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... [23,28,29] This idea of protein flexibility being crucial for catalysis is not new and has been present in enzymology for decades, [30][31][32][33][34][35][36] and the specific role of different protein motions occurring in different timescales is being intensely debated. [37][38][39][40][41] Following Pauling's [42,43] and Warshel's [44] postulates, our working hypothesis is that installing and maintaining the optimal arrangement of catalytic groups able to lower the energy of the rate-limiting transition state (TS) in a stable protein scaffold, will result in enzymatic catalysis for any given reaction. [45] By combining computational design with random or directed evolution, groundbreaking examples from the Baker, [11][12][13][14] Hilvert, [16][17][18] Mayo, [19] Tawfik, [46][47][48] and DeGrado [49,50] labs have demonstrated the validity of this hypothesis. ...
... [23] Using a broad selection of designed and evolved enzymes for the Kemp elimination as benchmark, we have found that the geometric similarity to the computed theozymes is maintained for a chemically meaningful time (100 ns) in the ground state, only when using polarized substrates with a charge distribution resembling the transition state. These observations are fully consistent with the principle that enzymes are optimized to provide maximum stabilization of the polar TS, [43] not the nonpolar reactant(s). [75] This principle is perfectly exemplified by enzymes that did not evolve under the specific pressure to reduce K M , such as Kemp eliminases, ketoreductases [76] and P450s, [7,77] all of which show flawed binding properties towards their typically small, nonpolar substrates. ...
Extensive molecular dynamics (MD) simulations on designed and evolved enzymes for the Kemp Elimination were performed. Over thirty different systems including combinations of three protein scaffolds used in computational design (KE70, KE59 and HG3), several directed evolution variants and ligands (5‐nitrobenzo[d]isoxazole, and 6‐nitro‐1H‐benzo[d][1,2,3]triazole), were screened computationally. The study was focused mostly on computational and evolved variants for which X‐ray structures are available. MD simulations were used to monitor the organization of the active sites over time periods of 100 ns. A general trend was observed in the simulations: the optimal organization of active site residues, corresponding to the most active catalysts, is only maintained with polarized, TS‐like substrates, reinforcing the crucial role of protein dynamics and electrostatic preorganization for efficient biocatalysis. Implications for enzyme design protocols are discussed.
... In 1948, Linus Pauling proposed that enzymes stabilize the transition state to a larger extent than reagents by means of noncovalent interactions between the active functional groups in the confined space and the substrate inside it. 3 Enzyme functionality is defined by the molecular organization within and around the confined space. [4][5][6][7][8][9][10][11][12][13] Enzyme activity is a topic of active discussion, while the "lock and key" model provides a favorable conformation involving binding of a substrate to the enzyme-active site followed by its activation. ...
... Zn II binding confers significant stability (T m increase of 24 °C), homodimer binding affinity (>100-fold increase), and catalytic function (hydrolytic rate acceleration of 10 5 ). The naturally occurring enzyme CA II contains a Zn(His) 3 O site and features a buried tunnel-like active site, and hydrolysis of p-NPA, a nonnatural substrate, proceeds with a k cat of 53 s −1 and k cat /K M of 2,550 M −1 s −1 . The differences in reactions between naturally occurring enzymes and the synthetic MIDI-Zn catalyst are likely to be attributed to more than just the structure of the cleft. ...
Enzymes with well-defined three-dimensional structure have in-built information for molecular organization in the near vicinity of the active sites—popularly known as enzyme architecture. Over the past few years, molecular assembly has been exploited in creating artificial enzyme or catalyst architectures. Emergent spatiotemporal structure and catalytic activity can be achieved through controlled assembly of suitable molecular building blocks. The programmed molecular assembly governed by the scheme of molecule architectonics can generate enzyme-mimetic catalyst assembly architecture. Apart from the conventional ligand-metal interaction in the first coordination sphere of a catalyst, a second coordination sphere plays a key role in the catalytic activity of enzymes. This review attempts to unravel the balancing act between molecular architectonics and second coordination spheres in catalyst assembly architecture development. Judicious design and exploitation of state-of-the-art biomimetic catalyst architecture derived from small molecules, sugars, nucleic acids, peptides, and proteins are discussed under the above-mentioned framework. Metal-coordinated molecular assembly architectures of specific catalytic properties are considered with respect to the nature of molecular assembly and experimental conditions. The concise and critical discussion provides a holistic view of enzyme-mimetic architectures and their second coordination spheres through a reductionistic approach based on the molecular architectonics of simple and modular molecular building blocks.
... The catalytic efficiency of biological enzymes is controlled by the intricate interplay between the shapes and characteristics of the active site and the substrates throughout the reaction being catalyzed. The general mode of action of enzyme catalysts can be summarized with reference to comparably simple principles introduced by Pauling [1] and Jencks, [2] according to which reaction barriers are lowered through transition state stabilization originating from shapecomplementarity between catalyst and transition structure, and ground state destabilization resulting from non-complementarity between reactant and catalyst. ...
... Aside from TS stabilization, destabilization of the reactant complex due to geometric distortions in host and guest resulting from the binding of the less complementary reactant is expected to contribute to overall reaction barrier lowering according to the Pauling-Jencks model of enzyme catalysis. [1,2] In order to quantify this ground state destabilization in our systems of interest, we calculate the differences in electronic energies (ΔE e ) between free equilibrium host and guest structures and the corresponding geometries of the isolated host and guest molecules, respectively, in reactant, transition structure, and product complexes. Figure 8a,b gives the destabilization energies resulting for the host and guest moieties of all complexes. ...
