Uncovering Structure-Activity Relationships of Phenethylamines: Paving the Way for Innovative Mental Health Treatments.

The rapidly evolving psychedelic industry has garnered considerable attention due to 3,4-methylenedioxymethamphetamine-assisted psychotherapy's ground-breaking success in treating moderate-to-severe Post-traumatic Stress Disorder in two Phase 3 clinical trials. This has opened Pandora's box for the development of innovative therapeutic modalities. Of particular interest are the phenethylamines and their ability to inhibit monoamine transporters. In this study, we employed the quantitative structure-activity relationship methodology to develop three vigorous models for the reuptake of serotonin, dopamine, and norepinephrine through monoamine transporters. These models were thoroughly validated using various criteria, including fitting (R2DAT = 0.869, R2SERT = 0.828, and R2NET = 0.887), internal (Q2looDAT = 0.795, Q2looSERT = 0.784, and Q2looNET = 0.820), and external (RMSEextDAT = 0.373, R2extDAT = 0.831, RMSEextSERT = 0.200, R2extSERT = 0.955, RMSEextNET = 0.318, and R2extNET = 0.711) criteria.

QM/MM investigation of the discriminatory pre-transfer editing mechanism operated by Lysyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that remarkable facilitate the aminoacylation process during translation. With a high fidelity, the mischarged tRNA is prevented through implementing pre- and post-transfer proofreading mechanisms. For instance, Lysine-tRNA synthetase charges the native substrate, lysine, to its cognate tRNA. In spite of the great structural similarity between lysine to the noncognate and toxic ornithine, with the side chain of lysine being only one methylene group longer, LysRS is able to achieve this discrimination with a high efficiency. In this work, the hybrid quantum mechanics/molecular mechanics (QM/MM) investigation was applied to probe the pre-transfer editing mechanism catalyzed by lysyl-tRNA synthetase to reject the noncognte aminoacyl, L-ornityl (Orn), compared to the cognate substrate, L-lysyl. Particularly, the self-cyclization pre-transfer editing mechanism was explored for the two substrates. The substrate-assisted self-cyclization editing of Orn-AMP, where its phosphate moiety acts as the catalytic base, is found to be the rate-determining step with an energy barrier of 101.2 kJ mol-1. Meanwhile, the corresponding rate-limiting pathway for the native Lys-AMP lies at 140.2 kJ mol-1. This observation clearly indicated the infeasibility of this catalytic scenario in the presence of the native substrate. Interestingly, a thermodynamically favorable cyclic product of -92.9 kJ mol-1 with respect to the aminoacyl reactant complex demonstrated evidence of a successful pre-transfer editing. This reaction resulted in the discharge of the on-cognate -ornithine derivative from LysU's active site. These valuable mechanistic insights are valuable to enrich our knowledge of this extremely efficient and specific catalytic machinery of LysRS.Communicated by Ramaswamy H. Sarma.

Sulfilimine bond formation in collagen IV.

The collagen IV network plays a crucial role in providing structural support and mechanical integrity to the basement membrane and surrounding tissues. A key aspect of this network is the formation of intra- and inter-collagen fibril crosslinks. One particular crosslink, an inter-residue sulfilimine bond, has been found, so far, to be unique to collagen IV. More specifically, these crosslinks are primarily formed between methionine and lysine or hydroxylysine residues and can occur within a single collagen fibril or between different collagen fibrils. Due to its significance as the major crosslink in the collagen IV network, the sulfilimine bond plays critical roles in tissue development and various human diseases. While the proposed reaction mechanism for sulfilimine bond formation is supported by experimental evidence, the precise nature of this bond remained uncertain until computational studies were conducted. The process involves the reaction of hypohalous acids (e.g., HOBr, HOCl), produced by a peroxidasin enzyme in the basement membrane, with the sidechain sulfur of methionine or sidechain nitrogen of lysine/hydroxylysine residues in collagen IV, to form halosulfonium or haloamine intermediates, respectively. The halosulfonium/haloamine then reacts with the sidechain amine/sulfide of the lysine (or hydroxylysine) or methionine respectively, eventually resulting in the formation of the sulfilimine (MetSNLys/Hyl) crosslink. The sulfilimine product formed not only plays a crucial role in physiological processes but also finds applications in various industrial and pharmaceutical contexts. In this review, we provide a comprehensive summary of existing studies, including our own research, aimed at understanding the reaction mechanism, protonation states, characteristic nature, and dynamic behavior of the sulfilimine bond in collagen IV. The goal is to offer readers an overview of this critically important biochemical bond.

