Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.     

Wiring of redox enzymes on three dimensional self-assembled molecular scaffold

The integration of biological molecules and nanoscale components provides a fertile basis for the construction of hybrid materials of synergic properties and functions. Stable protein 1 (SP1), a highly stable ring shaped protein, was recently used to display different functional domains, to bind nanoparticles (NPs), and to spontaneously form two and three-dimensional structures. Here we show an approach to wire redox enzymes on this self-assembled protein-nanoparticle hybrid. Those hybrids are genetically engineered SP1s, displaying glucose oxidase (GOx) enzymes tethered to the protein inner pore. Moreover, the Au-NP-protein hybrids self-assembled to multiple enzymatic layers on the surface. By wiring the redox enzymes to the electrode, we present an active structure for the bioelectrocatalytic oxidation of glucose. This system demonstrates for the first time a three-dimensional assembly of multiple catalytic modules on a protein scaffold with an efficient electrical wiring of the enzyme units on an electrode surface, thus implementing a hybrid electrically active unit for nanobioelectronic applications.     

Michaelis-Menten relations for complex enzymatic networks

Most biological processes are controlled by complex systems of enzymatic chemical reactions. Although the majority of enzymatic networks have very elaborate structures, there are many experimental observations indicating that some turnover rates still follow a simple Michaelis-Menten relation with a hyperbolic dependence on a substrate concentration. The original Michaelis-Menten mechanism has been derived as a steady-state approximation for a single-pathway enzymatic chain. The validity of this mechanism for many complex enzymatic systems is surprising. To determine general conditions when this relation might be observed in experiments, enzymatic networks consisting of coupled parallel pathways are investigated theoretically. It is found that the Michaelis-Menten equation is satisfied for specific relations between chemical rates, and it also corresponds to a situation with no fluxes between parallel pathways. Our results are illustrated for a simple model. The importance of the Michaelis-Menten relationship and derived criteria for single-molecule experimental studies of enzymatic processes are discussed.     

Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions

Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate‐controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition‐state structure and bonding optimally requires the use of theoretical models. In this work, we apply density‐functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2′‐O‐transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well‐studied series of phosphate and phosphorothioate mono‐, di‐ and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non‐enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate–KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.     

Predictions of Enzymatic Parameters: A Mini-Review with Focus on Enzymes for Biofuel

Enzymatic reactions are very basic processes in biological systems, and parameters related to enzymatic reactions always provide good indicators for understanding of mechanisms underlined in enzymatic reactions, for better controlling of enzymatic reactions, and for comparison of different enzymes. In this mini-review: first, parameters in enzymatic reactions were briefly reviewed from three different standpoints; second, predictions of parameters in enzymatic reactions without information on enzyme structure were shortly reviewed from viewpoints of geometric approach, graphic approach and compartmental approach; third, predictions of parameters in enzymatic reaction with information on enzyme structure were reviewed from the points of view of modeling, with 19 currently available databases, and 17 software packages and web servers; fourth, the current state of prediction on parameters in enzymatic reaction in biofuel industry with respect to cellulolytic enzymes were reviewed; fifth, the pros and cons for future development were discussed; and finally, a worked example was given in the Appendix to describe the whole procedures of prediction of enzymatic parameters in reactions.     

Macromolecular Coating Enables Tunable Selectivity in a Porous PDMS Matrix

Whether for laboratory use or clinical practice, many fields in Life Sciences require selective filtering. However, most existing filter systems lack the ability to easily tune their filtration behavior. Two key elements for efficient filtering are a high surface‐to‐volume ratio and the presence of suitable chemical groups which establish selectivity. In this study, an artificial PDMS‐based capillary system with highly tunable selectivity properties is presented. The high surface‐to‐volume ratio of this filter system is generated by first embedding sugar fibers into a synthetic polymer matrix and then dissolving these fibers from the cured polymer. To functionalize this filter, the inner surface of the capillaries is coated with purified or synthetic macromolecules. Depending on the type of macromolecule used for filter functionalization, selective sieving is observed based on steric hindrance, electrostatic binding, electrostatic repulsion, or specific binding interactions. Furthermore, it is demonstrated that enzymes can be immobilized in the capillary system which allows for performing multiple cycles of enzymatic reactions with the same batch of enzymes and without the need to separate the enzymes from their reaction products. In addition to lab‐scale filtration and enzyme immobilization applications demonstrated here, the functionalized porous PDMS matrix may also be used to test binding interactions between different molecules.     

