the alkaline tolerance of lipases. Communicated by Ramaswamy H. Sarma