Spectroscopic Characterization of an Eight‐Iron Nitrogenase Cofactor Precursor that Lacks the “9th Sulfur”

Nitrogenases catalyze the reduction of N₂ to NH₄⁺ at its cofactor site. Designated the M‐cluster, this [MoFe₇S₉C(R‐homocitrate)] cofactor is synthesized via the transformation of a [Fe₄S₄] cluster pair into an [Fe₈S₉C] precursor (designated the L‐cluster) prior to insertion of Mo and homocitrate. We report the characterization of an eight‐iron cofactor precursor (designated the L*‐cluster), which is proposed to have the composition [Fe₈S₈C] and lack the “9^th sulfur” in the belt region of the L‐cluster. Our X‐ray absorption and electron spin echo envelope modulation (ESEEM) analyses strongly suggest that the L*‐cluster represents a structural homologue to the l ‐cluster except for the missing belt sulfur. The absence of a belt sulfur from the L*‐cluster may prove beneficial for labeling the catalytically important belt region, which could in turn facilitate investigations into the reaction mechanism of nitrogenases.     

Bioelectrocatalytic Activity of Immobilized Alcohol Oxidase on 4‐(pyrrole‐1‐yl) Benzoic Acid Modified Carbon Nanotubes

A novel bioelectrocatalytic system was prepared by immobilizing alcohol oxidase (AOx) onto multiwalled carbon nanotubes (MWCNT) modified with 4‐(pyrrole‐1‐yl) benzoic acid (PyBA). Functional carboxylic groups from PyBA create covalent amide linkages with amine groups from the enzyme molecule and provide an anchor for the effective immobilization of AOx improving the stability of the whole system. The immobilized enzyme displayed a pair of reversible redox peaks of flavin adenine dinucleotide (FAD) cofactor with the formal potential E^0’=?0.451 V. The response showed a surface‐controlled electrode process with the heterogeneous electron transfer rate constant k_s=2.7 s^?1. Under aerobic conditions AOx(FADH₂) can be oxidized to AOx(FAD) by oxygen, which then reacts with ethanol decreasing the cathodic response, which could be used for ethanol detection with a high sensitivity 13.1 μA mM^?1 cm^?2. The lack of bioactivity towards ethanol in anaerobic conditions suggests the presence of two types of AOx molecules in the system: active with oxygen maintaining the direct electron transfer feature and not active without a redox mediator, due to the deeply embedded FAD cofactor. The polarization curve showed that the electrooxidation current of ethanol appears at ?410 mV and reaches 2.0 µA cm^?1 at ?300 mV. In this case, the bioactivity of AOx to ethanol can be observed offering promising solution for the development of mediatorless systems for application to biosensors and biofuel cells.     

The Bacterial [Fe]‐Hydrogenase Paralog HmdII Uses Tetrahydrofolate Derivatives as Substrates

[Fe]‐hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl‐tetrahydromethanopterin (methenyl‐H₄MPT⁺) with H₂. H₄MPT is a C1‐carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA–G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron–guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H₄MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1‐carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog.     

Mutation of Tyr-218 to Phe in <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD⁺). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD⁺ by mutating Tyr²¹⁸ to Phe (Y218F 2° ADH). This mutation increased the K _m(app) for NADP⁺ 200-fold while decreasing the K _m(app) for NAD⁺ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD⁺, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD⁺. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD⁺ was electrically recycled 6.7 × 10⁴ times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.     

Formation of the iron-oxo hydroxylating species in the catalytic cycle of aromatic amino acid hydroxylases

The first part of the catalytic cycle of the pterin‐dependent, dioxygen‐using nonheme‐iron aromatic amino acid hydroxylases, leading to the Fe^IV?O hydroxylating intermediate, has been investigated by means of density functional theory. The starting structure in the present investigation is the water‐free Fe? O₂ complex cluster model that represents the catalytically competent form of the enzymes. A model for this structure was obtained in a previous study of water‐ligand dissociation from the hexacoordinate model complex of the X‐ray crystal structure of the catalytic domain of phenylalanine hydroxylase in complex with the cofactor (6R)‐L ‐erythro‐5,6,7,8‐tetrahydrobiopterin (BH₄) (PAH‐Fe^II‐BH₄). The O? O bond rupture and two‐electron oxidation of the cofactor are found to take place via a Fe‐O‐O‐BH₄ bridge structure that is formed in consecutive radical reactions involving a superoxide ion, O₂^?. The overall effective free‐energy barrier to formation of the Fe^IV?O species is calculated to be 13.9 kcal mol^?1, less than 2 kcal mol^?1 lower than that derived from experiment. The rate‐limiting step is associated with a one‐electron transfer from the cofactor to dioxygen, whereas the spin inversion needed to arrive at the quintet state in which the O? O bond cleavage is finalized, essentially proceeds without activation.     

