Understanding Of Enzyme Catalysis Research Articles

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat (ΔH‡\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\Delta {{{{{\\rm{H}}}}}}}^{{{\\ddagger}} }$$\\end{document} = 54 kJ mol−1), significant loss in entropy (−TΔS‡\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$-T{\\Delta {{{{{\\rm{S}}}}}}}^{{{\\ddagger}} }$$\\end{document} = 20 kJ mol−1; 298 K) and negative activation heat capacity (ΔCp‡\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\Delta {{{{{\\rm{C}}}}}}}_{{{{{{\\rm{p}}}}}}}^{{{\\ddagger}} }$$\\end{document} = −0.64 kJ mol−1 K−1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

A unifying electrostatic basis for designing enzymes faster than natural ones.

Rationally designing proteins that outperform natural enzymes and de novo design of enzymes remain grand challenges after a decades-long effort. Achieving this goal would be a significant test for our understanding of enzyme catalysis and take us beyond the blackbox of directed evolution. In the course of studying liver alcohol dehydrogenase, we found that changing serine to threonine (S48T), the hydrogen bond donor at the active site, or replacing Zn2+ with Co2+, the catalytic metal site, both resulted in higher reaction rates of hydride transfer than that of the wild type. Using vibrational Stark effect spectroscopy, an experimental method enabling us to measure the electric field at the enzyme's active site, we found that both Co2+ and S48T mutations increase the active-site electric field, which shows a linear correlation with the reaction's free energy barrier. Given the correlation established by those single-point mutants, we designed double-point mutants and made quantitative predictions for both their field and activity—those predictions were found in close agreement with the experimental results. In particular, the S48T-Co2+ mutant shows a 50-fold increase in the hydride transfer rate compared to the wild type. We therefore demonstrate that the electrostatic interactions not only drive the catalysis of natural enzymes, but also can be leveraged for improving a natural enzyme. Furthermore, the observation that both serine and Zn2+ mutants follow the same linear correlation indicates that, despite their distinct chemical identities, the catalytic effects of hydrogen bonds and metal ions have a common electrostatic nature and can be quantified in a unifying manner. Taken together, we demonstrate electrostatic catalysis as a physical (and thus rational), quantitative, and predictive framework that unifies different intermolecular forces, for designing enzymes beyond the natural ones.

Ensemble-function relationships to connect structure to mechanism: application of EnsemblePDB to the serine protease reaction coordinate and its catalytic features

Classic proposals for catalysis by enzymes focused on their ability to position active site residues and substrates for reaction, yet atomic motions are central to the transformation of substrates to products, and more recent studies have started to reveal the role of dynamics in enzyme function. Understanding enzyme catalysis requires both perspectives: knowing how restricted reactants and catalytic groups are; knowing what motions are coupled to the reaction coordinate; and relating these features to the underlying energetics that describe catalysis.

Chemical Mapping Exposes the Importance of Active Site Interactions in Governing the Temperature Dependence of Enzyme Turnover.

Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, ΔCP‡, can reveal links between the protein free energy landscape, global protein dynamics, and enzyme turnover, suggesting that subtle changes in molecular interactions at the active site can affect long-range protein dynamics and link to enzyme temperature activity. Here, we use a model promiscuous enzyme (glucose dehydrogenase from Sulfolobus solfataricus) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, red edge excitation shift (REES) spectroscopy, and computational simulation, we explore the complex relationship between enzyme–substrate interactions and the global dynamics of the protein. We find that changes in ΔCP‡ and protein dynamics can be mapped to specific substrate–enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.

Enzyme Models-From Catalysis to Prodrugs.