Enzymes actuate catalysis through a combination of transition state stabilization and ground state destabilization, inducing enantioselectivity through chiral binding sites. Here, we present a supramolecular model system which employs these basic principles to catalyze the enantiomerization of [5]helicene. Catalysis is hereby mediated not through a network of functional groups but through π−π catalysis exerted from the curved aromatic framework of a chiral perylene bisimide (PBI) cyclophane offering a binding pocket that is intricately complementary with the enantiomerization transition‐state structure. Although transition state stabilization originates simply from dispersion and electrostatic interactions, enantiomerization kinetics are accelerated by a factor of ~700 at 295 K. Comparison with the meso‐congener of the catalytically active cyclophane shows that upon configurational inversion in only one PBI moiety the catalytic effect is lost, highlighting the importance of precise transition‐state structure recognition in supramolecular enzyme mimics.
... The catalytic efficiency of biological enzymes is controlled by the intricate interplay between the shapes and characteristics of the active site and the substrates throughout the reaction being catalyzed. The general mode of action of enzyme catalysts can be summarized with reference to comparably simple principles introduced by Pauling [1] and Jencks, [2] according to which reaction barriers are lowered through transition state stabilization originating from shapecomplementarity between catalyst and transition structure, and ground state destabilization resulting from non-complementarity between reactant and catalyst. ...
... Aside from TS stabilization, destabilization of the reactant complex due to geometric distortions in host and guest resulting from the binding of the less complementary reactant is expected to contribute to overall reaction barrier lowering according to the Pauling-Jencks model of enzyme catalysis. [1,2] In order to quantify this ground state destabilization in our systems of interest, we calculate the differences in electronic energies (ΔE e ) between free equilibrium host and guest structures and the corresponding geometries of the isolated host and guest molecules, respectively, in reactant, transition structure, and product complexes. Figure 8a,b gives the destabilization energies resulting for the host and guest moieties of all complexes. ...
Enzymes actuate catalysis through a combination of transition state stabilization and ground state destabilization, inducing enantioselectivity through chiral binding sites. Here, we present a supramolecular model system which employs these basic principles to catalyze the enantiomerization of [5]helicene. Catalysis is hereby mediated not through a network of functional groups but through π‐π catalysis exerted from the curved aromatic framework of a chiral perylene bisimide (PBI) cyclophane offering a binding pocket that is intricately complementary with the enantiomerization transition structure. Although transition state stabilization originates simply from dispersion and electrostatic interactions, enantiomerization kinetics are accelerated by a factor of ca. 700 at 295 K. Comparison with the meso‐congener of the catalytically active cyclophane shows that upon configurational inversion in only one PBI moiety the catalytic effect is lost, highlighting the importance of precise transition structure recognition in supramolecular enzyme mimics.
... [4] In early 1948, Pauling proposed a mechanism for how enzymes stabilize transition states through noncovalent interactions between functional groups within the enzyme cavity and the compounds it contains. [5] Since direct utilization of enzyme catalysts usually subjects to complex structure and intricate biological processes, the non-covalent interactions involved in enzymatic catalysis, which mainly inculde hydrogen-bond interactions, van der Waals forces, ionic interactions, and π-π stacking interactions, have becoming essential factors in the design and development of enzyme-mimic catalytic systems due to their unique property in regulating the reactivity and selectivity in organic transformations. Along this line, the well-organized artificial supramolecular reactors, [6] e.g., the self-assembled metal-organic cages (MOCs), [7] have emerged as promising supramolecular catalysts as enzyme blueprints. ...
The self‐assembled metal‐organic cages (MOCs) have been evolved as a paradigm of enzyme‐mimic catalysts since they are able to synergize multifunctionalities inherent in metal and organic components and constitute microenvironments characteristic of enzymatic spatial confinement and versatile host–guest interactions, thus facilitating unconventional organic transformations via unique driving‐forces such as weak noncovalent binding and electron/energy transfer. Recently, MOC‐based photoreactors emerged as a burgeoning platform of supramolecular photocatalysis, displaying anomalous reactivities and selectivities distinct from bulk solution. This perspective recaps two decades journey of the photoinduced radical reactions by using photoactive metal‐organic cages (PMOCs) as artificial reactors, outlining how the cage‐confined photocatalysis was evolved from stoichiometric photoreactions to photocatalytic turnover, from high‐energy UV‐irradiation to sustainable visible‐light photoactivation, and from simple radical reactions to multi‐level chemo‐ and stereoselectivities. We will focus on PMOCs that merge structural and functional biomimicry into a single‐cage to behave as multi‐role photoreactors, emphasizing their potentials in tackling current challenges in organic transformations through single‐electron transfer (SET) or energy transfer (EnT) pathways in a simple, green while feasible manner.
... They are elicited by immunization with haptens whose structure resembles the transition state of a reaction of interest, for example ester hydrolysis or an aldol coupling. As predicted by Pauling's theory that native enzymes function by stabilizing transition states, [16] antibodies that bind tightly to these transition state analogues can act as catalysts for that transformation. What may be less broadly appreciated by the chemistry community is that several native antibodies have also been found to have catalytic activity. ...
Many of the highest priority targets in a wide range of disease states are difficult‐to‐drug proteins. The development of reversible small molecule inhibitors for the active sites of these proteins with sufficient affinity and residence time on‐target is an enormous challenge. This has engendered interest in strategies to increase the potency of a given protein inhibitor by routes other than further improvement in gross affinity. Amongst these, the development of catalytic protein inhibitors has garnered the most attention and investment, particularly with respect to protein degraders, which catalyze the destruction of the target protein. This article discusses the genesis of the burgeoning field of catalytic inhibitors, the current state of the art, and exciting future directions.
... To efficiently use attractive noncovalent interactions in catalysis, substrate and catalyst need to be geometrically compatible with each other. [143] While this compatibility is usually achieved by utilizing functional groups, Karton et al. [144] recently demonstrated that simple pristine graphene catalyzes the racemization chiral 1,1'-binaphthyl-2,2'-diol (BINOL) via large area noncovalent contacts. By utilizing a single-layer graphene (R)-BINOL (> 99 : 1 e.r.) could be completely racemized. ...