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase’s biology, with wide-reaching implications for drug development.     

Transient DNA‐Based Nanostructures Controlled by Redox Inputs

Synthetic DNA has emerged as a powerful self‐assembled material for the engineering of nanoscale supramolecular devices and materials. Recently dissipative self‐assembly of DNA‐based supramolecular structures has emerged as a novel approach providing access to a new class of kinetically controlled DNA materials with unprecedented life‐like properties. So far, dissipative control has been achieved using DNA‐recognizing enzymes as energy dissipating units. Although highly efficient, enzymes pose limits in terms of long‐term stability and inhibition of enzyme activity by waste products. Herein, we provide the first example of kinetically controlled DNA nanostructures in which energy dissipation is achieved through a non‐enzymatic chemical reaction. More specifically, inspired by redox signalling, we employ redox cycles of disulfide‐bond formation/breakage to kinetically control the assembly and disassembly of tubular DNA nanostructures in a highly controllable and reversible fashion.     

Enzymatic Reactions using Ionic Liquids for Green Sustainable Chemical Process; Stabilization and Activation of Lipases

The enzymatic reaction is highly respected from an environmentally-friendly point-of-view. Optimization of the reaction media and supporting materials of enzymes must be investigated in parallel with the effort to develop new enzymes. Lipases are frequently used for organic syntheses as synthetic tools even industry because of their acceptance of having a broad range of substrates, stability, and availability. We have investigated the possibility of ILs as both a solvent and activating or stabilization agent of enzymes, in particular, lipase as a model enzyme. ILs allowed recyclable use of a lipase and significant acceleration of transesterification, and also enhanced the stability and reaction activity of a lipase by immobilization through a lyophilization process. We discuss how we enhanced the enzyme capability using the IL engineering focusing on lipase-catalyzed reactions, i. e., realization of the recyclable use of an enzyme, how ILs mediated the enhanced reaction rate, and improved the stability of the enzyme.     

Microbial and Enzymatic Processes for the Production of Biologically and Chemically Useful Compounds [New Synthetic Methods (69)]

In recent years, the most significant development in the field of synthetic organic chemistry has been the application of biological systems to chemical reactions. Reactions catalyzed by enzymes and enzyme systems display far greater specificities than more conventional organic reactions. Biological and/or enzymatic syntheses and transformations, that is, “microbial transformations,” have great potential. Some of these reactions have already been shown to have useful applications in the fields of synthetic organic chemistry and biotechnology. This article reviews the current status of the rapidly developing field of microbial transformation, the methodology, available technological procedures, and fields of application being described especially in relation to conventional organic synthesis methods.     

Three-way partial least-squares regression for the simultaneous kinetic-enzymatic determination of xanthine and hypoxanthine in human urine

The performance of three-way principal component analysis and three-way partial least-squares regression when applied to a complex kinetic-enzymatic system is studied, in order to investigate the analytical potential of the combined use of these chemometric technologies for non-selective enzymatic systems. A enzymatic-kinetic procedure for the simultaneous determination of hypoxanthine and xanthine in spiked samples of human urine is proposed. The chemical system involves two consecutive reactions catalyzed by xanthine oxidase (EC 1.17.3.2). This enzyme catalyzes the oxidation of hypoxanthine, first to xanthine and then to uric acid, a competitive inhibitor of the reactions. The influence of uric acid during quantitative determination was considered in the design of the calibration set. The sample and enzyme solution were mixed in a stopped-flow module and the reaction was monitored using a diode array spectrophotometer. The recorded data have an intrinsical three-component structure (samples, time and wavelength). This data array was studied via three-way principal component analysis and was modeled for quantitative purposes using a three-way partial least-squares calibration procedure. Results are compared with those obtained by applying classical bilinear PLS to the previously unfolded data matrix.     

