Studies Of Enzymatic Reactions Research Articles

XFEL structure of carbonic anhydrase II: a comparative study of XFEL, NMR, X-ray and neutron structures.

The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.

NMR spectroscopy for metabolomics in the living system: recent progress and future challenges.

Metabolism is a fundamental process that underlies human health and diseases. Nuclear magnetic resonance (NMR) techniques offer a powerful approach to identify metabolic processes and track the flux of metabolites at the molecular level in living systems. An in vitro study through in-cell NMR tracks metabolites in real time and investigates protein structures and dynamics in a state close to their most natural environment. This technique characterizes metabolites and proteins involved in metabolic pathways in prokaryotic and eukaryotic cells. In vivo magnetic resonance spectroscopy (MRS) enables whole-organism metabolic monitoring by visualizing the spatial distribution of metabolites and targeted proteins. One limitation of these NMR techniques is the sensitivity, for which a possible improved approach is through isotopic enrichment or hyperpolarization methods, including dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP). DNP involves the transfer of high polarization from electronic spins of radicals to surrounding nuclear spins for signal enhancements, allowing the detection of low-abundance metabolites and real-time monitoring of metabolic activities. PHIP enables the transfer of nuclear spin polarization from parahydrogen to other nuclei for signal enhancements, particularly in proton NMR, and has been applied in studies of enzymatic reactions and cell signaling. This review provides an overview of in-cell NMR, in vivo MRS, and hyperpolarization techniques, highlighting their applications in metabolic studies and discussing challenges and future perspectives.

Systematic QM Region Construction in QM/MM Calculations Based on Uncertainty Quantification.

While QM/MM studies of enzymatic reactions are widely used in computational chemistry, the results of such studies are subject to numerous sources of uncertainty, and the effect of different choices by the simulation scientist that are required when setting up QM/MM calculations is often unclear. In particular, the selection of the QM region is crucial for obtaining accurate and reliable results. Simply including amino acids by their distance to the active site is mostly not sufficient as necessary residues are missing or unimportant residues are included without evidence. Here, we take a first step toward quantifying uncertainties in QM/MM calculations by assessing the sensitivity of QM/MM reaction energies with respect to variations of the MM point charges. We show that such a point charge variation analysis (PCVA) can be employed to judge the accuracy of QM/MM reaction energies obtained with a selected QM region and devise a protocol to systematically construct QM regions that minimize this uncertainty. We apply such a PCVA to the example of catechol O-methyltransferase and demonstrate that it provides a simple and reliable approach for the construction of the QM region. Our PCVA-based scheme is computationally efficient and requires only calculations for a system with a minimal QM region. Our work highlights the promise of applying methods of uncertainty quantification in computational chemistry.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2017-2018.

This review is the tenth update of the original article published in 1999 on the application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2018. Also included are papers that describe methods appropriate to glycan and glycoprotein analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, new methods, matrices, derivatization, MALDI imaging, fragmentation and the use of arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Most of the applications are presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and highlights the impact that MALDI imaging is having across a range of diciplines. MALDI is still an ideal technique for carbohydrate analysis and advancements in the technique and the range of applications continue steady progress.

ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY: AN UPDATE FOR 2015-2016.

This review is the ninth update of the original article published in 1999 on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2016. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented over 30 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show no sign of deminishing. © 2020 Wiley Periodicals, Inc.

Supramolecular Construction of Biohybrid Nanozymes Based on the Molecular Chaperone GroEL as a Promiscuous Scaffold.