Screening a library of potential competitive inhibitors against bacterial threonyl-tRNA synthetase: DFT calculations.

Due to the growing interest in directing aminoacyl-tRNA synthetases for antimicrobial therapies, evaluating the binding proficiency of potential inhibitors against this target holds significant importance. In this work, we proposed potential ligands that could properly bind to the crucial Zn(II) cofactor located in the active site of Threonyl-tRNA synthetases (ThrRS), potentially functioning as competitive inhibitors. Initially, detailed DFT quantum chemical study was conducted to examine the binding ability of threonine against unnatural amino acids to cofactor Zn(II). Then, the binding energy value for each suggested ligand has been determined and compared to the value determined for the native substrate, threonine. Our screening investigation showed that the native threonine should coordinate in a bidentate fashion to this Zn(II) which lead to the highest (binding energy) BE Thereby, the synthetic site of ThrRS rejects unnatural amino acids that cannot afford this type of coordination to Zn(II) ion which has been supported by our calculations. Moreover, based on their binding to the Zn(II) and the obtained BE values compared to the cognate threonine, many potent ligands have been suggested. Importantly, ligands with deprotonated warheads showed the highest binding ability amongst a list of potential hits. Further investigation on the selected ligands using molecular docking and QM/MM calculations confirmed our findings of the suggested ligands being able to bind efficiently in the active site of ThrRS. The suggested hits from this study should be valuable in paving routs for developing candidates as competitive inhibitors against the bacterial ThrRS.Communicated by Ramaswamy H. Sarma.

Applications and Potential of In Silico Approaches for Psychedelic Chemistry.

Molecular-level investigations of the Central Nervous System have been revolutionized by the development of computational methods, computing power, and capacity advances. These techniques have enabled researchers to analyze large amounts of data from various sources, including genomics, in vivo, and in vitro drug tests. In this review, we explore how computational methods and informatics have contributed to our understanding of mental health disorders and the development of novel drugs for neurological diseases, with a special focus on the emerging field of psychedelics. In addition, the use of state-of-the-art computational methods to predict the potential of drug compounds and bioinformatic tools to integrate disparate data sources to create predictive models is also discussed. Furthermore, the challenges associated with these methods, such as the need for large datasets and the diversity of in vitro data, are explored. Overall, this review highlights the immense potential of computational methods and informatics in Central Nervous System research and underscores the need for continued development and refinement of these techniques and more inclusion of Quantitative Structure-Activity Relationships (QSARs).

Computational Insights into the Formation and Structure of S-N Containing Cyclic Peptides.

Cyclic peptides are known to have biologically important roles and may also be applicable to the pharmaceutical and other industries. Furthermore, thiols and amines, which are found throughout biological systems, can react to form S-N bonds and to date, ∼100 biomolecules containing such a bond have been identified. However, while there are in principle numerous S-N containing peptide-derived rings possible, only a few are presently known to occur in biochemical systems. Density functional theory-based calculations have been used to consider the formation and structure of S-N containing cyclic peptides from systematic series of linear peptides in which a cysteinyl has first been oxidized to a sulfenic or sulfonic acid. In addition, the possible effect of the cysteine's vicinal residue on the free energy of formation has also been considered. In general, when the cysteine is first oxidized to a sulfenic acid, only the formation of smaller S-N containing rings is calculated to be exergonic in aqueous solution. In contrast, when the cysteine is first oxidized to a sulfonic acid, the formation of all rings considered (with one exception) is calculated to be endergonic in aqueous solution. The nature of vicinal residue can influence ring formation through stabilizing or destabilizing intramolecular interactions.