London dispersion (LD) interactions are the main contribution of the attractive part of the van der Waals potential. Even though LD effects are the driving force for molecular aggregation and recognition, the role of these omnipresent interactions in structure and reactivity had been largely underappreciated over decades. However, in the recent years considerable efforts have been made to thoroughly study LD interactions and their potential as a chemical design element for structures and catalysis. This was made possible through a fruitful interplay of theory and experiment. This review highlights recent results and advances in utilizing LD interactions as a structural motif to understand and utilize intra‐ and intermolecularly LD‐stabilized systems. Additionally, we focus on the quantification of LD interactions and their fundamental role in chemical reactions.
... The stronger FDH inhibition by azide can be rationalized by the structural differences between the two tested inhibitor molecules that determine their specific interactions with the EcFDH-H active site. I.e., nitrate has a planar trigonal geometry and binds to the Mo ion in a competitive manner while azide is a linear molecule resembling a transition analogue of the FDH reaction that, in accordance with Pauling's induced fit model, tightly binds to the active site [44,45] (Fig. 1B). In addition, non-competitive binding to FDH has been also reported for azide [46] and its higher electron donor strength has been proposed as a basis for the strong binding to Mo(VI) as a strong electron acceptor [27]. ...
Metal-dependent formate dehydrogenases (Me-FDHs) are highly active CO 2-reducing enzymes operating at low redox potentials and employ either molybdenum or tungsten to reduce the bound substrate. This makes them suitable for electrochemical applications such as fossil-free production of commodity chemicals utilizing renewable energy. Electrocatalytic CO 2 reduction by cathode-immobilized Me-FDHs has been recently demonstrated and rational protein engineering can be used to optimize Me-FDHs for various carbon reduction reactions. In the present study, CO 2 reduction by soluble monomeric Escherichia coli formate dehydrogenase H (EcFDH-H) was demonstrated and the function of its nucleophilic selenocysteine residue as a transient ligand of a centrally bound molybdenum atom was investigated. Kinetic analysis of the wildtype enzyme revealed maximum CO 2 reduction rates of 44 ± 6 s − 1 at pH 5.8 that was decreased to 19% and 0% in the case of selenocysteine substitution with the structural homologues cysteine and serine, respectively. Further selenocysteine-to-cysteine substitution effects included an increased acid tolerance as well as stronger inhibition by nitrate and azide indicating a shift of the Mo oxidation state from IV to VI. Conversely, a destabilizing effect on the oxidized Mo (VI) center could be assigned to the native selenocysteine residue that may facilitate the observed efficient CO 2 reduction by rapid transition between Mo oxidation states. Taken together, the performed characterization of EcFDH-H as a catalyst for CO 2 reduction and the selenocysteine substitution analysis furthers the understanding of the active-site structure of Me-FDHs and thereby supports the development of more efficient biocatalysts for CO 2 reduction.
... Transition state analogs (TSAs) have been used by nature to control enzyme function (1,2), in research to provide insights into the properties of enzyme active sites and enzymatic catalysis (3)(4)(5)(6), and in medicine to provide highly effective drugs (6)(7)(8)(9)(10). Catalysis can be defined as preferential stabilization of a transition state over a reaction's ground state (3,(11)(12)(13)(14)(15)(16). Thus, it is expected-and has been observed-that compounds with electrostatic and geometric features resembling the transition state but not the ground state bind more strongly to enzymes than substrates or standard inhibitors. ...
Using high-throughput microfluidic enzyme kinetics (HT-MEK), we measured over 9,000 inhibition curves detailing impacts of 1,004 single-site mutations throughout the alkaline phosphatase PafA on binding affinity for two transition state analogs (TSAs), vanadate and tungstate. As predicted by catalytic models invoking transition state complementary, mutations to active site and active-site-contacting residues had highly similar impacts on catalysis and TSA binding. Unexpectedly, most mutations to more distal residues that reduced catalysis had little or no impact on TSA binding and many even increased tungstate affinity. These disparate effects can be accounted for by a model in which distal mutations alter the enzyme's conformational landscape, increasing the occupancy of microstates that are catalytically less effective but better able to accommodate larger transition state analogs. In support of this ensemble model, glycine substitutions (rather than valine) were more likely to increase tungstate affinity (but not more likely to impact catalysis), presumably due to increased conformational flexibility that allows previously disfavored microstates to increase in occupancy. These results indicate that residues throughout an enzyme provide specificity for the transition state and discriminate against analogs that are larger only by tenths of an Ångström. Thus, engineering enzymes that rival the most powerful natural enzymes will likely require consideration of distal residues that shape the enzyme's conformational landscape and fine-tune active-site residues. Biologically, the evolution of extensive communication between the active site and remote residues to aid catalysis may have provided the foundation for allostery to make it a highly evolvable trait.
... Early studies on enzymatic catalysis proposed that the residues at the enzyme's active site interact with the transition state (TS) rather than the substrate to catalyse the reaction [1][2][3]. The TS represents a specific intermediate configuration between the substrate and the enzyme, which is attained immediately before the completion of the reaction and corresponds to the highest potential energy along the reaction coordinate (see Fig. 1(A)) [4]. ...
The Near Attack Conformation (NAC) approach states that the efficiency of an enzyme-catalyzed reaction depends on the prior attainment of optimal conditions for substrate atom organization and positioning for bond formation. These conditions are prerequisites for the transition state (TS) in which the involved atoms are within the van der Waals range of contact and positioned at an angle similar to that achieved after bond formation. The successful application of this approach to investigate the reactivation mechanism of acetylcholinesterase inhibited by nerve agents has contributed to a better understanding of this mechanism and demonstrated consistent corroboration with experimental data. In this article, we summarize the accomplishments achieved thus far and outline future perspectives.
