Impact of Static-Oriented Electric Fields on the Kinetics of Some Representative Suzuki-Miyaura and Metal-Cluster Mediated Reactions.

In order to examine the effect of oriented (static) electric fields (OEF) on the kinetics of some representative Suzuki-Miyaura and metal-cluster mediated reactions at ambient temperatures, density functional theory-based calculations are reported herein. Results indicate that, in general, OEF can facilitate the kinetics of the concerned reactions when applied along the suitable direction (parallel or anti-parallel with respect to the reaction axis). The reverse effect happens if the direction of the OEF is flipped. OEF (when applied along the 'right' direction) helps to polarize the transition states in the desired direction, thereby facilitating favorable bonding interactions. Given the growing need for finding appropriate catalysts among the scientific community, OEF can prove to be a vital route for the same.

Supramolecular Strategies for Controlling Reactivity within Confined Nanospaces.

Nanospaces are ubiquitous in the realm of biological systems and are of significant interest among supramolecular chemists. Understanding chemical behavior within nanospaces offers new perspectives on biological phenomena in nature and opens the way to highly unusual and selective forms of catalysis. Supramolecular chemistry exploits weak, yet effective, intermolecular interactions such as hydrogen bonding, metal-ligand coordination, and the hydrophobic effect to assemble nano-sized molecular architectures, providing reactions with remarkable rate acceleration, substrate specificity, and product selectivity. In this minireview, the focus is on the strategies that supramolecular chemists use to emulate the efficiency of biological processes, and elucidating how chemical reactivity is efficiently controlled within well-defined nanospaces. Approaches such as orientation and proximity of substrate, transition-state stabilization, and active-site incorporation will be discussed.

Evolution of a Catalytic Mechanism.

The means by which superfamilies of specialized enzymes arise by gene duplication and functional divergence are poorly understood. The escape from adaptive conflict hypothesis, which posits multiple copies of a gene encoding a primitive inefficient and highly promiscuous generalist ancestor, receives support from experiments showing that resurrected ancestral enzymes are indeed more substrate-promiscuous than their modern descendants. Here, we provide evidence in support of an alternative model, the innovation-amplification-divergence hypothesis, which posits a single-copied ancestor as efficient and specific as any modern enzyme. We argue that the catalytic mechanisms of plant esterases and descendent acetone cyanohydrin lyases are incompatible with each other (e.g., the reactive substrate carbonyl must bind in opposite orientations in the active site). We then show that resurrected ancestral plant esterases are as catalytically specific as modern esterases, that the ancestor of modern acetone cyanohydrin lyases was itself only very weakly promiscuous, and that improvements in lyase activity came at the expense of esterase activity. These observations support the innovation-amplification-divergence hypothesis, in which an ancestor gains a weak promiscuous activity that is improved by selection at the expense of the ancestral activity, and not the escape from adaptive conflict in which an inefficient generalist ancestral enzyme steadily loses promiscuity throughout the transition to a highly active specialized modern enzyme.

Computational perspective and evaluation of plausible catalytic mechanisms of peptidyl-prolyl cis-trans isomerases.

BACKGROUND: Peptidyl prolyl cis-trans isomerization of the protein backbone is involved in the regulation of many biological processes. Cis-trans isomerization is notoriously slow and is catalyzed by a family of cis-trans peptidyl prolyl isomerases (PPIases) that have been implicated in many diseases. A general consensus on how these enzymes speed up prolyl isomerization has not been reached after decades of both experimental and computational studies. SCOPE OF REVIEW: Computational studies carried out to understand the catalytic mechanism of the prototypical FK506 binding protein 12, Cyclophilin A and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) are reviewed. A summary and an evaluation of the implications of the proposed mechanisms from computational studies are presented. MAJOR CONCLUSIONS: The analysis of computational studies and evaluation of the proposed mechanisms provide a general consensus and a better understanding of PPIase catalysis. The speedup of the rate of peptidyl-prolyl isomerization by PPIases can be best described by a catalytic mechanism in which the substrate in transition state configuration is stabilized. The enzymes preferentially bind the transition state configuration of the substrate relative to the cis conformation, which in most cases is bound better than the trans conformation of the substrate. Stabilization of the transition state configuration of the substrate leads to a lower free energy barrier and a faster rate of isomerization when compared to the uncatalyzed isomerization reaction. GENERAL SIGNIFICANCE: Fully understanding the catalytic mechanism of PPIases has broad implications for drug design, elucidation of the molecular basis of many diseases, protein engineering, and enzyme catalysis in general. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Thermodynamic framework for identifying free energy inventories of enzyme catalytic cycles.

