Substrate‐Selective Catalysis

Substrate selectivity is an important output function for the validation of different enzyme models, catalytic cavity compounds, and reaction mechanisms as demonstrated in this review. In contrast to stereo‐, regio‐, and chemoselective catalysis, the field of substrate‐selective catalysis is under‐researched and has to date generated only a few, but important, industrial applications. This review points out the broad spectrum of different reaction types that have been investigated in substrate‐selective catalysis. The present review is the first one covering substrate‐selective catalysis and deals with reactions in which the substrates involved have the same reacting functionality and the catalysts is used in catalytic or in stoichiometric amounts. The review covers real substrate‐selective catalysis, thus only including cases in which substrate‐selective catalysis has been observed in competition between substrates.     

A Cofactor‐Substrate‐Based Supramolecular Fluorescent Probe for the Ultrafast Detection of Nitroreductase under Hypoxic Conditions

Identifying the location and expression levels of enzymes under hypoxic conditions in cancer cells is vital in early‐stage cancer diagnosis and monitoring. By encapsulating a fluorescent substrate, L‐NO₂ , within the NADH mimic‐containing metal–organic capsule Zn‐ MPB , we developed a cofactor‐substrate‐based supramolecular luminescent probe for ultrafast detection of hypoxia‐related enzymes in solution in vitro and in vivo. The host–guest structure fuses the coenzyme and substrate into one supramolecular probe to avoid control by NADH, switching the catalytic process of nitroreductase from a double‐substrate mechanism to a single‐substrate one. This probe promotes enzyme efficiency by altering the substrate catalytic process and enhances the electron transfer efficiency through an intra‐molecular pathway with increased activity. The enzyme content and fluorescence intensity showed a linear relationship and equilibrium was obtained in seconds, showing potential for early tumor diagnosis, biomimetic catalysis, and prodrug activation.     

Enzymatic radical catalysis: coenzyme B12-dependent diol dehydratase

A peculiar function resides in a peculiar structure. Coenzyme B₁₂ or adenosylcobalamin, a naturally occurring organometallic compound, serves as a cofactor for enzymatic radical reactions. How do the enzymes form catalytic radicals at the active sites? How do the enzymes utilize and control the high reactivity of the radicals for catalysis? Recently, three‐dimensional structures of several radical‐containing or radical‐forming enzymes including B₁₂ enzymes have been reported, enabling the analysis of the fine mechanisms of the action of these interesting enzymes. Our biochemical, mutational, and crystallographic studies as well as theoretical calculations on diol dehydratase, an adenosylcobalamin–dependent enzyme, revealed that its structure is adapted for its function—that is, activation of the Co? C bond toward homolysis, abstraction of a specific hydrogen atom from the substrate and its recombination to a particular product, and transition state stabilization in the hydroxyl group migration of a substrate‐derived radical. The functions of K⁺ and the active‐site amino acid residues in enzyme catalysis are also investigated. Based on the results, the fine mechanism of the enzyme and the energetic feasibility of enzymatic radical catalysis are described here. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 352–366, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10035     

Polyfunctional Bis‐Lewis‐Acid‐/Bis‐Triazolium Catalysts for Stereoselective 1,4‐Additions of 2‐Oxindoles to Maleimides

Achieving enzyme‐like catalytic activity and stereoselectivity without the typically high substrate specificity of enzymes is a challenge in the development of artificial catalysts for asymmetric synthesis. Polyfunctional catalysts are considered to be a promising tool for achieving excellent catalytic efficiency. A polyfunctional catalyst system was developed, which incorporates two Lewis acidic/Brønsted basic cobalt centers in combination with triazolium moieties that are crucial for high reactivity and excellent stereoselectivity in the direct 1,4‐addition of oxindoles to maleimides. The catalyst is assembled through click chemistry and is readily recyclable through precipitation by making use of its charges. Kinetic studies support a cooperative mode of action. Diastereodivergency is achievable with either Boc‐protected or unprotected maleimide.     