Enzymes are highly specific biological catalysts that accelerate the rate of chemical reactions within the cell. Our knowledge of how enzymes work remains incomplete. Computational methodologies such as molecular mechanics (MM) and quantum mechanical (QM) methods play an important role in elucidating the detailed mechanisms of enzymatic reactions where experimental research measurements are not possible. Theories invoked by a variety of scientists indicate that enzymes work as structural scaffolds that serve to bring together and orient the reactants so that the reaction can proceed with minimum energy. Enzyme models can be utilized for mimicking enzyme catalysis and the development of novel prodrugs. Prodrugs are used to enhance the pharmacokinetics of drugs; classical prodrug approaches focus on alternating the physicochemical properties, while chemical modern approaches are based on the knowledge gained from the chemistry of enzyme models and correlations between experimental and calculated rate values of intramolecular processes (enzyme models). A large number of prodrugs have been designed and developed to improve the effectiveness and pharmacokinetics of commonly used drugs, such as anti-Parkinson (dopamine), antiviral (acyclovir), antimalarial (atovaquone), anticancer (azanucleosides), antifibrinolytic (tranexamic acid), antihyperlipidemia (statins), vasoconstrictors (phenylephrine), antihypertension (atenolol), antibacterial agents (amoxicillin, cephalexin, and cefuroxime axetil), paracetamol, and guaifenesin. This article describes the works done on enzyme models and the computational methods used to understand enzyme catalysis and to help in the development of efficient prodrugs.

Design and SAR Analysis of Covalent Inhibitors Driven by Hybrid QM/MM Simulations.

Quantum mechanics/molecular mechanics (QM/MM) hybrid technique is emerging as a reliable computational method to investigate and characterize chemical reactions occurring in enzymes. From a drug discovery perspective, a thorough understanding of enzyme catalysis appears pivotal to assist the design of inhibitors able to covalently bind one of the residues belonging to the enzyme catalytic machinery. Thanks to the current advances in computer power, and the availability of more efficient algorithms for QM-based simulations, the use of QM/MM methodology is becoming a viable option in the field of covalent inhibitor design. In the present review, we summarized our experience in the field of QM/MM simulations applied to drug design problems which involved the optimization of agents working on two well-known drug targets, namely fatty acid amide hydrolase (FAAH) and epidermal growth factor receptor (EGFR). In this context, QM/MM simulations gave valuable information in terms of geometry (i.e., of transition states and metastable intermediates) and reaction energetics that allowed to correctly predict inhibitor binding orientation and substituent effect on enzyme inhibition. What is more, enzyme reaction modelling with QM/MM provided insights that were translated into the synthesis of new covalent inhibitor featured by a unique combination of intrinsic reactivity, on-target activity, and selectivity.

The role of ligand-gated conformational changes in enzyme catalysis.

Structural and biochemical studies on diverse enzymes have highlighted the importance of ligand-gated conformational changes in enzyme catalysis, where the intrinsic binding energy of the common phosphoryl group of their substrates is used to drive energetically unfavorable conformational changes in catalytic loops, from inactive open to catalytically competent closed conformations. However, computational studies have historically been unable to capture the activating role of these conformational changes. Here, we discuss recent experimental and computational studies, which can remarkably pinpoint the role of ligand-gated conformational changes in enzyme catalysis, even when not modeling the loop dynamics explicitly. Finally, through our joint analyses of these data, we demonstrate how the synergy between theory and experiment is crucial for furthering our understanding of enzyme catalysis.

Expanding the Scope of Functionalized Small Nonprotein Components for Holoabzyme 27C1

AbstractThe creation of artificial enzymes that achieve high selectivity, and turnovers equivalent to natural enzymes remains a challenging aspect of biocatalyst research. The design of new catalytic proteins presents a straightforward test of our understanding of enzyme catalysis. The holoabzyme 27C1 possesses binding pockets for substrates, as well as for a large variety of functionalized small nonprotein components. Site‐directed chemical mutations have optimized the β‐elimination activity of antibody 27C1, resulting in a 106 fold rate enhancement. In this study, we incorporate synthetic catalytic components with different steric properties into the binding site, and investigate the substrate scope of the 27C1‐catalyzed β‐elimination reaction. Our results indicate that the flexibility and tunability of the binding pocket toward synthetic small molecules may enable incorporation of newly devised components, and thus, introduce novel reactivities.