The Nitrogenase FeMo‐Cofactor Precursor Formed by NifB Protein: A Diamagnetic Cluster Containing Eight Iron Atoms

The biological activation of N₂ occurs at the FeMo‐cofactor, a 7Fe–9S–Mo–C–homocitrate cluster. FeMo‐cofactor formation involves assembly of a Fe_6–8–S_X–C core precursor, NifB‐co, which occurs on the NifB protein. Characterization of NifB‐co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe–4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB‐co and deliver it to the NifEN protein, upon which FeMo‐cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB‐co‐loaded form provided an opportunity for Mössbauer analysis of NifB‐co. The results indicate that NifB‐co is a diamagnetic (S=0) 8‐Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the ⁵⁷Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB‐co is either a 4Fe²⁺–4Fe³⁺ or 6Fe²⁺–2Fe³⁺ cluster having valence‐delocalized states.     

Spata‐13,17‐diene Synthase—An Enzyme with Sesqui‐, Di‐, and Sesterterpene Synthase Activity from Streptomyces xinghaiensis

A terpene synthase from the marine bacterium Streptomyces xinghaiensis has been characterised, including a full structure elucidation of its products from various substrates and an in‐depth investigation of the enzyme mechanism by isotope labelling experiments, metal cofactor variations, and mutation experiments. The results revealed an interesting dependency of Mn²⁺ catalysis on the presence of Asp‐217, a residue that is occupied by a highly conserved Glu in most other bacterial terpene synthases.     

Combining a Nitrogenase Scaffold and a Synthetic Compound into an Artificial Enzyme

Nitrogenase catalyzes substrate reduction at its cofactor center ([(Cit)MoFe₇S₉C]^n?; designated M‐cluster). Here, we report the formation of an artificial, nitrogenase‐mimicking enzyme upon insertion of a synthetic model complex ([Fe₆S₉(SEt)₂]^4?; designated Fe₆^RHH) into the catalytic component of nitrogenase (designated NifDK^apo). Two Fe₆^RHH clusters were inserted into NifDK^apo, rendering the conformation of the resultant protein (designated NifDK^Fe) similar to the one upon insertion of native M‐clusters. NifDK^Fe can work together with the reductase component of nitrogenase to reduce C₂H₂ in an ATP‐dependent reaction. It can also act as an enzyme on its own in the presence of Eu^IIDTPA, displaying a strong activity in C₂H₂ reduction while demonstrating an ability to reduce CN^? to C₁–C₃ hydrocarbons in an ATP‐independent manner. The successful outcome of this work provides the proof of concept and underlying principles for continued search of novel enzymatic activities based on this approach.     

Modulation of the F1FO‐ATPase function by butyltin compounds

Among butyltin compounds, tributyltin (TBT), widely exploited in the past in antifouling paints for its biocidal properties, is long known as one of the most harmful sea contaminants. Among the ascertained and universal toxicity mechanisms, TBT targeting F₁F_O‐ATPase and thus impairing cell bioenergetics, is here reviewed. While TBT effects on F₁F_O‐ATPase have been investigated for decades, the possible impact of the derivatives dibutyltin (DBT) and monobutyltin (MBT), produced by abiotic and/or biotic dealkylation of TBT and usually considered far less toxic, have been poorly explored up until now. Butyltin effects on F₁F_O‐ATPase and their underlying action mechanism seem to be tightly structure dependent. Butyltins are membrane‐active toxicants. Owing to its more pronounced lipophilicity TBT targets the transmembrane F_O sector, blocks ionic translocation and causes a dose‐dependent loss of sensitivity to F_O inhibitors such as oligomycin and N,N′‐dicyclohexylcarbodiimide. DBT strongly inhibits F₁F_O‐ATPase activity by competing with the Mg⁺² cofactor in the F₁ catalytic site but is ineffective on the enzyme sensitivity to F_O inhibitors. MBT is apparently ineffective. The possible contribution of DBT to the overall butyltin toxicity on membrane systems may not be neglectable since usually TBT coexists with its derivatives in organotin‐exposed animal tissues. Copyright © 2013 John Wiley & Sons, Ltd.     