Calixarenes and resorcinarenes as scaffolds for supramolecular metallo-enzyme mimicry

Metallo-enzymes are natural catalysts carrying out highly selective and demanding reactions under mild conditions, using readily available metal ions (iron, copper, zinc, etc.). Understanding the factors explaining these performances is thus of fundamental and applicative interest. Classical model complexes displaying significant limitations in reactivity led to the development of supramolecular systems associating a reactive complex to a molecular cavity receptor, in order to not only mimic the first coordination sphere of the metal, but also the supramolecular enzymatic environment (access channel and binding pocket) responsible for their remarkable kinetics and selectivities. Calixarenes and resorcinarenes are particularly suited to this goal due to their wide range of sizes and flexibility. This review illustrates several specific aspects of enzymatic systems that were successfully mimicked with such supramolecular model systems. This toolbox of supramolecular effects could be used for future developments of bioinorganic supramolecular catalysts.     

Catalytic palladium nanoparticles supported on nanoscale MOFs: a highly active catalyst for Suzuki–Miyaura cross-coupling reaction

Pd nanoparticles have been successfully supported on nanoscale metal-organic frameworks (NMOFs) by using a simple and effective microwave-assisted impregnation process. The resulting composite, representing as a highly active NMOFs supported metal nanoparticles catalyst for the Suzuki cross-coupling reaction between aryl/heteroaryl halides and arylboronic acids, is well characterized, and its high activity and good recyclability are discussed in details. It reveals that, compared to the corresponding bulk MOFs and conventional active carbon materials, nanoscale MOFs as novel support materials for heterogeneous catalysts can exhibit superior performance in the catalytic reactions.     

二苯乙烯类低聚物的仿生合成

天然二苯乙烯低聚物是一类自然界分布广泛的多酚化合物,因其结构复杂且生物活性多样而受到密切关注,但此类化合物天然资源的稀少极大限制了其构效关系的调查及活性药物的筛选。近年来许多化学家对此类低聚物的仿生合成方法做了广泛而深入的研究,已形成一个新的研究热点。本文详尽综述了迄今三十多年来二苯乙烯类低聚物的仿生合成研究进展,包括在不同介质中的酶催化或金属氧化剂催化的氧化偶联方法、光催化的异构化及强酸催化下的环合反应,由不同的二苯乙烯前体通过仿生合成途径,构建出结构多样的二苯乙烯低聚物。此外,本文对该类低聚物的仿生合成研究前景做了展望。     

An addressable antibody nanoarray produced on a nanostructured surface

The ability to address specific nanoscale features is required to produce diverse biological nanoarrays or perform local assembly using biological building blocks and is an important unsolved problem in nanotechnology. In this work, we describe the use of a novel nanofabricated gold surface, with regions of distinct topography and chemical functionalities, to solve this problem. First, nanoarrays of IgG antibodies were produced by selective immobilization in nanoholes on the surface. The smallest feature size was determined by the hole size (fwhm 90 nm) and not surface diffusion. Using holes of 300 nm diameter, we selectively addressed specific features in the array by nanopipet delivery of a functional antibody, anti-IgG. To our knowledge, this is the first example of addressing specific biologically functional features on a surface at the nanoscale.     

Nucleotide-Selective Templated Self-Assembly of Nanoreactors under Dissipative Conditions

Nature adopts complex chemical networks to finely tune biochemical processes. Indeed, small biomolecules play a key role in regulating the flux of metabolic pathways. Chemistry, which was traditionally focused on reactions in simple mixtures, is dedicating increasing attention to the network reactivity of highly complex synthetic systems, able to display new kinetic phenomena. Herein, we show that the addition of monophosphate nucleosides to a mixture of amphiphiles and reagents leads to the selective templated formation of self-assembled structures, which can accelerate a reaction between two hydrophobic reactants. The correct matching between nucleotide and the amphiphile head group is fundamental for the selective formation of the assemblies and for the consequent up-regulation of the chemical reaction. Transient stability of the nanoreactors is obtained under dissipative conditions, driven by enzymatic dephosphorylation of the templating nucleotides. These results show that small molecules can play a key role in modulating network reactivity, by selectively templating self-assembled structures that are able to up-regulate chemical reaction pathways.     