Interfacial catalysis by phospholipases at conjugated lipid vesicles: colorimetric detection and NMR spectroscopy

Background: Self-assembled conjugated polymers are rapidly finding biological and biotechnological applications. This work describes a synthetic membrane system based on self-assembled polydiacetylenes, which are responsive to the enzymatic activity of phospholipases - a ubiquitous class of enzymes that catalyze the hydrolysis of phospholipid molecules embedded in cell membranes.Results: We show that phospholipases are active at bilayer vesicles composed of the natural enzyme substrate, dimyristoylphosphatidylcholine (DMPC), and a synthetic π-conjugated polymerized lipid based on polydiacetylene (PDA). In addition, the enzymatic reaction induces an optical transition in the surrounding PDA matrix, visible to the naked eye. Nuclear magnetic resonance spectroscopy confirms the occurrence of enzymatic catalysis and reveals the fate of the cleavage products.Conclusions: The results indicate that the structural and color changes of the PDA matrix are directly related to interfacial catalysis by phospholipase. This novel biocatalytic method of inducing optical transitions in conjugated polymers might lead to new approaches towards rapidly screening new enzyme inhibitor compounds.     

A High‐Throughput Assay for Arylamine Halogenation Based on a Peroxidase‐Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high‐throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho‐benzoquinone‐amine adduct, which is produced by the peroxidase‐catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non‐halogenated arylamine. This assay is sensitive, rapid and amenable to high‐throughput screening platforms. We have also shown this assay to be easily coupled to a flavin‐dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one‐pot” workflow.     

Nonspecific catalysis by protein surfaces

Catalytic antibodies are the best availablea llaround enzyme mimics. They provide a unique experimental approach and some special insights into general questions about catalysis by enzymes. They offer enantiospecific reactions and levels of substrate binding that compare well with typical enzyme reactions, but not—so far—comparable catalytic efficiency. We and others have used the Kemp elimination as a probe of catalytic efficiency in antibodies. We compare these reactions with nonspecific catalysis by other proteins, and with catalysis by enzymes. Several simple reactions are catalyzed by theserum albumins with Michaelis-Menten kinetics, and can be shown to involve substrate binding and catalysis by local functional groups. Here, we report the details of one investigation, which implicate known binding sites on the protein surface and discuss implications for catalyst design and efficiency.     

Solution‐State 17O Quadrupole Central‐Transition NMR Spectroscopy in the Active Site of Tryptophan Synthase

Oxygen is an essential participant in the acid–base chemistry that takes place within many enzyme active sites, yet has remained virtually silent as a probe in NMR spectroscopy. Here, we demonstrate the first use of solution‐state ¹⁷O quadrupole central‐transition NMR spectroscopy to characterize enzymatic intermediates under conditions of active catalysis. In the 143 kDa pyridoxal‐5′‐phosphate‐dependent enzyme tryptophan synthase, reactions of the α‐aminoacrylate intermediate with the nucleophiles indoline and 2‐aminophenol correlate with an upfield shift of the substrate carboxylate oxygen resonances. First principles calculations suggest that the increased shieldings for these quinonoid intermediates result from the net increase in the charge density of the substrate–cofactor π‐bonding network, particularly at the adjacent α‐carbon site.     

Pathway from N‐Alkylglycine to Alkylisonitrile Catalyzed by Iron(II) and 2‐Oxoglutarate‐Dependent Oxygenases

N‐alkylisonitrile, a precursor to isonitrile‐containing lipopeptides, is biosynthesized by decarboxylation‐assisted ‐N≡C group (isonitrile) formation by using N‐alkylglycine as the substrate. This reaction is catalyzed by iron(II) and 2‐oxoglutarate (Fe/2OG) dependent enzymes. Distinct from typical oxygenation or halogenation reactions catalyzed by this class of enzymes, installation of the isonitrile group represents a novel reaction type for Fe/2OG enzymes that involves a four‐electron oxidative process. Reported here is a plausible mechanism of three Fe/2OG enzymes, Sav607, ScoE and SfaA, which catalyze isonitrile formation. The X‐ray structures of iron‐loaded ScoE in complex with its substrate and the intermediate, along with biochemical and biophysical data reveal that ‐N≡C bond formation involves two cycles of Fe/2OG enzyme catalysis. The reaction starts with an Fe^IV‐oxo‐catalyzed hydroxylation. It is likely followed by decarboxylation‐assisted desaturation to complete isonitrile installation.     