Influence of Cysteine 440 on the Active Site Properties of 3-Deoxy-d-Arabino-Heptulosonate 7-Phosphate Synthase in Mycobacterium tuberculosis (MtDAHPS).

The shikimate pathway, which produces aromatic amino acids and key intermediates, is critical to the viability of the tuberculosis-causing pathogen Mycobacterium tuberculosis. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first committed step of this pathway and possesses regulatory functions. Its active site contains two cysteinyls: one (Cys87) bound to a metal ion, while the other (Cys440) is in proximity to the first but is located on a connecting loop. This arrangement seemingly appeared as a disulfide linkage. However, Cys440 is not metal binding, and its positioning indicates that it could collapse the disulfide linkage. Hence, its potential role may be more than simply structural support of the active site fold. Using a multiscale computational approach, molecular dynamics (MD) simulations, and DFT-based calculations, the influence of Cys440 on the active site properties has been investigated. MD simulations reveal an unusually long disulfide bond, more than 3.0 Å, whereas DFT calculations identified two stable active site conformers in the triplet and quintet spin states. Analysis of group spin density distribution identified antiferromagnetic coupling in each conformer, which suggests their relatively low potential energy and stable conformations. The conformer in the triplet spin state could favor enzyme reactivity due to its low HOMO-LUMO energy gap. In addition, reduction of the Cys440 thiolate group results in collapse of the active site metal-ligand configuration with large exothermicity. Hence, Cys440 could activate and inactivate the enzyme. For the first time, the study revealed the role of Cys440 as being vital for the catalytic activity of the enzyme rather than solely for the structural stabilization of its active site. Thus, the findings may lead to a novel basis for antituberculosis drug design and development that would disrupt the contributions of the Cys440.

Fundamentals behind the specificity of Cysteinyl-tRNA synthetase: MD and QM/MM joint investigations.

Cysteinyl-tRNA synthetase (CysRS) catalyzes the aminoacylation reaction of cysteine to its cognate tRNACys in the first step of protein translation. It is found that CysRS is different from other aaRSs as it transfers cysteine without the need for an editing reaction, which is not applicable in the case of serine despite the similarity in their structures. Surprisingly, the reasons why CysRS has high amino acid specificity are not clear yet. In this research, the binding configurations of Cys-AMP and its near-cognate amino acid Ser-AMP with CysRS are compared by Molecular Dynamics (MD). The results reveal that CysRS screens the substrate Cys-AMP to a certain extent in the process of combination and recognition, thus providing a guarantee for the high selectivity of the next reaction. While Ser-AMP is in a folded state in CysRS. In the meanwhile, the interaction between Cys-AMP and Zn963 in CysRS is much stronger than Ser-AMP. The substrate-assisted aminoacylation mechanism in CysRS is also explored by Quantum Mechanics/Molecular Mechanics (QM/MM) modeling. According to the QM/MM potential energies, the energy barrier of TSCys-AMP is 91.75 kJ/mol, while that of TSSer-AMP is close to 150 kJ/mol. Based on thermochemistry calculations, it is found that the product of Cys-AMP is more stable than the reactant. In contrast, Ser-AMP has a reactant that is more stable than its product. As a result, it reflects that the specificity of CysRS originates from both the kinetic and thermodynamical perspectives of the reaction. Our investigations demonstrate comprehensively on how CysRS recognizes and catalyzes the substrate Cys-AMP, hoping to provide some guidance for researchers in this area.