Pauling's suggestion that enzymes are complementary in structure to the activated complexes of the reactions they catalyze has provided the conceptual basis to explain how enzymes obtain their fantastic catalytic prowess, and has served as a guiding principle in drug design for over 50 y. However, this model by itself fails to predict the magnitude of enzymes' rate accelerations. We construct a thermodynamic framework that begins with the classic concept of differential binding but invokes additional terms that are needed to account for subtle effects in the catalytic cycle's proton inventory. Although the model presented can be applied generally, this analysis focuses on ketosteroid isomerase (KSI) as an example, where recent experiments along with a large body of kinetic and thermodynamic data have provided strong support for the noncanonical thermodynamic contribution described. The resulting analysis precisely predicts the free energy barrier of KSI's reaction as determined from transition-state theory using only empirical thermodynamic data. This agreement is suggestive that a complete free energy inventory of the KSI catalytic cycle has been identified.

Transition State Stabilization by SCF2 -H⋠⋠⋠O Bifurcated Hydrogen Bond.

The difluoromethylthio group (SCF2 H), which is generally considered a highly lipophilic weak hydrogen bonding donor, has attracted special interest from the pharmaceutical and agrochemical industry. Remarkably, there have been relatively few literature investigations of SCF2 H hydrogen bonding interactions. Here, we report the determination of the hydrogen bond acidity parameter A of the SCF2 H in the most popularly used electrophilic difluoromethylthiolating reagent. We present kinetic and computational evidence of the RSCF2 -H⋅⋅⋅O2 bifurcated hydrogen bond for stabilizing the SCF2 H-transferring transition state, which could cause a reversal of apparent electrophilic reactivity of difluoromethylthiolating and trifluoromethylthiolating reagents. Solvent effects on the RSCF2 -H⋅⋅⋅O2 bifurcated hydrogen bonds will also be discussed.

Solvent and solvation effects on reactivities and mechanisms of phospho group transfers from phosphate and phosphinate esters to nucleophiles.

Organophosphorus esters fulfil many industrial, agricultural, and household roles. Nature has deployed phosphates and their related anhydrides as energy carriers and reservoirs, as constituents of genetic materials in the form of DNA and RNA, and as intermediates in key biochemical conversions. The transfer of the phosphoryl (PO3) group is thus a ubiquitous biological process that is involved in a variety of transformations at the cellular level such as bioenergy and signals transductions. Significant attention has been paid in the last seven decades to understanding the mechanisms of uncatalyzed (solution) chemistry of the phospho group transfer because of the notion that enzymes convert the dissociative transition state structures in the uncatalyzed reactions into associative ones in the biological processes. In this regard, it has also been proposed that the rate enhancements enacted by enzymes result from the desolvation of the ground state in the hydrophobic active site environments, although theoretical calculations seem to disagree with this position. As a result, some attention has been paid to the study of the effects of solvent change, from water to less polar solvents, in uncatalyzed phospho transfer reactions. Such changes have consequences on the stabilities of the ground and the transition states of reactions which affect reactivities and, sometimes, the mechanisms of reactions. This review seeks to collate and evaluate what is known about solvent effects in this domain, especially their effects on rates of reactions of different classes of organophosphorus esters. The outcome of this exercise shows that a systematized study of solvent effects needs to be undertaken to fully understand the physical organic chemistry of the transfer of phosphates and related molecules from aqueous to substantially hydrophobic environments, since significant knowledge gaps exist.

Capturing the free energy of transition state stabilization: insights from the inhibition of mandelate racemase.

Mandelate racemase (MR) catalyses the Mg2+-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 (-ΔGtx), 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 ΔGtx 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 ΔGtx 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 ΔGtx. The boronic acid group interacts with the Mg2+ 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'.

Reciprocally-Coupled Gating: Strange Loops in Bioenergetics, Genetics, and Catalysis.

Bioenergetics, genetic coding, and catalysis are all difficult to imagine emerging without pre-existing historical context. That context is often posed as a "Chicken and Egg" problem; its resolution is concisely described by de Grasse Tyson: "The egg was laid by a bird that was not a chicken". The concision and generality of that answer furnish no details-only an appropriate framework from which to examine detailed paradigms that might illuminate paradoxes underlying these three life-defining biomolecular processes. We examine experimental aspects here of five examples that all conform to the same paradigm. In each example, a paradox is resolved by coupling "if, and only if" conditions for reciprocal transitions between levels, such that the consequent of the first test is the antecedent for the second. Each condition thus restricts fluxes through, or "gates" the other. Reciprocally-coupled gating, in which two gated processes constrain one another, is self-referential, hence maps onto the formal structure of "strange loops". That mapping uncovers two different kinds of forces that may help unite the axioms underlying three phenomena that distinguish biology from chemistry. As a physical analog for Gödel's logic, biomolecular strange-loops provide a natural metaphor around which to organize a large body of experimental data, linking biology to information, free energy, and the second law of thermodynamics.

GH47 and Other Glycoside Hydrolases Catalyze Glycosidic Bond Cleavage with the Assistance of Substrate Super-arming at the Transition State.

