Enzyme Catalysis Today and the Chemistry of the 21st Century

This chapter is a brief overview of the general information about the main classes of enzymes and its applications. The key enzymatic systems involved in the conversion of solar energy into chemical energy, the formation of energy-rich molecules, carbon dioxide, and atmospheric nitrogen fixation, the synthesis and degradation of amino acids, lipids, proteins, nucleotides, and nucleic acids. Enzymes provide vital chemical reactions like oxidative phosphorylation, Krebs cycle, Glycolysis Calvin-Benson cycle. Several paragraphs are devoted to miscellaneous enzymes including cytochromes P450, methane monooxygenases, hydrolases, kinases, and phosphatases. Enzymes play a vital role for all living things and control all normal and pathological processes. Enzymes in healthcare and medicine are used for treating disorders, assisting metabolism, medical device cleaning, the manufacture of medicines, regulation of digestion, detection of diseases, addressing food quality, treating heart, brain, lung, digestive tract, and mental diseases. Separate sections refer to the crucial role of enzymes in cancer and in COVID-19. Enzymes are widely used in such practically important areas as pharmaceutical industries, yogurt, beer and cheese production, apple wine fermentation, baking, brewing, production of cakes, preparation of digestive aids, fruit juices, and starch syrups and addition to the dough of bread, biodiesel production, enzymatic degumming, textile and paper industry, textile desizing, laundry detergents, the enzyme-mediated ceramic synthesis, skincare and cosmetics industry, the enzyme-mediated ceramic synthesis, the enzymatic decomposition of organic matter in soil. Thus, modern enzymology represents an inexhaustible source of knowledge and ideas for modern chemistry to initiate and bring about the real industrial revolution of the twenty-first century.

To monitor the kinetics of enzymatic processes, a whole cascade of physicochemical methods is used. This chapter is devoted to a brief review of the physical foundations of these methods, the application of which is illustrated by typical examples. The most traditional and widely used methods are conventional absorption, infrared, Raman and luminescence spectroscopies with timescales from 10⁻¹⁴ s to 10⁻² s. Much less commonly used are such developed physical methods as dielectric relaxation (DR), depolarized light scattering (DLS), optical Kerr-effect (OKE), ultrasound, terahertz (THz) spectroscopies, femtosecond pump–probe, photon-echo, optical Kerr effect, sum-frequency generation, two-dimensional infrared spectroscopic two-dimensional sum-frequency generation, three-dimensional infrared, and two-dimensional Raman terahertz spectroscopy.

The main goals of studying the kinetics of enzymatic processes are to establish their elementary mechanisms, namely, the succession and rate of intermediate of elementary stages as well as their chemical nature. To determine the parameters of the Michaelis–Menton equation, a whole arsenal of classical and modern physical methods described in Chap. 2 is used. In parallel, the following groups of methods are also successfully applied: (1) Continuous flow, temperature, pH and pressure jumps, kinetic isotope effect (KIE), artificial intermediates, and transition state analogs methods. Of particular importance are theoretical computation approaches. Foundation of these approaches, combined quantum mechanics/molecular mechanics (QM/MM) methods in computational enzymology, in particular, and examples illustrated theoretical approaches are subject of consideration in separate sections.

The outstanding catalytic, regulatory, and self-preserving properties of enzymes have been developed over the course of several million years of molecular evolution. Proximity and Oriental Effects are an important part of these mechanisms due to the pre-binding of the substrate by unique orientation relative to the catalytic groups. For example, cyclization of aliphatic molecule provides decrease of entropy of DS₀ = – 3.6 − 4.7 eu. Difference in rates is 3–4 orders of magnitude (intramolecular versus intermolecular). In a concerted reaction, when a substrate is simultaneously attracted by different active donor and acceptor reagents such as acid and basic groups, nucleophile and electrophile, or reducing and oxidizing agents a significant decrease in the activation energy is expected. General theoretical considerations in favor of synchronous or sequential mechanisms and specific cases are discussed in a separate section. In the case of effective concerted mechanism, the decrease in the synchronization probability with increasing number of atoms or groups, participating in an elementary stage, can be compensated for by an appreciable decrease in the activation energy due to the inclusion of nuclei of donor and acceptor groups in the process. This consideration has led us to the formulation of the principle of optimum motion (POM). In 1975, the author of this monograph has suggested that rapid electron transfer in photosynthetic reaction centers in the forward direction and significantly slower transfer in the reverse direction may account for the cascade structure of RC which provides tunneling (long-distance) mechanism of the photoseparated charges. The discovery and experimental confirmation of Long-Range Electron Transfer (LRET) between donor (D) and acceptor (A) centers in model and biological systems were described. Data on Photosystem I and photosystem II which are the two multi-protein complexes that contain the pigments necessary to harvest photons and use light energy to catalyze the primary photosynthetic endergonic reactions producing high energy compounds were briefly reviewed. Magnetic isotope effects arise when a chemical reaction involves spin-selective processes proceeded via the radical pair mechanism was described.