The study of enzymatic reactions in a confined space can provide valuable insight into the natural selection of nanocompartments for biocatalytic processes. Design of nanozyme capsules with the barrel-shaped protein cage of GroEL has been proposed as a promising means to constrain chemical reactions in a spatiotemporally controllable manner. Herein, we further demonstrate with hemin that the open GroEL cavity can provide a favorable microenvironment for shielding hydrophobic catalytically active species. Meanwhile, it is shown that the GroEL-caged hemin nanozyme not only has a significantly higher catalytic activity than merely dispersed hemin but also exhibits substrate specificity in the model oxidation reactions, which is a merit lacking in natural hemoproteins. To understand the underlying mechanism behind this supramolecular assembly, molecular docking and molecular dynamics simulations were performed to study the detailed interactions of hemin with the protein cage. This revealed the most likely binding mode and preferred binding residues in the paired hydrophobic α-helices lining the GroEL cavity which are genetically encoded for substrate capture. Finally, we demonstrate that the hemin-GroEL nanozyme has great potential in label-free fluorometric molecular detection when combined with suitable substrates such as homovanillic acid. We believe that our strategy is an advantageous tool for studying confined biocatalytic kinetics as simple mimics of protein-based organelles found in nature and for designing diverse nanozymes or bio-nanoreactors with the promiscuous GroEL binding cavity.

How inverting β-1,4-galactosyltransferase-1 can quench a high charge of the by-product UDP3- in catalysis: a QM/MM study of enzymatic reaction with native and UDP-5' thio galactose substrates.

The catalysis of inverting glycosyltransferases consists of several biophysical and biochemical processes during which the transfer of a sugar residue from the purine phosphate donor substrate to an acceptor substrate occurs with stereo-inversion of the anomeric C1 center at a product. During catalysis a highly charged phosphate by-product (UDP3-) is formed and a mechanism of how the enzyme stabilizes it back to the UDP2- form is not known. Using methods of molecular modeling (hybrid DFT-QM/MM calculations) we proposed and validated a catalytic mechanism of bovine inverting β-1,4-galactosyltransferase-1 (β4Gal-T1) with native (UDP-galactose) and thio donor substrates (UDP-5' thio galactose). We focused on three aspects of the mechanism not yet investigated: (i) the formation of an oxocarbenium ion intermediate, which was only found for the retaining glycosyltransferases for the time being; (ii) the mechanism of stabilization of a highly charged phosphate by-product (UDP3-) back to its standard in vivo form (UDP2-); (iii) explanation for why in experimental measurements the rate of catalysis with the thio donor substrate is only 8% of the rate of that with the natural substrate. To understand the differences in the interaction patterns between the complexes enzyme : UDP-Gal and enzyme : UDP-5S-Gal, fragmented molecular orbital (FMO) decomposition energy analysis was carried out at the DFT level.

Study of Enzymatic Reaction with the Combination of Bioinformatics and Organic Chemistry: An Introduction and Discussion of an Undergraduate Experiment Abroad

Study of Enzymatic Reaction with the Combination of Bioinformatics and Organic Chemistry: An Introduction and Discussion of an Undergraduate Experiment Abroad

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2013-2014.

This review is the eighth update of the original article published in 1999 on the application of Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly- saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 37:353-491, 2018.

Importance of MM Polarization in QM/MM Studies of Enzymatic Reactions: Assessment of the QM/MM Drude Oscillator Model.

Comput. 2014, 10, 1795-1809), we now report a comprehensive investigation of the effects of MM polarization on two enzymatic reactions, namely the Claisen rearrangement in chorismate mutase and the hydroxylation reaction in p-hydroxybenzoate hydroxylase, using the QM/CHARMM-DO model and two QM methods (B3LYP, OM2). We compare the results from extensive geometry optimizations and free energy simulations at the QM/MM-DO level to those obtained from analogous calculations at the conventional QM/MM level.

Enhancing Paradynamics for QM/MM Sampling of Enzymatic Reactions.

Despite the enormous increase in computer power, it is still extremely challenging to obtain computationally converging sampling of ab initio QM/MM (QM(ai)/MM) free energy surfaces in condensed phases. The sampling problem can be significantly reduced by the use of the reference potential paradynamics (PD) approach, but even this approach still requires major computer time in studies of enzymatic reactions. To further reduce the sampling problem we developed here a new PD version where we use an empirical valence bond reference potential that has a minimum rather than a maximum at the transition state region of the target potential (this is accomplished conveniently by shifting the EVB of the product state). Hence, we can map the TS region in a more efficient way. Here, we introduce and validate the inverted EVB PD approach. The validation involves the study of the S(N)2 step of the reaction catalyzed by haloakene dehalogenase (DhlA) and the GTP hydrolysis in the RasGAP system. In addition, we have also studied the corresponding reaction in water for each of the systems described here and the reaction involving trimethylsulfonium and dimethylamine in solution. The results are encouraging and the new strategy appears to provide a powerful way of evaluating QM(ai)/MM activation free energies.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2011-2012.