Photoenzymatic Catalysis Enables Radical‐Mediated Ketone Reduction in Ene‐Reductases

Flavin‐dependent ene‐reductases (EREDs) are known to stereoselectively reduce activated alkenes, but are inactive toward carbonyls. Demonstrated here is that in the presence of photoredox catalysts, these enzymes will reduce aromatic ketones. Mechanistic experiments suggest this reaction proceeds through ketyl radical formation, a reaction pathway that is distinct from the native hydride‐transfer mechanism. Furthermore, this reactivity is accessible without modification of either the enzyme or cofactors, allowing both native and non‐natural mechanisms to occur simultaneously. Based on control experiments, we hypothesize that binding to the enzyme active site attenuates the reduction potential of the substrate, enabling single‐electron reduction. This reactivity highlights opportunities to access new catalytic manifolds by merging photoredox catalysis with biocatalysis.     

Excited‐State Charge Separation in the Photochemical Mechanism of the Light‐Driven Enzyme Protochlorophyllide Oxidoreductase

The unique light‐driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride‐ and proton‐transfer chemistry, have so far proven difficult to detect. We have used a combination of time‐resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond–microsecond time range, to propose a new mechanism for the photochemistry. Excited‐state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so‐called “reactive” intramolecular charge‐transfer state creates an electron‐deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited‐state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light‐activated chemical and biological catalysts.     

An Elastic Monolithic Catalyst: A Microporous Metalloporphyrin‐Containing Framework‐Wrapped Melamine Foam for Process‐Intensified Acyl Transfer

The advent of conjugated microporous polymers (CMPs) has had significant impact in catalysis. However, the presence of only micropores in these polymers often imposes diffusion limitations, which has resulted in the low utilization of CMPs in catalytic reactions. Herein, the preparation of a foam‐supporting CMP composite with interconnective micropores and macropores and elastic properties is reported. Metalloporphyrin‐based CMP organogels are synthesized within the melamine foam by a room‐temperature oxidative homocoupling reaction of terminal alkynes. Upon drying, the CMP‐based xerogels tightly wrap the framework skeletons of the foam, while the foam cells are still open to allow for the preservation of elasticity and macroporosity. Such a hierarchical structure is efficient for acyl transfer, facilitates substrate diffusion within interpenetrative macropores and micropores, and could be used to intensify catalytic processes.     

Quantum Mechanics/Molecular Mechanics Studies on the Mechanism of Action of Cofactor Pyridoxal 5′‐Phosphate in Ornithine 4,5‐Aminomutase

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5′‐phosphate (PLP)‐dependent D ‐ornithine 4,5‐aminomutase (OAM)‐catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff‐base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial “strain” energy on the orientation of the cyclic intermediate to control its trajectory. In addition the “strain” energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP‐dependent reactions.     

A Phospholipase with a Novel Catalytic Triad

Catalytic triads are a long-standing paradigm of enzyme catalysis. By using a “matched mutation” approach, that is, a simultaneous exchange of the protein (through mutagenesis) and the substrate (through substitution of oxygen atoms by sulfur atoms) followed by enzyme kinetic analysis, a novel catalytic triad (see figure) with an unusual amino acid composition is now proposed for a phospholipase that fulfills a dual function in catalysis.     

Expression,Purification and Characterization of a Bifunctional α‐L‐Arabinofuranosidase/β‐D‐Xylosidase from Trichoderma Koningii G‐39

A gene of α‐L ‐arabinofuranosidase (Abf) from Trichoderma koningii G‐39 was successfully expressed in Pichia pastoris. The recombinant enzyme was purified to > 90% homogeneity by a cation‐exchanged chromatography. The purified enzyme exhibits both α‐L ‐arabinofuranosidase and β‐D ‐xylosidase (Xyl) activities with p‐nitrophenyl‐α‐L ‐arabionfuranoside (pNPAF) and 2,4‐dinitrophenyl‐β‐D ‐xylopyanoside (2,4‐DNPX) as substrate, respectively. The stability and the catalytic feature of the bifunctional enzyme were characterized. The enzyme was stable for at least 2 h at pH values between 2 and 8.3 at room temperature when assayed for Abf and Xyl activities. Enzyme activity decreased dramatically when the pH exceeded 9.5 or dropped below 1.5. The enzyme lost 35% of Abf activity after incubation at 55 °C for 2 h, but retained 95% of Xyl activity, with 2,4‐DNXP as substrate, under the same conditions. Further investigation of the active site topology of both enzymatic functions was performed with the inhibition study of enzyme activities. The results revealed that methyl‐α‐L ‐arabinofuranoside inhibition is noncompetitive towards 2,4‐DNPX as substrate but competitive towards pNPAF. Based on the thermal stability and the inhibition studies, we suggest that the enzymatic reactions of Abf and Xyl are performed at distinct catalytic sites. The recombinant enzyme possesses both the retaining transarabinofuranosyl and transxylopyranosyl activities, indicating both enzymatic reactions proceed through a two‐step, double displacement mechanism.     