Dynamical origins of heat capacity changes in enzyme-catalysed reactions

Heat capacity changes are emerging as essential for explaining the temperature dependence of enzyme-catalysed reaction rates. This has important implications for enzyme kinetics, thermoadaptation and evolution, but the physical basis of these heat capacity changes is unknown. Here we show by a combination of experiment and simulation, for two quite distinct enzymes (dimeric ketosteroid isomerase and monomeric alpha-glucosidase), that the activation heat capacity change for the catalysed reaction can be predicted through atomistic molecular dynamics simulations. The simulations reveal subtle and surprising underlying dynamical changes: tightening of loops around the active site is observed, along with changes in energetic fluctuations across the whole enzyme including important contributions from oligomeric neighbours and domains distal to the active site. This has general implications for understanding enzyme catalysis and demonstrating a direct connection between functionally important microscopic dynamics and macroscopically measurable quantities.

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Purification, Characterization, and Biochemical Assays of Biotin Synthase From Escherichia coli.

Biotin synthase (BioB) catalyzes the oxidative insertion of a sulfur atom between the C6 methylene and the C9 methyl positions in dethiobiotin. The enzyme couples oxidation of each carbon position to reduction of the S-adenosyl-l-methionine (SAM) sulfonium center, generating 5'-deoxyadenosine and l-methionine, products that are characteristic of enzymes from the radical SAM superfamily. In bacteria, biotin biosynthesis is tightly regulated by the dual-function BirA repressor/holocarboxylase synthetase, resulting in very low levels of all biotin biosynthetic enzymes such that activity-based purification of BioB from the native organism is virtually impossible. However, overexpression and purification of recombinant BioB from E. coli are straight forward and, in contrast with many radical SAM enzymes, can be carried out under aerobic conditions. The active enzyme contains two iron-sulfur clusters, and the characterization and manipulation of these clusters are essential for a thorough understanding of enzyme catalysis and stability. An optimized in vitro assay for BioB is described herein that requires use of an auxiliary protein reducing system and must be carried out under anaerobic conditions to prevent oxidative damage to the reduced iron-sulfur clusters. Three methods for detection of biotin are described, with discussion of the advantages and limitations of each method. Challenges that may be encountered in adapting these assays to other organisms are also discussed.

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 ((β/α)7β-barrel and N-terminal α + β 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.

Whither Enzymology in the Twenty First Century?

This “grand challenge” article marks the creation of a new specialty section shared between Frontiers in Chemistry and Frontiers in Molecular Biology that is dedicated to enzymology and protein chemistry. My aim in this article is to provide a personal perspective on the challenges and opportunities for field of enzymology and the directions in which it could and should move in the coming years. I hope this will challenge the reader to think more deeply about this discipline and its broader impact on science. But before gazing into the future, it is worthwhile briefly reviewing how our field has evolved until now, as it is this perspective that frames our outlook. Whither Enzymology? was the title of a symposium held in 1990 to honor the late Jeremy Knowles on the occasion of his Sixtieth birthday (Raines, 2008). Knowles was in the vanguard of a pioneering group of enzymologists who first employed the tools of physical organic chemistry to study enzyme reactions. In the 1970's his group was the first to determine the full set of elementary rate constants defining the free energy profile for an enzyme-catalyzed reaction—that of triose phosphate isomerase (Albery and Knowles, 1976). This information, put together with the crystal structure for this enzyme, allowed us to understand enzyme catalysis with the kind of precision previously only possible for simple organic molecules. Subsequently, his lab was among the first to use site-specific mutagenesis to make precisely defined changes to enzyme active sites, which combined with detailed measurements of their effect on catalysis, allowed the relationships between enzyme structure and mechanism to be elucidated. Studies such as these blew away the “fluffy clouds” obscuring the view of earlier generations of enzymologists, revealing enzymes to be exquisitely evolved, highly complex and incredibly efficient organic catalysts, but nevertheless subject to, and understood through, the basic principles of chemistry—in Knowles' words: “not different, just better” (Knowles, 1991). This view has shaped enzymology for the past 25 years, and by extension our view of cellular biology. It directly underpins the burgeoning discipline of synthetic biology, a field that involves tinkering with enzymes on a scale unimaginable in Knowles' era. The tools available to enzymologists have advanced immeasurably in the last 25 years, resulting in a paradigm shift in our approach to studying them. Not least, much of the drudgery (and perhaps also some of the fun!) of obtaining enzymes for study has been removed and the accessibility of enzymes has expanded enormously. The advent of genomic sequencing, combined with cheap, commercially available gene synthesis and protein affinity tags, allows one to select essentially any enzyme for study and produce large quantities of pure protein with minimal effort. What would once have taken many person years to accomplish can now be achieved in a few days. (Although some classes of proteins, e.g., integral membrane proteins still remain challenging to express.) Advances in structural biology mean that determining the 3-D structure of most enzymes, if not trivial, is now routine; indeed, thanks to structural proteomics initiatives, the structure is increasingly the first information to emerge for a new enzyme. Moreover, techniques for studying enzymes have become increasingly powerful: instrumentation more sensitive (for some techniques to the level of single molecules) and data analysis more sophisticated and rapid due to exponential increases in computing power. With such powerful tools and so many enzymes to explore, it seems reasonable some 25+ years on from Knowles' apotheosis, once again to pose the question: whither enzymology? In attempting to answer this question, I propose the three “grand challenges” discussed below. The first two I have chosen because they fundamentally challenge our understanding of enzyme catalysis. The first challenge is to predict enzyme catalytic activity from structure and dynamic information; the second challenge to design enzymes from scratch that approach the activity of naturally evolved enzymes. The third challenges focuses on expanding the frontiers of enzymology by discovering new enzymes that catalyze new reactions.