Fabrication of interdigitated micropatterns of self-assembled polymer nanofilms containing cell-adhesive materials

Micropatterns of different biomaterials with micro- and nanoscale features and defined spatial arrangement on a single substrate are useful tools for studying cellular-level interactions, and recent reports have highlighted the strong influence of scaffold compliance in determining cell behavior. In this paper, a simple yet versatile and precise patterning technique for the fabrication of interdigitated micropatterns of nanocomposite multilayer coatings on a single substrate is demonstrated through a combination of lithography and layer-by-layer (LbL) assembly processes, termed polymer surface micromachining (PSM). The first nanofilm pattern is constructed using lithography, followed by LbL multilayer assembly and lift-off, and the process is repeated with optical alignment to obtain interdigitated patterns on the same substrate. Thus, the method is analogous to surface micromachining, except that the deposition materials are polymers and biological materials that are used to produce multilayer nanocomposite structures. A key feature of the multilayers is the capability to tune properties such as stiffness by appropriate selection of materials, deposition conditions, and postdeposition treatments. Two- and four-component systems on glass coverslips are presented to demonstrate the versatility of the approach to construct precisely defined, homogeneous nanofilm patterns. In addition, an example of a complex system used as a testbed for in vitro cell adhesion and growth is provided: micropatterns of poly(sodium 4-styrenesulfonate)/poly-L-lysine hydrobromide (PSS/PLL) and secreted phospholipase A(2)/poly(ethyleneimine) (sPLA(2)/PEI) multilayers. The interdigitated square nanofilm array patterns were obtained on a single coverslip with poly(diallyldimethylammonium chloride) (PDDA) as a cell-repellent background. Cell culture experiments show that cortical neurons respond and bind specifically to the sPLA(2) micropatterns in competition with PLL micropatterns. The fabrication and the initial biological results on the nanofilm micropatterns support the usefulness of this technique for use in studies aimed at elucidating important biological structure-function relationships, but the applicability of the fabrication method is much broader and may impact electronics, photonics, and chemical microsystems.     

Mimicking heme enzymes in the solid state: metal-organic materials with selectively encapsulated heme

To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal-organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a "ship-in-a-bottle" fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.     

Surface organometallic chemistry on metals: a novel and effective route to custom-designed bimetallic catalysts

The synthesis, characterization and catalytic properties of new materials obtained by reaction of organometallic complexes of groups IIb, IVa, and VIa with the surface of metallic particles are reviewed. Two types of materials may be obtained by surface organometallic chemistry on metals: metal particles covered with organometallic fragments, and bimetallic particles of predetermined composition. Characterization of the organometallic fragments on the metal particles has demonstrated their thermal stability. These particles covered with surface organometallic fragments are new catalytic materials, highly selective in several reactions such as the hydrogenation of α,β-unsaturated aldehydes, ethyl pyruvate, nitrobenzene, acrylonitrile, and olefins. The bimetallic particles without organometallic fragments are also highly active and selective for a variety of reactions such as hydrogenolysis of various alkanes and hydrogenolysis of esters. For these systems, the concept of “site isolation” has been advanced to account for the high selectivity of the reactions.     

Thermodynamic Feasibility of Enzymatic Reduction of Carbon Dioxide to Methanol

Production of valuable chemicals from CO₂ is highly desired for the purpose of controlling CO₂ emission. Toward that, enzymatic reduction of CO₂ for the production of methanol appeared to be especially promising. That has been achieved by reversing the biological metabolic reaction pathways. However, hitherto, there has been little discussion on the thermodynamic feasibility of reversing such biological pathways. The reported yields of methanol have been generally very low under regular reaction conditions preferred by naturally evolved enzymes. The current work examines the sequential enzymatic conversion of CO₂ into methanol from a thermodynamic point of view with a focus on factors that control the reaction equilibrium. Our analysis showed that the enzymatic conversion of carbon dioxide is highly sensitive to the pH value of the reaction solution and, by conducting the reactions at low pHs (such as pH 6 or 5) and ionic strength, it is possible to shift the biological methanol metabolic reaction equilibrium constants significantly (by a factor of several orders of magnitude) to favor the synthesis of methanol.