Molecular Dynamics Investigation on the Effects of Protonation and Lysyl Hydroxylation on Sulfilimine Cross-links in Collagen IV.

Collagen IV networks are an essential component of basement membranes that are important for their structural integrity and thus that of an organism's tissues. Improper functioning of these networks has been associated with several diseases. Cross-links, such as sulfilimine bonds interconnecting NC1 domains, are critical for forming and mechanically stabilizing these collagen IV networks. More specifically, the sulfilimine cross-links form between methionine (Met93) and lysine/hydroxylsine (Lys211/Hyl211) residues of NC1 domains. Therefore, the dynamic nature of the sulfilimine bond in collagen IV is crucial for network formation. To understand the dynamic nature of a neutral and protonated sulfilimine bond in collagen IV, we performed molecular dynamics (MD) simulations on four sulfilimine cross-linked systems (i.e., Met93S-NLys211, Met93S-NHLys211 +, Met93S-NHyl211, and Met93S-NHHyl211 +) of collagen IV. The MD results showed that the neutral Met93S-NLys211 system has the smallest protein backbone and showed the cross-linked residues' RMSD value. The conformational change analyses showed that the conformations of the sulfilimine cross-linked residues take on a U-shape for the Met93S-NHyl211 and Met93S-HNHyl211 + systems, whereas the conformations of the sulfilimine cross-linked residues are more open for the Met93S-NLys211, and Met93S-NHLys211 + systems. Protonation is a crucial biochemical process to stabilize the protein structure or the biological cross-links. Furthermore, the protonation of the sulfilimine bond could potentially influence hydrogen bond interaction with near amino acid residues, and according to water distribution analyses, the sulfilimine bond can potentially exist in one or more protonation states.

Conjugated Polymer Nanoparticles as a Universal High-Affinity Probe for the Selective Detection of Microplastics.

Microplastic (MP) pollution is a global challenge that requires immediate mitigation practices. Monitoring is crucial for quantifying MPs, but their mitigation remains very challenging due to several factors, including the lack of selective materials to specific polymers, and the low sensitivity of the current detection techniques. In this work, we introduce a novel design for the selective detection of MPs through fluorescence spectroscopy by exploiting conjugated polymer nanoparticles (CPNs). Fluorescent diketopyrrolopyrrole nanoparticles were prepared by nanoprecipitation to incorporate peripheral hyaluronic acid to increase their affinity for various plastics. The affinity of the new ligand for various types of MPs was examined through several characterization techniques, including fluorescence spectroscopy and microscopy, nanoparticle tracking analysis and computational studies. The new CPN were shown to be highly fluorescent in the presence of typically abundant MPs, achieving very strong binding constants in the picomolar range. This very strong affinity for a broad family of plastics was found to be the results of cooperative supramolecular effects and topographical affinity, as probed by advanced microscopy and in silico studies. Furthermore, the new affinity probes were shown to be highly selective for MPs, allowing for their detection in heterogeneous samples, including soil debris and other organic contaminants. The new materials design introduced in this work constitute a promising platform for the development of novel MP detection devices directly useable at the point of collection. Moreover, it opens new avenue for the mitigation of this environmental hazard through tailorable materials.

The Hydrolysis Rate of Paraoxonase-1 Q and R Isoenzymes: An In Silico Study Based on In Vitro Data.

Human serum paraoxonase-1 (PON1) is an important hydrolase-type enzyme found in numerous tissues. Notably, it can exist in two isozyme-forms, Q and R, that exhibit different activities. This study presents an in silico (QSAR, Docking, MD and QM/MM) study of a set of compounds on the activity towards the PON1 isoenzymes (QPON1 and RPON1). Different rates of reaction for the Q and R isoenzymes were analyzed by modelling the effect of Q192R mutation on active sites. It was concluded that the Q192R mutation is not even close to the active site, while it is still changing the geometry of it. Using the combined genetic algorithm with multiple linear regression (GA-MLR) technique, several QSAR models were developed and relative activity rates of the isozymes of PON1 explained. From these, two QSAR models were selected, one each for the QPON1 and RPON1. Best selected models are four-variable MLR models for both Q and R isozymes with squared correlation coefficient R2 values of 0.87 and 0.83, respectively. In addition, the applicability domain of the models was analyzed based on the Williams plot. The results were discussed in the light of the main factors that influence the hydrolysis activity of the PON1 isozymes.