The observation of oxygen—oxygen interactions in ice

In this article, we present a series of first principle density functional theory simulation results which have, reproduced the vibrational spectra for ice Ih and VIII obtained by neutron scattering. Detailed analysis of the pressure-dependent simulation results shows that there is an oxygen–oxygen (O–O) interaction between the nearest water molecules participating in the H-bonding and this interaction is 1.5 times stronger than the H-bond at an O–O distance Roo of 2.7 Å observed in ice I. The O–O interaction strength increases rapidly to 2.3 times the H-bond at Roo = 2.5 Å. Analysis of the simulation results for ice VIII, shows that the strong O–O interaction only exists between H-bonded water molecules (i.e. O-H—O) and not for non-H-bonded water molecules, even when the O—O distances are similar. This discovery completely changed our view of H-bonding in water and ice, redefining the term of hydrogen-bonding between water–water molecules. Incorporation of this term may also help us to explain water anomalies. In addition short H-bonds (Roo < 2.7 Å) exist in DNA and proteins, and are thought to play a role crucial in enzyme catalysis and function. Hence this result could have major implications in our understanding of enzyme-catalysis and biology.

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes.

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Valence bond and enzyme catalysis: a time to break down and a time to build up.

Understanding enzyme catalysis and developing ability to control of it are two great challenges in biochemistry. A few successful examples of computational-based enzyme design have proved the fantastic potential of computational approaches in this field, however, relatively modest rate enhancements have been reported and the further development of complementary methods is still required. Herein we propose a conceptually simple scheme to identify the specific role that each residue plays in catalysis. The scheme is based on a breakdown of the total catalytic effect into contributions of individual protein residues, which are further decomposed into chemically interpretable components by using valence bond theory. The scheme is shown to shed light on the origin of catalysis in wild-type haloalkane dehalogenase (wt-DhlA) and its mutants. Furthermore, the understanding gained through our scheme is shown to have great potential in facilitating the selection of non-optimal sites for catalysis and suggesting effective mutations to enhance the enzymatic rate.

Role of substrate dynamics in protein prenylation reactions.