... Moreover, chirally pure polymers, folding into supramolecular structures, may sustain the strain needed for catalysis [53] better than mixed-handed polymers in the same manner as pure substances maintain structure upon heating better than mixtures. Conversely, like other standards, chirality standards will be lost in circumstances where they no longer provide superior free energy consumption over random processes. ...
The prevalence of chirally pure biological polymers is often assumed to stem from some slight preference for one chiral form at the origin of life. Likewise, the predominance of matter over antimatter is presumed to follow from some subtle bias for matter at the dawn of the universe. However, rather than being imposed from the start, handedness standards in societies emerged to make things work. Since work is the universal measure of transferred energy, it is reasoned that standards at all scales and scopes emerge to consume free energy. Free energy minimization, equal to entropy maximization, turns out to be the second law of thermodynamics when derived from statistical physics of open systems. This many-body theory is based on the atomistic axiom that everything comprises the same fundamental elements known as quanta of action; hence, everything follows the same law. According to the thermodynamic principle, the flows of energy naturally select standard structures over less-fit functional forms to consume free energy in the least time. Thermodynamics making no distinction between animate and inanimate renders the question of life’s handedness meaningless and deems the search for an intrinsic difference between matter and antimatter pointless.
... For example, transition and intermediate states along the reaction path can be used as templates for the development of new enzymatic inhibitors. 13 However, the standard mechanistic proposal for cysteine proteases presents some difficulties in the case of caspases. The overall mechanism presented in Figure 1 surmises a thiolate− imidazolium ion-pair configuration for the catalytic dyad in the noncovalent enzyme−substrate or Michaelis complex. ...
Caspases are cysteine proteases in charge of breaking a peptide bond next to an aspartate residue. Caspases constitute an important family of enzymes involved in cell death and inflammatory processes. A plethora of diseases, including neurological and metabolic diseases and cancer, are associated with the poor regulation of caspase-mediated cell death and inflammation. Human caspase-1 in particular carries out the transformation of the pro-inflammatory cytokine pro-interleukin-1β into its active form, a key process in the inflammatory response and then in many diseases, such as Alzheimer's disease. Despite its importance, the reaction mechanism of caspases has remained elusive. The standard mechanistic proposal valid for other cysteine proteases and that involves the formation of an ion pair in the catalytic dyad is not supported by experimental evidence. Using a combination of classical and hybrid DFT/MM simulations, we propose a reaction mechanism for the human caspase-1 that explains experimental observations, including mutagenesis, kinetic, and structural data. In our mechanistic proposal, the catalytic cysteine, Cys285, is activated after a proton transfer to the amide group of the scissile peptide bond, a process facilitated by hydrogen-bond interactions with Ser339 and His237. The catalytic histidine does not directly participate in any proton transfer during the reaction. After formation of the acylenzyme intermediate, the deacylation step takes place through the activation of a water molecule by the terminal amino group of the peptide fragment formed during the acylation step. The overall activation free energy obtained from our DFT/MM simulations is in excellent agreement with the value derived from the experimental rate constant, 18.7 vs 17.9 kcal·mol-1, respectively. Simulations of the H237A mutant support our conclusions and agree with the reported reduced activity observed for this caspase-1 variant. We propose that this mechanism can explain the reactivity of all cysteine proteases belonging to the CD clan and that differences with respect to other clans could be related to the larger preference showed by enzymes of the CD clan for charged residues at position P1. This mechanism would avoid the free energy penalty associated with the formation of an ion pair. Finally, our structural description of the reaction process can be useful to assist in the design of inhibitors of caspase-1, a target in the treatment of several human diseases.
... The presented model for protein dynamics (Fig. 6) is very different from previous textbook descriptions of enzyme catalysis that are primarily focused on static enzyme structures and are rooted in the principle of "enhanced transition state binding" (97,98). Although this particular study is focused on an enzyme reaction that proceeds via deep hydrogen tunneling, a combination of distributed protein conformational substates and embedded thermal networks within a protein scaffold is likely to play a key physical role in enzymatic rate enhancements independent of the reaction catalyzed (99) The dominant role of the protein scaffold has been visualized through a combination of conformational selection (Fig. 6A) and subsequent conformational sampling that occurs subsequent to formation of the ES complex (Fig. 6B). ...
The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen-deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.
... It was Henry Eyring (1901À1981) who developed the transition state theory soon thereafter [60] almost simultaneously with Meredith Gwynne Evans (1904À52) and Michael Polanyi (1891À1976) [61]. Linus Carl Pauling (1901À94) did suggest during 1946À48 that transition state formation powers the enzyme catalysis [62,63], but according to Kraut, his ideas did not get traction immediately in the context of how enzymes work [58]. Kurtz in 1963 developed the theoretical framework and William Platt Jencks (1927À2007) around 1966 advanced the idea of transition state-analog inhibitors [64À66]. ...
The modern view of enzymes as complex biological machines with unique structures, complex catalytic mechanisms, and specific conformational dynamics and the wide spread of their industrial and pharmaceutical applications are the result of extensive efforts of a big army of scientists with very different backgrounds. In this chapter, we provide a brief admittedly subjective overview of the most important steps that led to the current understanding of the enzyme structure and function. We describe developments in applied enzymology and white biotechnology, talk about enzymes in neat solvents, and also describe some common myths about applications of enzymes.
... According to Linus Pauling [99] who wrote, "Enzymes are molecules that are complementary in structure to the activated complexes of the reactions that they catalyze, [rather than] entering into reactions." This model came with impact in drug design. ...
Compounds containing an S−F bond have garnered intense interest in the chemical and biological literature. In particular, sulfonyl fluorides (RSO2F) are commonly used as covalent protein inhibitors and biological probes. The introduction of the fluorine atom into drugs often leads to significantly promoted medicinal properties, which revolutionized the development of pharmaceuticals and gained attention because of the beneficial properties of these small and highly electro-negative halogens. The sulfonyl fluoride functional group has also been widely adopted throughout the field of chemical biology due to its unique balance between reactivity and stability under physiological conditions. This comprehensive review highlights the recent developments of sulfonyl fluorides based compounds in a massive range of therapeutic applications. We believe this review article will be helpful to inspire new ideas for structural design and development of less toxic and potent Sulfur based drugs against the numerous death-causing diseases.