Cytochromes P450 (CYPs), nitric oxide synthases (NOS )and methane monooxigenase, (MMO) are powerful enzyme hydroxylation systems of organic substrates (CYPs) and nitric oxide synthesis (NOS ). Despite the huge diversity of substrates, catalysis is carried out by a single fundamental mechanism. The mechanism includes the following key stages: activation of molecular oxygen by a two-molecular mechanism, decomposition of the product into a radical-like particle that attacks the substrate.

Two electron mechanism may involve the direct transport of two electrons from a mononuclear transition complex to a substrate. The probability of two electron processes, however, increases sharply if two electrons are transferred together with a proton as a hydride or the change in electric charge is compensated by the simultaneous shift of the electron cloud from neighboring groups. Hydrogenases are a diverse group of metalloenzymes that catalyze the conversion of dihydrogen into protons and electrons and the reverse reaction, the generation of dihydrogen. The [NiFe] hydrogenase and the [FeFe] hydrogenase contain sulfur bridged bimetallic centers. The concept of four-electron mechanism of N₂ reduction at mild conditions was first introduced by Likhtenshtein and Shilov in 1970. It was suggested that the four-electron process can occur in a multinuclear cluster of transition metals. The multielectron nature of the energetically favorable processes in clusters does not evidently impose any new additional restrictions. The need for a synchronous four-electronic elementary act with the formation of a hydrazine-like intermediate is dictated by thermodynamics. The central enzyme of biological nitrogen fixation catalyzes in the nitrogen-fixing bacteria the reduction of molecular nitrogen to ammonia by a reductant with the assistance of ATP hydrolysis: The nitrogenase active has iron-sulfur F cluster (F₄S₄), P-clusters (F₈S₇) and FeMo cofactor (F₇S₉Mo). After the accumulation of four and six reduction equivalents in, the reduction of u occurs first to the hydrozine derivative and then to ammonia, respectively, the reduction of u occurs first to the hydrozine derivative and then to ammonia, respectively. The role of ATP in the nitrogenase reaction was discussed. Subsequent absorption of four light quanta by Photosystem I (PS)I and photosystem II (PS II) results in evaluation of dioxygen from a two-water molecule. Photosynthetic water oxidation occurs at the oxygen-evolving complex (OEC) of (PSII) which contains a Mn₄CaO₅ inorganic cluster ligated by oxides, waters and amino acid residues. In 1976, Semenov, Shilov, and Likhtenstein suggested that in plant photosynthesis, the splitting of a water molecule with the release of molecular oxygen can occur only according the thermodynamically allowed four-electron mechanism.

This chapter presents a brief overview of physical and computer methods for studying the mechanisms and frequencies of the molecular dynamics of proteins and enzymes, illustrated with typical examples. The list of methods used includes the following: spin and Mössbauer labels and probes, fluorescence quenching, fluorescence dynamic Stokes shift, Förster resonance energy transfer (FRET), nuclear magnetic resonance, differential scanning calorimetry, small-wide-angle X-ray scattering, femtosecond time-resolved X-ray solution scattering, and neutron scattering. Protein dynamics occur at time scales from millisecond to subfemtosecond. The range of time-scales involved in substrate turnover step of enzyme catalyzed reactions and internal protein dynamics are similar. The protein motions necessary for catalysis are an intrinsic property of the enzyme and may limit the overall turnover rate. Recent works on selected protein systems have addressed the role of dynamics in enzyme evolution. In some cases, connections among hydration layer dynamics, solvation shell structure, protein surface structure and their function has been revealed. Particular attention was paid to works in which a relationship is established between specific stages of the enzymatic process and specific molecular dynamic modes.