Electrode Reactions of Catechol at Tyrosinase‐Immobilized Latex Suspensions

Tyrosinase was immobilized on polystyrene latex particles in order to control amounts of the enzyme. The tyrosinase‐coated latex particles were composed of the core polystyrene and four successive coating layers: polystyrene sulfonate, polyallylamine, tyrosinase and polyallylamine again, built up by the layer‐by‐layer technique. They showed catalytic currents for the enzymatic oxidation of catechol to o‐quinone. The enzyme activity per particle was evaluated as 2.3×10^?7 units from UV absorption of o‐quinone. The relation between the catalytic current and the concentration of catechol leads to a Michaelis‐Menten type kinetic equation. The layer‐by‐layer method was found to have a deactivating effect on enzyme catalysis. In spite of this, the catechol oxidation current was larger than the current from free tyrosinase at a common value of enzyme units per volume. This is ascribed to strong adsorption of the latex particles on the electrode, leading to the enhancement of the local concentration of tyrosinase.     

Hollow‐Shell‐Structured Nanospheres: A Recoverable Heterogeneous Catalyst for Rhodium‐Catalyzed Tandem Reduction/Lactonization of Ethyl 2‐Acylarylcarboxylates to Chiral Phthalides

Chiral organorhodium‐functionalized hollow‐shell‐structured nanospheres were prepared by immobilization of a chiral N‐sulfonylated diamine‐based organorhodium complex within an ethylene‐bridged organosilicate shell. Structural analysis and characterization reveal its well‐defined single‐site rhodium active center, and transmission electron microscopy images reveal a uniform dispersion of hollow‐shell‐structured nanospheres. As a heterogenous catalyst, it exhibits excellent catalytic activity and enantioselectivity in synthesis of chiral phthalides by a tandem reduction/lactonization of ethyl 2‐acylarylcarboxylates in aqueous medium. The high catalytic performance is attributed to the synergistic effect of the high hydrophobicity and the confined chiral organorhodium catalytic nature. The organorhodium‐functionalized nanospheres could be conveniently recovered and reused at least 10 times without loss of catalytic activity. This feature makes it an attractive catalyst in environmentally friendly organic reactions. The results of this study offer a new approach to immobilize chiral organometal functionalities within the hollow‐shell‐structured nanospheres to prepare materials with high activity in heterogeneous asymmetric catalysis.     

Short Self‐Assembling Peptides Are Able to Bind to Copper and Activate Oxygen

We have shown that de novo designed peptides self‐assemble in the presence of copper to create supramolecular assemblies capable of carrying out the oxidation of dimethoxyphenol in the presence of dioxygen. Formation of the supramolecular assembly, which is akin to a protein fold, is critical for productive catalysis since peptides possessing the same functional groups but lacking the ability to self‐assemble do not catalyze substrate oxidation. The ease with which we have discovered robust and productive oxygen activation catalysts suggests that these prion‐like assemblies might have served as intermediates in the evolution of enzymatic function and opens the path for the development of new catalyst nanomaterials.     

Structural Snapshots of α‐1,3‐Galactosyltransferase with Native Substrates: Insight into the Catalytic Mechanism of Retaining Glycosyltransferases

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double‐displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α‐1,3‐galactosyltransferase (α3GalT) from Bos taurus , which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP‐Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front‐side substrate‐assisted S_Ni‐type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.     

载酶纳米催化体系用于疾病诊疗的研究进展

酶作为一种具有高度特异性和高效性的催化剂, 可在细胞器中通过复杂有序的生化反应调节细胞的代谢过程. 受细胞区隔化结构的启发, 仿生设计纳米酶催化体系、 构筑限域酶催化微环境从而提高酶催化活性的研究为酶催化应用开辟了新思路. 纳米催化体系保留了小尺寸、 大比表面积、 肿瘤部位选择性富集等优势, 在疾病的诊疗方面发挥了巨大的优势. 本文首先总结了天然酶、 模拟酶和级联酶体系的催化机理, 对仿生构筑的纳米酶催化材料的载体体系进行了概述, 介绍了纳米酶催化体系在生物成像方面的应用, 讨论了其在相关代谢类疾病的作用途径, 并对纳米酶催化体系用于生物诊疗的发展前景进行了展望.