... For instance, desaturated hemoglobin changes conformation when binding with oxygen, favoring affinity with further oxygen atoms and allowing to deliver oxygen when the difference in partial pressure would be insufficient without this change in conformation. Again, "the configuration of antibody molecules is very closely complementary to that of the surface of the homologous antigen"[Pau48]. In particular biological macromolecules with variable conformations are the key elements in the regulation of the activity of organisms[Mon65].Biological macromolecules can also play a role as envelope for certain organisms. ...
A new application for a laser-cooled trapped ion ensemble is proposed for the non-destructive detection of giant molecules. In this application a molecular ion is injected into a Ca+ ion ensemble. The detection signal is expected in the fluorescence of the ion set which may vary after the perturbation induced by the molecule. In this manuscript, the necessary elements for the design and handling of a prototype are presented, as well as experimental and numerical results to validate the concept and to study the operating conditions. The thesis is organised in three parts. The first part presents the fundamental aspects of ion trapping and laser cooling, with an emphasis on the non-adiabatic case the behaviour of more than one ions. Experimental principles and results are also exhibited. The second part deals with the fundamental and experimental aspects of the electrospray ionisation molecular source and its attached guiding elements for charged particle. This part is the opportunity to sum up the recent advances in similar systems. The third part presents the molecular dynamics simulations conducted in order to numerically reproduce the interaction as well as to study the behaviour of the trapped ions. With the simulations, we highlight the key roles of the Coulomb interaction combined with radio-frequency heating in the cloud destabilisation mechanism leading to detection. The conditions favouring detection are studied, as well as certain aspects of ion trapping such as radio-frequency heating.
The observation of multiple conformations of a functional loop (termed M20) in the Escherichia coli dihydrofolate reductase (ecDHFR) enzyme triggered the proposition that large-scale motions of protein structural elements contribute to enzyme catalysis. The transition of the M20 loop from a closed conformation to an occluded conformation was thought to aid the rate-limiting release of the products. However, the influence of charged species in the solution environment on the observed M20 loop conformations, independent of charged ligands bound to the enzyme, had not been considered. Molecular dynamics simulations of ecDHFR in model CaCl2 solutions of varying molar ionic strengths IM reveal a substantial free energy barrier between occluded and closed M20 loop states at IM exceeding the E. coli threshold (∼0.24 M). This barrier may facilitate crystallization of ecDHFR in the occluded state, consistent with ecDHFR structures obtained at IM exceeding 0.3 M. At lower IM (≤0.15 M), the M20 loop can explore the occluded state, but prefers an open/partially closed conformation, again consistent with ecDHFR structures. Our findings caution against using ecDHFR structures obtained at nonphysiological ionic strengths in interpreting catalytic events or in structure-based drug design.
Enzymes can be optimized to accelerate chemical transformations via a range of methods. In this review, we showcase how protein engineering and computational design techniques can be interfaced to develop highly efficient and selective biocatalysts.
The self‐assembled metal‐organic cages (MOCs) have been evolved as a paradigm of enzyme‐mimic catalysts since they are able to synergize multifunctionalities inherent in metal and organic components and constitute microenvironments characteristic of enzymatic spatial confinement and versatile host‐guest interactions, thus facilitating unconventional organic transformations via unique driving‐forces such as weak noncovalent binding and electron/energy transfer. Recently, MOC‐based photoreactors emerged as a burgeoning platform of supramolecular photocatalysis, displaying anomalous reactivities and selectivities distinct from bulk solution. This perspective recaps two decades journey of the photoinduced radical reactions by using photoactive metal‐organic cages (PMOCs) as artificial reactors, outlining how the cage‐confined photocatalysis was evolved from stoichiometric photoreactions to photocatalytic turnover, from high‐energy UV‐irradiation to sustainable visible‐light photoactivation, and from simple radical reactions to multi‐level chemo‐ and stereoselectivities. We will focus on PMOCs that merge structural and functional biomimicry into a single‐cage to behave as multi‐role photoreactors, emphasizing their potentials in tackling current challenges in organic transformations through single‐electron transfer (SET) or energy transfer (EnT) pathways in a simple, green while feasible manner.
12 Favored biological characteristics can evolve by a subtle path, but beneficial selection has 13 predictable qualities to guide thought. These favored pathways are paths of "least selection". 14 Faster evolution is least selection, more probable because earlier evolutionary success is simply, 15 "success". A more likely path also requires least selection in the form of least selected change. 16 Truncation selections, accepting only extreme values of a distributed quantity, produce greater 17 change in population means. Truncation selection therefore readily offers a least selection. 18
In 1965, Woodward and Hoffmann proposed a theory to predict the stereochemistry of electrocyclic reactions, which, after expansion and generalization, became known as the Woodward–Hoffmann Rules. Subsequently, Longuet-Higgins and Abrahamson used correlation diagrams to propose that the stereoselectivity of electrocyclizations could be explained by the correlation of reactant and product orbitals with the same symmetry. Immediately thereafter, Hoffmann and Woodward applied correlation diagrams to explain the mechanism of cycloadditions. We describe these discoveries and their evolution. We now report an investigation of various electrocyclic reactions using DFT and CASSCF. We track the frontier molecular orbitals along the intrinsic reaction coordinate and modeled trajectories and examine the correlation between HOMO and LUMO for thermally forbidden systems. We also investigate the electrocyclizations of several highly polarized systems for which the Houk group had predicted that donor–acceptor substitution can induce zwitterionic character, thereby providing low-energy pathways for formally forbidden reactions. We conclude with perspectives on the field of pericyclic reactions, including a refinement as the meaning of Woodward and Hoffmann’s “Violations. There are none!” Lastly, we comment on the burgeoning influence of computations on all fields of chemistry.