Enthalpy–entropy compensation (EEC) referred to the behavior of a series of closely related enzyme reactions exhibits a linear relationship between one of the following kinetic or thermodynamic parameters and isokinetic or thermodynamic temperatures. The enthalpy–entropy compensation effect in enzymatic catalysis was first described by Likhtenshtein in 1966. In this chapter, separate sections are devoted to statistical artifacts at apparent enthalpy–entropy compensation, theoretical consideration of EEC, EES for small molecules, EES for peptides and proteins, and miscellaneous examples of EEC in enzyme catalysis. The following compensation pairs of energy–entropy activation were considered: hydrolysis catalyzed by carboxypepsidase, transformation fumarate to malate catalyzed be fumarase, α-amylase reaction of starch hydrolysis, reactions catalyzed by unvertase, urease, lipase as well as enthalpy–entropy compensation at binding various inhibitors with catalase, and at inhibition bacterial luminescence. The predominant contribution of water reorganization to the thermodynamic and kinetic parameters of enzymatic reactions was stressed.

Artificial enzymes are a class of catalysts being potentially viable alternatives to natural enzymes. Enzyme mimics can have the important advantages including tunable structures and catalytic efficiencies, stability in experimental conditions, lower cost, and simple synthetic routes to their preparation. Cyclodextrins, metal complexes, porphyrins, polymers, dendrimers, membranes, nanoparticles, organic and metalorganic compounds and biomolecules can serve as the basis for an artificial catalyst. On the path of mimicry, seven stages have been formulated. Typical examples of enzyme mimetic catalysts, performance, the reactions they catalyze, and the main source of their catalytic efficiency were considered. Catalytically active nanomaterials (nanozymes) also show several advantages over natural enzymes. A wide range of materials are used as nanoenzymes, such as nanoparticles of Fe₃O₄, Prussian blue, Mn₃O₄, CeO₂, WO₂, NiCo₂O₄@MnO₂, graphene oxide, graphene-hemin nanocomposites, carbon nanotubes, carbon nanodots, mesoporous silica-encapsulated gold nanoparticles, gold nanoclusters, Pep–Au-NP = peptide–gold nanoparticle conjugate gold nanoparticles, ferrous ferrocyanide, nonheme iron, vanadium, and copper complexes bearing hemicryptophane. Metal atom (M = K, Ti, Fe, Co, Ni, Cu, Rh) doping on a Mo6S8 cluster and mononuclear rhodium species anchored on a zeolite or titanium dioxide illustrate single-atom catalysis. Nanozymes can exhibit activity of oxidase, catalase, superoxide dismutase, DNAse, alkaline phosphatase phospholipase, topoisomerase and can display antioxidant and biofilm activity. Molecular imprinting is a technique to create template (analogs of substrates, transition states, or products) shaped cavities in polymer matrices with predetermined selectivity and high affinity in a specific function. Schemes of the principle of molecular imprinting recognition, a molecularly imprinted catalyst scheme for selective catalysis, and, as an illustration, imprinted catalytic system based on methacrylic layer polymerization were presented. Despite the obvious advantages of artificial enzymes, it is still not possible to achieve the activity of natural catalysts.