Computational insights into the formation and nature of the sulfilimine bond in collagen-IV.

Collagen IV is essential component of basement membrane in the tissues. It provides proper cellular structure by the formation of sulfilimine bond (S[double bond, length as m-dash]N) between methionine and lysine or hydroxylysine (cross-links) residues which can be formed with or without post-translational modification. The sulfilimine bond has critical roles in tissue development and human diseases. Peroxidasin, a basement membrane peroxidase, generates reactive halogen species including hypobromous (HOBr) acid and hypochlorous (HOCl) acid which help to form halosulfonium or haloamine. The sulfilamine bond can be formed either by the formation of halosulfonium or by the formation of halomine. The aim of the study is the investigation of the formation of sulfilimine bond and its nature in collagen IV using multi-scale approach that included MD, QM-cluster, systematic series of small models, and NBO analysis. These results suggest that sulfilimine bond can be formed either via brominated/chlorinated halosulfonium or haloamine pathway. The results of systematic series of small model indicate that the formation of sulfilimine complex from halosulfonium happens through the formation of positively charged halosulfonated sulfilimine complex. It also suggests that the formation of sulfilimine complex from haloamine occurs through the formation of positively charged sulfilimine complex where the S and N bond forms and halogen goes off at the same time. Furthermore, the NBO analysis suggest the S and N bond is strongly polarized toward nitrogen in both single protonated and neutral system, N δ- ← S δ+ and also indicate the existence of a coordinate covalent (i.e. dative) bond.

Comparative QM/MM study on the inhibition mechanism of ß-Hydroxynorvaline to Threonyl-tRNA synthetase.

ß-Hydroxynorvaline (ßHNV) is unnatural amino acid structurally identical to the threonine amino acid with branched ethyl group instead of threonine's methyl. It is a known competitive inhibitor that readily bind to Threonyl-tRNA synthetase's (ThrRS) catalytic site and blocks its function. In this work, we utilized a combination of Molecular Dynamics simulation (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) methodologies to provide mechanistic insights into its inhibition reaction for ThrRS. Due to the presence of Zn(II) with its Lewis acidity character, only the ionized form of ßHNV gives an enzymatically feasible energy barrier. Furthermore, in consistence with the homochirality behavior of this active site, we observed only one conformation of ßHNV that could be acylated in the active site of ThrRS. Considering these new findings together with the recent search for new antibacterial agents, our findings should guide pharmaceutical scientists with further knowledge regarding the chemical nature of this drug. Moreover, benchmarking analysis of the utilized DFT functional has also been performed to identify the impact of various DFT functionals on representing the geometry and kinetics of our system. Notably, our Zn(II) containing chemical models are found to be responsive to the %HF contribution included together with the dispersion correction. Importantly, the BP86(0%HF)-D3 functional is found to display the greatest impact on the rate-limiting step kinetically. The crucial role played by Zn(II) is further enriched when its mutation with the chemically similar Cd(II) led to dramatic difference via obtaining less feasible reaction mechanism from thermodynamic and kinetic perspectives.

Hydrolysis Mechanism of the Linkers by Matrix Metalloproteinase-9 Using QM/MM Calculations.