Synthetic chemistry has traditionally relied on reactions between reactants of high chemical potential and transformations that proceed energetically downhill to either a global or local minimum (thermodynamic or kinetic control). Catalysts can be used to manipulate kinetic control, lowering activation energies to influence reaction outcomes. However, such chemistry is still constrained by the shape of one-dimensional reaction coordinates. Coupling synthesis to an orthogonal energy input can allow ratcheting of chemical reaction outcomes, reminiscent of the ways that molecular machines ratchet random thermal motion to bias conformational dynamics. This fundamentally distinct approach to synthesis allows multi-dimensional potential energy surfaces to be navigated, enabling reaction outcomes that cannot be achieved under conventional kinetic or thermodynamic control. In this Review, we discuss how ratcheted synthesis is ubiquitous throughout biology and consider how chemists might harness ratchet mechanisms to accelerate catalysis, drive chemical reactions uphill and programme complex reaction sequences.
Many of the highest priority targets in a wide range of disease states are difficult‐to‐drug proteins. The development of reversible small molecule inhibitors for the active sites of these proteins with sufficient affinity and residence time on‐target is an enormous challenge. This has engendered interest in strategies to increase the potency of a given protein inhibitor by routes other than further improvement is gross affinity. Amongst these, the development of catalytic protein inhibitors has garnered the most attention and investment, particularly with respect to protein degraders, which catalyze the destruction of the target protein. This article discusses the genesis of the burgeoning field of catalytic inhibitors, the current state of the art and exciting future directions.
London dispersion (LD) interactions are the main contribution of the attractive part of the van der Waals potential. Even though LD effects are the driving force for molecular aggregation and recognition, the role of these omnipresent interactions in structure and reactivity had been largely underappreciated over decades. However, in the recent years considerable efforts have been made to thoroughly study LD interactions and their potential as a chemical design element for structures and catalysis. This was made possible through a fruitful interplay of theory and experiment. This review highlights recent results and advances in utilizing LD interactions as a structural motif to understand and utilize intra‐ and intermolecularly LD‐stabilized systems. Additionally, we focus on the quantification of LD interactions and their fundamental role in chemical reactions.
Propose Information Systems Biology as a model of systems biology in which information is a primary organizing principle and biosystems are considered as multiscalar entities of information creation, representation, and transfer. Discuss AdS/Biology and Chern-Simons Biology as models for testing the application of physics findings to biophysics in the context of complex multiscalar biosystems, genomic medicine, and Alzheimer’s Disease.
Magnetism plays a pivotal role in many biological systems. However, the intensity of the magnetic forces exerted between magnetic bodies is usually low, which demands the development of ultra-sensitivity tools for proper sensing. In this framework, magnetic force microscopy (MFM) offers excellent lateral resolution and the possibility of conducting single-molecule studies like other single-probe microscopy (SPM) techniques. This comprehensive review attempts to describe the paramount importance of magnetic forces for biological applications by highlighting MFM’s main advantages but also intrinsic limitations. While the working principles are described in depth, the article also focuses on novel micro- and nanofabrication procedures for MFM tips, which enhance the magnetic response signal of tested biomaterials compared to commercial nanoprobes. This work also depicts some relevant examples where MFM can quantitatively assess the magnetic performance of nanomaterials involved in biological systems, including magnetotactic bacteria, cryptochrome flavoproteins, and magnetic nanoparticles that can interact with animal tissues. Additionally, the most promising perspectives in this field are highlighted to make the reader aware of upcoming challenges when aiming toward quantum technologies.
Directed evolution has transformed protein engineering offering a path to rapid improvement of protein properties. Yet, in practice it is limited by the hyper-astronomic protein sequence search space, and approaches to identify mutagenic hot spots, i.e., locations where mutations are most likely to have a productive impact, are needed. In this perspective, we categorize and discuss recent progress in the experimental approaches (broadly defined as structural, bioinformatic, and dynamic) to hot spot identification. Recent successes in harnessing protein dynamics and machine learning approaches provide new opportunities for the field and will undoubtedly help directed evolution reach its full potential.
A survey of protein databases indicates that the majority of enzymes exist in oligomeric forms, with about half of those found in the UniProt database being homodimeric. Understanding why many enzymes are in their dimeric form is imperative. Recent developments in experimental and computational techniques have allowed for a deeper comprehension of the cooperative interactions between the subunits of dimeric enzymes. This review aims to succinctly summarize these recent advancements by providing an overview of experimental and theoretical methods, as well as an understanding of cooperativity in substrate binding and the molecular mechanisms of cooperative catalysis within homodimeric enzymes. Focus is set upon the beneficial effects of dimerization and cooperative catalysis. These advancements not only provide essential case studies and theoretical support for comprehending dimeric enzyme catalysis but also serve as a foundation for designing highly efficient catalysts, such as dimeric organic catalysts. Moreover, these developments have significant implications for drug design, as exemplified by Paxlovid, which was designed for the homodimeric main protease of SARS-CoV-2.
The pressure to optimize enzymatic rate accelerations has driven the evolution of the induced-fit mechanism for enzyme catalysts where the binding interactions of nonreacting phosphodianion or adenosyl substrate pieces drive enzyme conformational changes to form protein substrate cages that are activated for catalysis. We report the results of experiments to test the hypothesis that utilization of the binding energy of the adenosine 5'-diphosphate ribose (ADP-ribose) fragment of the NAD cofactor to drive a protein conformational change activates Candida boidinii formate dehydrogenase (CbFDH) for catalysis of hydride transfer from formate to NAD+. The ADP-ribose fragment provides a >14 kcal/mol stabilization of the transition state for CbFDH-catalyzed hydride transfer from formate to NAD+. This is larger than the ca. 6 kcal/mol stabilization of the ground-state Michaelis complex between CbFDH and NAD+ (KNAD = 0.032 mM). The ADP, AMP, and ribose 5'-phosphate fragments of NAD+ activate CbFDH for catalysis of hydride transfer from formate to nicotinamide riboside (NR). At a 1.0 M standard state, these activators stabilize the hydride transfer transition states by ≈5.5 (ADP), 5.5 (AMP), and 4.4 (ribose 5'-phosphate) kcal/mol. We propose that activation by these cofactor fragments is partly or entirely due to the ion-pair interaction between the guanidino side chain cation of R174 and the activator phosphate anion. This substitutes for the interaction between the α-adenosyl pyrophosphate anion of the whole NAD+ cofactor that holds CbFDH in the catalytically active closed conformation.