This review is the seventh update of the original article published in 1999 on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2012. General aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, and fragmentation are covered in the first part of the review and applications to various structural types constitute the remainder. The main groups of compound are oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:255-422, 2017.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for 2009-2010.

This review is the sixth update of the original article published in 1999 on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2010. General aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, arrays and fragmentation are covered in the first part of the review and applications to various structural typed constitutes the remainder. The main groups of compound that are discussed in this section are oligo and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Many of these applications are presented in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 34: 268–422, 2015.

Multi-Scale Computational Biology

Cellular functions such as growth, programmed cell death, sensing, and metabolism, as well as the pathological processes underlying protein malfunctioning, involve molecular interactions that span from single protein to multi-protein complexes. Indeed, huge protein complexes are regulated through complex networks of transient molecular interactions. Consequently, the behaviour of a biological system is determined by inter-relationships between molecules, rather than by its individual components. The key technique for this kind of characterization, i.e. considering complete cellular pathways, is ‘systems biology’. This approach is mainly devoted to the unravelling at a mathematical level the functioning of cellular pathways or generegulatory networks. But, in the other hand, systems biology needs to be accompanied by a quantitative molecular description of pathways, so far most lacking. The characterization of these phenomena needs to be performed computationally and it is highly challenging as it requires bridging many orders of magnitude in spatial and temporal dimensions, as well as advanced techniques in data-mining. This calls for the development of computational approaches, able to deeply characterize complex molecular systems into their individual players. For each individual level many simulation methods and strategies exist: bioinformatics uses the information encoded in the protein sequences to predict structure and function, molecular dynamics (MD) simulations predicts structural, dynamical, and energetic (bio)molecular properties, quantum mechanics simulations allow the characterization of bond-forming and bond -breaking phenomena, the prediction of spectroscopic and vibration properties of drugs bound to their targets and the study of enzyme reaction. Finally, hybrid methods, i.e. quantum mechanics/molecular mechanics (QM/MM) and molecular mechanics/ coarse-grained (MM/CG) simulations, allow the characterization of events occurring at different temporal and spatial scales. The ultimate goal of computational biology is thus the development of multi-scale computational methods, able to extract properties computed from protein structures (such as kinetic parameters, affinities and binding energies) into macroscopic biochemical network models. This novel area of investigation will impact strongly on pharmaceutical sciences and toxicology, as drugs target (and mutations affect) pathways, rather than a single bio molecule.

1P097 酵素反応の時間分解分光解析を実現するマイクロ流路フローフラッシュ法の開発(02.ヘム蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

1P097 酵素反応の時間分解分光解析を実現するマイクロ流路フローフラッシュ法の開発(02.ヘム蛋白質,ポスター,日本生物物理学会年会第51回(2013年度))

Applications of Potential Energy Surfaces in the Study of Enzymatic Reactions

From a generated PES, one can determine the relative energies of species involved, the sequence in which they occur, and the activation barrier(s) associated with individual steps or the overall mechanism. Furthermore, they can provide more insights than a simple indication of a path of sequential mechanistic structures and their energetic relationships. The investigation into the activation of O2by alpha-ketoglutarate-dependent dioxygenase (AlkB) clearly shows the opportunity for spin inversion, where one can see that the lowest energy product may be formed via several possible routes. In the investigation of uroporphyrinogen decarboxylase III (UROD), the use of QM/MM methods allowed for the inclusion of the anisotropic protein environment providing greater insight into the rate-limiting barrier. Lastly, the mechanism of 6-phospho-α-glucosidase (GlvA) was discussed using different active site models. In particular, a continuum model PES was compared to the gas-phase PES.

Long-Range Electrostatic Effects in QM/MM Studies of Enzymatic Reactions: Application of the Solvated Macromolecule Boundary Potential.