Activatable cell-penetrating peptides (ACPPs) are known to be able to decrease the cytotoxicity of cell-penetrating peptide (CPP)-based drug delivery systems. Furthermore, they can improve the targeting of CPPs when specifically recognized and hydrolyzed by characteristic proteases. A comprehensive and profound understanding of the recognition and hydrolysis process will provide a better design of the ACPP-based drug delivery system. Previous studies have clearly described how ACPPs are recognized and bound by MMPs. However, the hydrolysis mechanism of ACPPs is still unsolved. This work focuses on a proteinase-sensitive cleavable linker of ACPPs (PLGLAG), the key structure for recognition and hydrolysis, trying to determine the mechanism by which MMP-9 hydrolyzes its substrate PLGLAG. The quantum mechanics/molecular mechanics (QM/MM) calculations herein show that MMP-9 proteolysis is a water-mediated four-step reaction. More specifically, it consists of (i) nucleophilic attack, (ii) hydrogen-bond rearrangement, (iii) proton transfer, and finally (iv) amide bond rupture. Considering the reversibility of multistep reaction, the second step (i.e., hydrogen-bond rearrangement) has the highest barrier and is the rate-limiting step in the hydrolysis of PLGLAG. The possible design and improvement of the key P1 and P1' sites are also explored through mutations. The present results indicate that, while the mutations affect the reaction energy barriers and the rate-limiting steps, all mutants considered could be hydrolyzed by MMP-9. To provide further insights, the hydrolysis mechanism of MMP-2, which has a similar hydrolysis process to that of MMP-9 but with different reaction barriers, is also studied and compared. As a result, this work provides detailed insights into the hydrolysis mechanism of ACPPs by MMP-9 and, thus, also possible insights for the development of new strategies for ACPP-based delivery systems.

Molecular Dynamics Simulations of a Cytochrome P450 from Tepidiphilus thermophilus (P450-TT) Reveal How Its Substrate-Binding Channel Opens.

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl's side chain tightens and relaxes the opening to the channel in conjunction with the arginyl's, though the latter's side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.

Antagonizing binding of cell cycle and apoptosis regulatory protein 1 (CARP-1) to the NEMO/IKKγ protein enhances the anticancer effect of chemotherapy.

NF-κB is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways. However, many NF-κB-mediated pathways for cell survival and apoptosis signaling in cancer remain to be elucidated. Cell cycle and apoptosis regulatory protein 1 (CARP-1 or CCAR1) is a perinuclear phosphoprotein that regulates signaling induced by anticancer chemotherapy and growth factors. Although previous studies have reported that CARP-1 is a part of the NF-κB proteome, regulation of NF-κB signaling by CARP-1 and the molecular mechanism(s) involved are unclear. Here, we report that CARP-1 directly binds the NF-κB-activating kinase IκB kinase subunit γ (NEMO or NF-κB essential modulator) and regulates the chemotherapy-activated canonical NF-κB pathway. Importantly, blockade of NEMO-CARP-1 binding diminished NF-κB activation, indicated by reduced phosphorylation of its subunit p65/RelA by the chemotherapeutic agent adriamycin (ADR), but not NF-κB activation induced by tumor necrosis factor α (TNFα), interleukin (IL)-1ß, or epidermal growth factor. High-throughput screening of a chemical library yielded a small molecule inhibitor of NEMO-CARP-1 binding, termed selective NF-κB inhibitor 1 (SNI)-1). We noted that SNI-1 enhances chemotherapy-dependent growth inhibition of a variety of cancer cells, including human triple-negative breast cancer (TNBC) and patient-derived TNBC cells in vitro, and attenuates chemotherapy-induced secretion of the pro-inflammatory cytokines TNFα, IL-1ß, and IL-8. SNI-1 also enhanced ADR or cisplatin inhibition of murine TNBC tumors in vivo and reduced systemic levels of pro-inflammatory cytokines. We conclude that inhibition of NEMO-CARP-1 binding enhances responses of cancer cells to chemotherapy.

Generation and Reactions of a Benzodehydrotropylium Ion-Co2(CO)6 Complex.