The environmental and health hazards created by industrial chemicals and consumer products must be minimized. For safer products to be designed, the relationships between structure and toxicity must be understood at the molecular level. Green chemistry combined with free radical research has the potential to offer innovative solutions to such problems.
Some solutions are "greener then others", and many necessitate significant financial investment. New technology will only be adopted if real benefit can be shown and sometimes adaptation of existing methods is the best option. The efficiency of processes must be assessed, not only in terms of the final yield, but also cost, environmental impact and waste toxicity.
This practical and concise guide showcases the sustainable methods offered by green free radical chemistry and summarizes the fundamental science involved. It discusses the pros and cons of free radical chemistry in aqueous systems for synthetic applications. All transformation steps are covered including initiation, propagation, and termination. Useful background knowledge is combined with examples, including industrial scale processes for pharmaceuticals and fine chemicals.
The book helps chemists to choose appropriate methods for achieving maximum output using a modern, environmentally conscious approach. It shows that, armed with an elementary knowledge of kinetics, an understanding of the mechanistic and technical aspects, and some common sense, it is possible to harness free radicals for use in a broad range of applications.
Streamlining Green Free Radical Chemistry is aimed at chemists, engineers, materials scientists, biochemists and biomedical experts, as well as undergraduate and postgraduate students. It encourages readers to question conventional methods and move towards the "Benign-by-Design" approach of the future. References to further reading are provided at the end of each chapter.
The most important difference between enzyme and small molecule catalysts is that only enzymes utilize the large intrinsic binding energies of nonreacting portions of the substrate in stabilization of the transition state for the catalyzed reaction. A general protocol is described to determine the intrinsic phosphodianion binding energy for enzymatic catalysis of reactions of phosphate monoester substrates, and the intrinsic phosphite dianion binding energy in activation of enzymes for catalysis of phosphodianion truncated substrates, from the kinetic parameters for enzyme-catalyzed reactions of whole and truncated substrates. The enzyme-catalyzed reactions so-far documented that utilize dianion binding interactions for enzyme activation; and, their phosphodianion truncated substrates are summarized. A model for the utilization of dianion binding interactions for enzyme activation is described. The methods for the determination of the kinetic parameters for enzyme-catalyzed reactions of whole and truncated substrates, from initial velocity data, are described and illustrated by graphical plots of kinetic data. The results of studies on the effect of site-directed amino acid substitutions at orotidine 5'-monophosphate decarboxylase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase provide strong support for the proposal that these enzymes utilize binding interactions with the substrate phosphodianion to hold the protein catalysts in reactive closed conformations.
This chapter explores the chemical nature of enzyme catalysis. It examines that both substrate specificity and rate acceleration result from the precise three‐dimensional structure of the substrate binding pocket within the enzyme molecule, known as the active site . The transition state stabilization associated with enzyme catalysis is the result of the structure and reactivity of the enzyme active site, and how these structural features interact with the bound substrate molecule. Enzymes use numerous detailed chemical mechanisms to achieve transition state stabilization and the resulting reaction rate acceleration. These can be grouped into five major categories: approximation of reactants, covalent catalysis, general acid–base catalysis, conformational distortion, and preorganization of the active site for transition state complementarity. The serine proteases are a family of enzymes that catalyze the cleavage of specific peptide bonds in proteins and peptides.
Mandelate racemase (MR) catalyses the Mg ²⁺ -dependent interconversion of ( R )- and ( S )-mandelate. To effect catalysis, MR stabilizes the altered substrate in the transition state (TS) by approximately 26 kcal mol –1 (–Δ G tx ), such that the upper limit of the virtual dissociation constant of the enzyme-TS complex is 2 × 10 –19 M. Designing TS analogue inhibitors that capture a significant amount of Δ G tx for binding presents a challenge since there are a limited number of protein binding determinants that interact with the substrate and the structural simplicity of mandelate constrains the number of possible isostructural variations. Indeed, current intermediate/TS analogue inhibitors of MR capture less than or equal to 30% of Δ G tx because they fail to fully capitalize on electrostatic interactions with the metal ion, and the strength and number of all available electrostatic and H-bond interactions with binding determinants present at the TS. Surprisingly, phenylboronic acid (PBA), 2-formyl-PBA, and para -chloro-PBA capture 31–38% of Δ G tx . The boronic acid group interacts with the Mg ²⁺ ion and multiple binding determinants that effect TS stabilization. Inhibitors capable of forming multiple interactions can exploit the cooperative interactions that contribute to optimum binding of the TS. Hence, maximizing interactions with multiple binding determinants is integral to effective TS analogue inhibitor design.
This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.
In order to efficiently assess mutant libraries encompassing genetic diversification, screening, and/or selection methods are highly required in directed enzyme evolution. High‐throughput gas chromatography (GC)‐ or high‐performance liquid chromatography (HPLC)‐based screening techniques are routinely employed. Growth‐coupled, UV/vis/fluorescence‐based, or molecular probe‐dependent screening systems can also be chosen based on the signals of enzyme‐catalysis. Exerting growth selection pressure on host strains, including antibiotic resistance, auxotrophy, or detoxification, is sometimes possible. This chapter summarizes critically the frequently‐used screening and selection systems and documents their applications, particularly when attempting to alter stereo‐ and/or regio‐selectivity.