ConspectusThe role dynamics plays in proteins is of intense contemporary interest. Fundamental insights into how dynamics affects reactivity and product distributions will facilitate the design of novel catalysts that can produce high quality compounds that can be employed, for example, as fuels and life saving drugs.We have used molecular dynamics (MD) methods and combined quantum mechanical/molecular mechanical (QM/MM) methods to study a series of proteins either whose substrates are too far away from the catalytic center or whose experimentally resolved substrate binding modes cannot explain the observed product distribution. In particular, we describe studies of farnesyl transferase (FTase) where the farnesyl pyrophosphate (FPP) substrate is ∼8 Å from the zinc-bound peptide in the active site of FTase. Using MD and QM/MM studies, we explain how the FPP substrate spans the gulf between it and the active site, and we have elucidated the nature of the transition state (TS) and offered an alternate explanation of experimentally observed kinetic isotope effects (KIEs). Our second story focuses on the nature of substrate dynamics in the aromatic prenyltransferase (APTase) protein NphB and how substrate dynamics affects the observed product distribution.Through the examples chosen we show the power of MD and QM/MM methods to provide unique insights into how protein substrate dynamics affects catalytic efficiency. We also illustrate how complex these reactions are and highlight the challenges faced when attempting to design de novo catalysts.While the methods used in our previous studies provided useful insights, several clear challenges still remain. In particular, we have utilized a semiempirical QM model (self-consistent charge density functional tight binding, SCC-DFTB) in our QM/MM studies since the problems we were addressing required extensive sampling. For the problems illustrated, this approach performed admirably (we estimate for these systems an uncertainty of ∼2 kcal/mol), but it is still a semiempirical model, and studies of this type would benefit greatly from more accurate ab initio or DFT models. However, the challenge with these methods is to reach the level of sampling needed to study systems where large conformational changes happen in the many nanoseconds to microsecond time regimes. Hence, how to couple expensive and accurate QM methods with sophisticated sampling algorithms is an important future challenge especially when large-scale studies of catalyst design become of interest.The use of MD and QM/MM models to elucidate enzyme catalytic pathways and to design novel catalytic agents is in its infancy but shows tremendous promise. While this Account summarizes where we have been, we also discuss briefly future directions that improve our fundamental ability to understand enzyme catalysis.

Flexibility analysis of activity-enhanced mutants of bacteriophage T4 lysozyme

Abstract Enzymes are essential biological molecules and widely used in industry. Therefore, investigating the mechanisms underlying how enzyme activity is changed by mutations may have widespread clinical and industrial applications as well as understanding of enzyme catalysis. In this paper, various reported mutants of bacteriophage T4 lysozyme were analyzed to investigate the relationship between enzyme activity and flexibility. The activity-enhanced mutants of bacteriophage T4 lysozyme demonstrated a tendency for mutations to localize to the edge of helices and to involve substitutions to flexible amino acids such as glutamic acid and aspartic acid. Additionally, the mutated residues were shown to demonstrate increased flexibility in B-factor. These findings suggest that flexibility modulation may be a feasible means to enhance enzyme activity.

Understanding enzyme catalysis by means of supramolecular artificial enzymes

Enzymes are biomacromolecules responsible for the abundant chemical biotransformations that sustain life. Recently, biochemists have discovered that multiple conformations and numerous parallel paths are involved during the processes catalyzed by enzymes. It is plausible that the entire macromolecular scaffold is involved in catalysis via cooperative motions that result in incredible catalytic efficiency. Moreover, some enzymes can very strongly bind the transition state with an association constant of up to 1024 M−1, suggesting that covalent bond formation is a possible process during the conversion of the transition state in enzyme catalysis, in addition to the concatenation of non-covalent interactions. Supramolecular chemistry provides fundamental knowledge about the relationships between the dynamic structures and functions of organized molecules. By taking advantage of supramolecular concepts, numerous supramolecular enzyme mimics with complex and hierarchical structures have been designed and investigated. Through the study of supramolecular enzyme models, a great deal of information to aid our understanding of the mechanism of catalysis by natural enzymes has been acquired. With the development of supramolecular artificial enzymes, it is possible to replicate the features of natural enzymes with regards to their constitutional complexity and cooperative motions, and eventually decipher the conformation-based catalytic mystery of natural enzymes.