A series of 7-methylenedehydrobenzo[7]annulen-5-ol hexacarbonyldicobalt complexes were generated by Hosomi-Sakurai reactions of allylsilanes containing o-alkynylarylaldehyde-Co2(CO)6 complexes. One of the cyclization products was converted into its corresponding dihydrobenzo[7]annulen-7-ol hexacarbonyldicobalt complex, an immediate precursor to a benzodehydrotropylium-Co2(CO)6. The cation was generated in situ and reacted with four nucleophiles, and its aromatic stabilization was determined by computational methods.

Evidence for an Allosteric S-Nitrosoglutathione Binding Site in S-Nitrosoglutathione Reductase (GSNOR).

Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill-Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn2+-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen-deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme.

Multiscale Computational Study on the Catalytic Mechanism of the Nonmetallo Amidase Maleamate Amidohydrolase (NicF).

Maleamate amidohydrolase (NicF) is a key enzyme in vitamin B3 metabolism that catalyzes the hydrolysis of maleamate to produce maleic acid and ammonia. Unlike most members from the amidohydrolase superfamily it does not require a metal ion. Here, we use multiscale computational enzymology to investigate the catalytic mechanism, substrate binding, oxyanion hole, and roles of key active site residues of NicF from Bordetella bronchiseptica. In particular, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) and QTAIM methods have been applied. The mechanism of the NicF-catalyzed reaction proceeds by a nucleophilic addition-elimination sequence involving the formation of a thioester enzyme intermediate (IC2 in stage 1) followed by hydrolysis of the thioester bond to form the products (stage 2). Consequently, the formation of IC2 in stage 1 is the rate-limiting step with a barrier of 88.8 kJ·mol-1 relative to the reactant complex, RC. Comparisons with related metal-dependent enzymes, particularly the zinc-dependent nicotinamidase from Streptococcus pneumonia (SpNic), have also been made to further illustrate unique features of the present mechanism. Along with -NH- donor groups of the oxyanion hole (i.e., HN-Thr146, HN-Cys150), the active site ß-hydroxyl of threonine (HO-ßThr146) is concluded to play a role in stabilizing the carbonyl oxygen of maleamate during the mechanism.

The K2(9-ethylguanine)122+ quadruplex is more stable to unimolecular dissociation than the K(9-ethylguanine)8+ quadruplex in the gas phase: a BIRD, energy resolved SORI-CID, IRMPD spectroscopic, and computational study.

A combination of experimental trapped-ion mass spectrometric studies and computational chemistry has been used in the present work to assess the intrinsic properties of the potassiated 9-ethylguanine (9eG) self-assembled quadruplex, K2(9eG)122+, in the gas phase. Infrared multiple photon dissociation (IRMPD) spectroscopy in the N-H/C-H stretching region (2700-3800 cm-1) revealed that this G-quadruplex is a sandwich-type structure with two G-tetrads sandwiching each of the two K+, very similar to the structure determined previously for the K(9eG)8+ complexes. The stability of K2(9eG)122+ toward unimolecular dissociation and its binding energy were examined using energy-resolved sustained off-resonance collision induced dissociation (SORI-CID) and blackbody infrared radiative dissociation (BIRD) kinetics experiments. SORI-CID experiments showed that the self-assembled K2(9eG)122+ complex undergoes charge separation forming K(9eG)8+ and K(9eG)4+ compared to K(9eG)8+ which loses neutral 9eG. More interestingly, K2(9eG)122+ is more stable toward unimolecular dissociation activated by SORI-CID than the K(9eG)8+ complex. Temperature dependent BIRD kinetics for K2(9eG)122+ were consistent with energy-resolved SORI-CID results showing K2(9eG)122+ to have an activation energy of 225 ± 15 kJ mol-1, approximately 50 kJ mol-1 greater than that determined for K(9eG)8+. The extra stability of K2(9eG)122+ is apparently not thermodynamic stability, but most likely due to an energy barrier for dissociation.