The studies of artificial photosynthesis use biomimetic techniques to replicate the process of natural photosynthesis, which employs abundant resources of sunlight, water, and carbon dioxide to produce oxygen and energy-rich carbohydrates. In this chapter, requirements for successive mimicking of these processes and means for fulfillment, these requirements were formulated. The compounds such as porphyrins or other cyclic tetrapyrroles, coratenoides, ruthenium and zinc complexes, fullerenes, borondipyrromethene, melanin, polyphenylene, go forth, can be light harvesters. Examples of charge separation in artificial donor–acceptor and pairs and photophysical and photochemical processes in dual flourophore-nitroxide molecules were presented and discussed in separate sections. The evolution dioxygen from water in a cluster of transition metals in the biological systems at the absorption of four light quanta of low energy occurs by a sequence of elementary steps: four one-electron steps of oxidation of the manganese complex and, most probably by one four-electron elementary step of O₂ evolution (Sect. 3.​5.​2). In approaching mimicking problem, a number of artificial manganese clusters and other transition metal clusters were synthesized and investigated. The reduction, or fixation, of carbon dioxide is a vital process associated with natural and artificial photosynthesis in which can generate hydrocarbon fuels such as formic acid, methanol, carbon monoxide, and methane, with the utilization of hydrogen. Employing of transition metal complexes catalyzed carboxylation reactions with CO₂ and carboxylation of preactivated substrates and examples of successive carboxylation were presented The chapter focused on advances in the field of direct carboxylation reactions of C(sp³)–H and C(sp²)–H bonds using CO₂ encompassing both transition metal catalysis and base-mediated approach and on applications of electrochemical and photochemical methods.

Iron–sulfur clusters occur in many biological systems, often as components of electron transfer proteins and as the H-cluster in hydrogenase and the P-cluster and FeMo-cofactor in nitrogenase. This chapter describes significant progress made in synthetical, compositional and functional aspects of the nitrogenase-like clusters through the recent decades. New synthesized clusters were found to react with dihydrogen and proton mimicking the hydrogenase reaction S (H₂ + A_ox → 2H⁺ + A_red and 2H⁺ + D_red → H₂ + D_ox). Number of synthetic rhombic and cuboidal clusters could convert N₂ to hydrazine and ammonium modeling the nitrogenase reaction. As an example of four electron mechanism of dinitrogen reduction, four nuclear complex V(II)-pyrocatechol (V^(II)₄) was utilized in a homogeneous reaction V^(II)₄N₂ + V^(II)₄ + 8H⁺ → 8V^(III) + 2NH₃ + H₂O. The Chatt Cycle, a hypothetical model for the mechanism of nitrogenase action at the atomic level based upon reactions of molybdenum compounds and scheme of the multi-stage nitrogenase process were considered.

In hydroxylation reactions: 2R₃C–H + O₂\(\to\) 2R₃C–OH and R₃C–H + O₂ + 2e^– + 2H⁺ \( \to\) R₃C–OH + H₂O a substrate C–H bond is converted to an alcohol group. A mechanism of abstracting a hydrogen atom from the substrate by a high active intermediate, an iron(IV)-oxo porphyrin radical intermediate (Compound I) (rebound mechanism) was proposed for cytochrome P450. A similar rebound mechanism was suggested for the methane monooxygenase (MMO). The large number of complexes mimicking cytochromes P450 including iron protoporphyrin IX were synthesized and their hydroxylation activity was tested. To avoid problems associated with indirect pathway for conversion methane to methanol, which widely used in chemical industry using 1000 °C and elevated pressure, numerous complexes mimicking the structure and enzymatic activity of MMO were prepared. Among them are the following compounds: (μ-oxo)(μ-hydroxo)diiron (III) core [Fe₂(O)(OH)(6TLA)₂(ClO₄)₃], [Fe₂(O)₂(6TLA)₂(ClO₄)₂] (II), a (-1,2-peroxo)bis(-carboxylato)diiron(III). According to the Shilov Cycle, CH₄ oxidation in mild condition is catalyzed by PtCl₂ in an aqueous solution, where PtCl₆ ²⁻ acts as the ultimate oxidizing agent. Artificial hydrolases was utilized to catalyze the cleavage a number of substrates such as p-nitrophenyl acetate, glycerophospholipids, the phosphodiester bond of DNA, phosphate diester model of RNA, RNA model substrate 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). As a successful modeling nature’s catalytic triad, consisting of a hydroxyl group of a serine residue, an imidazole group of a histidine residue, and a carboxyl group of an aspartic acid, the miniature organic model of chymotrypsin, was designed on the basis of cyclodextrin. A water-soluble fullerene functionalized with carboxylic acid moieties, C60-1, mono- and binuclear metalloenzymes, carbon-based nanozymes, peptide-based compounds are examples of successful mimicry of hydrolases.