Long-range electrostatic interactions are important in simulations of enzymatic reactions. They can be divided into the effects due to bulk solvent and those due to the electrostatic potential of the outer macromolecule. We study and quantify the importance of these two effects for two test systems by application of the solvated macromolecule boundary potential (SMBP) [J. Chem. Theory Comput. 2009, 5, 3114-3128]. We validate the accuracy of the SMBP for these test systems and present a transferable protocol for determination of optimal SMBP parameters as well as recommended default values for these parameters. Two enzymatic reactions with different characteristics are studied: the intramolecular Claisen rearrangement in chorismate mutase that is associated with little charge transfer and the hydroxylation reaction in p-hydroxybenzoate hydroxylase that corresponds to a formal "OH(+)" transfer and thus involves significant charge transfer. It is found that the effects of the electrostatic potential of the outer macromolecule and of bulk solvent are only important in the latter case, where their neglect causes deviations in the computed barriers on the order of 1-2 kcal/mol, respectively. Even larger deviations on the order of several kilocalories per mole are observed for the reaction energies in p-hydroxybenzoate hydroxylase if the electrostatic potential of the outer macromolecule is neglected.

Labeling and Purification of Cellulose-Binding Proteins for High Resolution Fluorescence Applications

The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino acids. However, these reactions are probabilistic and can yield mixtures of unlabeled and labeled enzymes with catalytic activities that can be modified by the addition of fluorophores. To have meaningful interpretations of results from the study of labeled enzymes, it is then necessary to reduce the variability in physical, chemical, and biological characteristics of the labeled products. In this paper, a solid phase labeling protocol is described as an advantageous alternative to free solution labeling of cellulose-binding proteins and is applied to tag cellulases with three different fluorophores. The products from the labeling reactions were purified to remove the unreacted dye and separate labeled and unlabeled enzymes. Characterization of the catalytic and spectroscopic properties of the isolated labeled species confirmed that highly homogeneous populations of labeled cellulases can be achieved. The protocol for the separation of labeled products is applicable to any mixture of labeled proteins, making this an attractive methodology for the production of labeled proteins suitable for single molecule fluorescence spectroscopy.

DFT Studies on Hydrolyses of Dimethylchlorothiophosphate

Many thiophosphates are widely used as substrate analogues in studies of enzymatic reactions as well as organic insecticides in agricultural pest control. These organophosphorus compounds yield toxic metabolites by several reactions before hydrolysis occurs. Therefore, the rates of hydrolyses of these compounds are important for green chemistry. More sluggish degradations of thiophosphate pesticides are often observed as compared with the corresponding phosphate compound: for example, the hydrolysis rate of diazinon (I, X=S) is slower than that of the diazoxon (I, X=O), where the sulfur atom is replaced by oxygen atom, by about 10 times at pH of 7.4, corresponding to the difference in the free energy of activation by about 1.1 kcal/ mol at 20 °C. However, an inverse thio effect (kS/kO > 1) was found occasionally for the hydrolyses of organophosphorus compounds including parathion (II, X=S) and paraoxon (II, X=O), and it was rationalized by the higher electronegativity of oxygen as compared to sulfur. Although, the differential electronic structure including the bond order and charge localization of P=O and P=S bond are discussed in detail, the relative bond order and charge localization are varied for the phosphorothioates and the origin of differential reactivity of those compounds is not clear.

Oscillatory reaction of catalase wrapped by liposome

To date we have been studying the enzymatic oscillatory reaction caused by gradual entry of substrate via semi-permeable membrane. It has been found that many enzymes cause oscillatory reaction. Here we present an oscillatory reaction of enzyme wrapped by liposome. We used catalase as an enzyme, since its oscillatory reaction has been already investigated in detail in the absence of liposome. Distinct oscillation with shorter period than without liposome was obtained. On the other hand, it was shown that the presence of liposome facilitated the permeation rate of hydrogen peroxide through semi-permeable membrane. This is thought to be factor of shortening the oscillation period compared with that in the absence of liposome. Oscillation was also temperature dependent. Our finding may provide an important insight into the study of enzyme reaction taking part in rhythms in living systems.