Super-armed glycosyl donors, whose substituents are predominantly held in pseudoaxial positions, exhibit strongly increased reactivity in glycosylation through significant stabilization of oxocarbenium-like transition states. Examination of X-ray crystal structures reveals that the GH47 family of glycoside hydrolases have evolved so as to distort their substrates away from the ground state conformation in such a manner as to present multiple C-O bonds in pseudoaxial positions and so benefit from conformational super-arming of their substrates, thereby enhancing catalysis. Through analysis of literature mutagenic studies, we show that a suitably placed aromatic residue in GHs 6 and 47 sterically enforces super-armed conformations on their substrates. GH families 45, 81, and 134 on the other hand impose conformational super-arming on their substrates, by maintaining the more active ring conformation through hydrogen bonding rather than steric interactions. The recognition of substrate super-arming by select GH families provides a further parallel with synthetic carbohydrate chemistry and nature and opens further avenues for the design of improved glycosidase inhibitors.

Exploring the Development of Ground-State Destabilization and Transition-State Stabilization in Two Directed Evolution Paths of Kemp Eliminases.

Computer-aided enzyme design presents a major challenge since in most cases it has not resulted in an impressive catalytic power. The reasons for the problems with computational design include the use of nonquantitative approaches, but they may also reflect other difficulties that are not completely obvious. Thus, it is very useful to try to learn from the trend in directed evolution experiments. Here we explore the nature of the refinement of Kemp eliminases by directed evolution, trying to gain an understanding of related requirements from computational design. The observed trend in the directed evolution refinement of KE07 and HG3 are reproduced, showing that in the case of KE07 the directed evolution leads to ground-state destabilization, whereas in the case of HG3 the directed evolution leads to transition-state stabilization. The nature of the different paths of the directed evolution is examined and discussed. The present study seems to indicate that computer-aided enzyme design may require more than calculations of the effect of single mutations and should be extended to calculations of the effect of simultaneous multiple mutations (that make a few residues preorganized effectively). However, the analysis of two known evolution paths can still be accomplished using the relevant sequences and structures. Thus, by comparing two directed evolution paths of Kemp eliminases we reached the important conclusion that the more effective path leads to transition-state stabilization.

Quantum chemical modeling of the reaction path of chorismate mutase based on the experimental substrate/product complex.

Chorismate mutase is a well-known model enzyme, catalyzing the Claisen rearrangement of chorismate to prephenate. Recent high-resolution crystal structures along the reaction coordinate of this enzyme enabled computational analyses at unprecedented detail. Using quantum chemical simulations, we investigated how the catalytic reaction mechanism is affected by electrostatic and hydrogen-bond interactions. Our calculations showed that the transition state (TS) was mainly stabilized electrostatically, with Arg90 playing the leading role. The effect was augmented by selective hydrogen-bond formation to the TS in the wild-type enzyme, facilitated by a small-scale local induced fit. We further identified a previously underappreciated water molecule, which separates the negative charges during the reaction. The analysis includes the wild-type enzyme and a non-natural enzyme variant, where the catalytic arginine was replaced with an isosteric citrulline residue.

A Paradigm for CH Bond Cleavage: Structural and Functional Aspects of Transition State Stabilization by Mandelate Racemase.

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg2+-dependent, 1,1-proton transfer reaction that racemizes (R)- and (S)-mandelate. MR shares a partial reaction (i.e., the metal ion-assisted, Brønsted base-catalyzed proton abstraction of the α-proton of carboxylic acid substrates) and structural features ((ß/α)7ß-barrel and N-terminal α + ß capping domains) with a vast group of homologous, yet functionally diverse, enzymes in the enolase superfamily. Mechanistic and structural studies have developed this enzyme into a paradigm for understanding how enzymes such as those of the enolase superfamily overcome kinetic and thermodynamic barriers to catalyze the abstraction of an α-proton from a carbon acid substrate with a relatively high pKa value. Structural studies on MR bound to intermediate/transition state analogues have delineated those structural features that MR uses to stabilize transition states and enhance reaction rates of proton abstraction. Kinetic, site-directed mutagenesis, and structural studies have also revealed that the phenyl ring of the substrate migrates through the hydrophobic cavity within the active site during catalysis and that the Brønsted acid-base catalysts (Lys 166 and His 297) may be utilized as binding determinants for inhibitor recognition. In addition, structural studies on the adduct formed from the irreversible inhibition of MR by 3-hydroxypyruvate revealed that MR can form and deprotonate a Schiff-base with 3-hydroxypyruvate to yield an enol(ate)-aldehyde adduct, suggesting a possible evolutionary link between MR and the Schiff-base forming aldolases. As the archetype of the enolase superfamily, mechanistic and structural studies on MR will continue to enhance our understanding of enzyme catalysis and furnish insights into the evolution of enzyme function.

Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation.

Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.