Learning lessons from basic research is not only a philosophical issue; it also has practical ramifications. This also applies especially to directed evolution of stereo‐ and/or regio‐selective enzymes that are used in synthetic organic and pharmaceutical chemistry, where two different types of lessons are learned: (i) Unraveling the reasons for altered catalytic profiles leads to knowledge of hitherto unnoticed mechanistic intricacies, which in turn aids subsequent protein engineering for further improvements, enabling practical applications. (ii) Discovering that the best mutagenesis methods and strategies generate multi‐mutational variants in which the effects of point mutations and sets of point mutations are more than additive (cooperative). This in turn speeds up the development of efficient protein engineering in directed evolution and in rational enzyme design and suggests further theoretical investigations.
The traditional approaches used in directed evolution, including mutator strains, error‐prone polymerase chain reaction, gene insertion and deletion, saturation mutagenesis, DNA shuffling and related recombinant gene mutagenesis, circular mutation and domain swapping, as well as solid‐phase gene synthesis techniques, are presented critically in this chapter. Computational tools and approaches, as well as machine learning algorithms established in rational enzyme design, are also described. As outlined in Section 3.18 of this chapter, the techniques in directed evolution and rational design no longer develop on separate tracks but have merged recently with the construction of powerful techniques such as Focused Rational Iterative Site‐specific Mutagenesis (FRISM).
Polymeric mesoporous monoliths were prepared via ring-opening metathesis polymerization (ROMP) from norbornene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene (DMN-H6), tris(norborn-2-enylmethylenoxy)methylsilane and the 1st-generation Grubbs catalyst [RuCl2(PCy3)2(CHC6H5)] in the presence of 2-propanol and toluene and...
The cytoplasm is an environment crowded by macromolecules and filled with metabolites and ions. Recent experimental and computational studies have addressed how this environment affects protein stability, folding kinetics, and protein-protein and protein-nucleic acid interactions, though its impact on metabolites remains largely unknown. Here we show how a simulated cytoplasm affects the conformation of adenosine triphosphate (ATP), a key energy source and regulatory metabolite present at high concentrations in cells. Analysis of our all-atom model of a small volume of the Escherichia coli cytoplasm when contrasted with ATP modeled in vitro or resolved with protein structures deposited in the Protein Data Bank reveals that ATP molecules bound to proteins in cell form specific pitched conformations that are not observed at significant concentrations in the other environments. We hypothesize that these interactions evolved to fulfill functional roles when ATP interacts with protein surfaces.
How life originated from the inanimate mixture of organic and inorganic compounds on the priomordial earth remains one of the great unknowns in science. This origin of life, or abiogenesis, continues to be examined in the context of the conditions and materials required for natural life to have begun on Earth both theoretically and experimentally. This book provides a broad but in-depth analysis of the latest discoveries in prebiotic chemsitry from the microscopic to the macroscopic scale; utilising experimental insight to provide a bottom up approach to plausibly explaining how life arose. With contributions from global leaders, this book is an ideal reference for postgraduate students and a single source of comprehensive information on the latest technical and theoretical advancements for researchers in a variety of fields from astrochemistry and astrophysics to organic chemistry and evolution.
Heterogeneous asymmetric catalysis, with the advantages of easy separation and recycling of the catalyst, easy product purification, and possible continuous-flow operation, has potential application prospects in fine chemical and pharmaceutical industries. Heterogeneous asymmetric catalysis is an intersection of homogeneous catalysis, heterogeneous catalysis, biocatalysis, and materials science. Therefore, the importance of interdisciplinary concepts from these fields in developing efficient heterogeneous asymmetric catalysts is increasingly recognized. Recently, concepts and strategies borrowed from other fields, such as synergistic activation, substrate activation, and Pickering emulsion interfacial catalysis, have been used for heterogeneous asymmetric catalysis. This perspective summarizes recent progress in the development of heterogeneous asymmetric catalysts with crossover concepts and provides an overview for future development.
It is not a coincidence that both chirality and noncovalent interactions are ubiquitous in nature and synthetic molecular systems. Noncovalent interactivity between chiral molecules underlies enantioselective recognition as a fundamental phenomenon
regulating life and human activities. Thus, noncovalent interactions represent the narrative
thread of a fascinating story which goes across several disciplines of medical, chemical,
physical, biological, and other natural sciences. This review has been conceived with the
awareness that a modern attitude toward molecular chirality and its consequences needs to be founded on multidisciplinary approaches to disclose the molecular basis of essential enantioselective phenomena in the domain of chemical, physical, and life sciences. With the primary aim of discussing this topic in an integrated way, a comprehensive pool of rational and systematic multidisciplinary information is provided, which concerns the fundamentals of chirality, a description of noncovalent interactions, and their implications in enantioselective processes occurring in different contexts. A specific focus is devoted to enantioselection in chromatography and electromigration techniques because of their unique feature as “multistep” processes. A second motivation for writing this review is to make a clear statement about the state of the art, the tools we have at our disposal, and what is still missing to fully understand the mechanisms underlying enantioselective recognition.
With the growing acceptance of the contribution of protein conformational ensembles to enzyme catalysis and regulation, research in the field of protein dynamics has shifted toward an understanding of the atomistic properties of protein dynamical networks and the mechanisms and time scales that control such behavior. A full description of an enzymatic reaction coordinate is expected to extend beyond the active site and include site-specific networks that communicate with the protein/water interface. Advances in experimental tools for the spatial resolution of thermal activation pathways are being complemented by biophysical methods for visualizing dynamics in real time. An emerging multidimensional model integrates the impacts of bound substrate/effector on the distribution of protein substates that are in rapid equilibration near room temperature with reaction-specific protein embedded heat transfer conduits.
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