Enantiocomplementary Epoxidation Reactions Catalyzed by an Engineered Cofactor‐Independent Non‐natural Peroxygenase

Peroxygenases are heme‐dependent enzymes that use peroxide‐borne oxygen to catalyze a wide range of oxyfunctionalization reactions. Herein, we report the engineering of an unusual cofactor‐independent peroxygenase based on a promiscuous tautomerase that accepts different hydroperoxides (t‐BuOOH and H₂O₂) to accomplish enantiocomplementary epoxidations of various α,β‐unsaturated aldehydes (citral and substituted cinnamaldehydes), providing access to both enantiomers of the corresponding α,β‐epoxy‐aldehydes. High conversions (up to 98 %), high enantioselectivity (up to 98 % ee), and good product yields (50–80 %) were achieved. The reactions likely proceed via a reactive enzyme‐bound iminium ion intermediate, allowing tweaking of the enzyme's activity and selectivity by protein engineering. Our results underscore the potential of catalytic promiscuity for the engineering of new cofactor‐independent oxidative enzymes.     

Discovery of a Fungal Multicopper Oxidase That Catalyzes the Regioselective Coupling of a Tricyclic Naphthopyranone To Produce Atropisomers

Atropisomeric dinapinones A1 and A2 (DPA1 and DPA2) were isolated from a culture of Talaromyces pinophilus FKI‐3864. Monapinone coupling enzyme (MCE), which dimerizes naphthopyranone monapinone A (MPA), was purified from a cell‐free extract of T. pinophilus FKI‐3864. MCE regioselectively dimerizes MPA at the 8,8′‐positions to synthesize the atropisomers DPA1 and DPA2 in a ratio of approximately 1:2.5 without a cofactor. The optimal pH value and temperature for MCE were 4.0 and 50 °C, and the apparent K_m and V_max values for MPA were (72.7±23.2) μm and (1.21±0.170) μmol min⁻¹ mg⁻¹ protein. The MCE polypeptide is significantly homologous with multicopper oxidases. Heterologous expression of MCE and functional analysis confirmed that MCE catalyzes the regioselective coupling reaction of MPA to produce DPA. No fungal multicopper oxidase has previously been reported to catalyze regioselective intermolecular oxidative phenol coupling to produce naphthopyranone atropisomers.     

Dioxygen Sensitivity of [Fe]‐Hydrogenase in the Presence of Reducing Substrates

Mono‐iron hydrogenase ([Fe]‐hydrogenase) reversibly catalyzes the transfer of a hydride ion from H₂ to methenyltetrahydromethanopterin (methenyl‐H₄MPT⁺) to form methylene‐H₄MPT. Its iron guanylylpyridinol (FeGP) cofactor plays a key role in H₂ activation. Evidence is presented for O₂ sensitivity of [Fe]‐hydrogenase under turnover conditions in the presence of reducing substrates, methylene‐H₄MPT or methenyl‐H₄MPT⁺/H₂. Only then, H₂O₂ is generated, which decomposes the FeGP cofactor; as demonstrated by spectroscopic analyses and the crystal structure of the deactivated enzyme. O₂ reduction to H₂O₂ requires a reductant, which can be a catalytic intermediate transiently formed during the [Fe]‐hydrogenase reaction. The most probable candidate is an iron hydride species; its presence has already been predicted by theoretical studies of the catalytic reaction. The findings support predictions because the same type of reduction reaction is described for ruthenium hydride complexes that hydrogenate polar compounds.     

Structure and Mechanism Leading to Formation of the Cysteine Sulfinate Product Complex of a Biomimetic Cysteine Dioxygenase Model

Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3‐His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme–substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work‐up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X‐ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys‐SO₂^? functional group was found to be bound in an η²‐O,O‐coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η²‐O,O‐binding mode for synthetic as well as the natural enzyme.     

Substrate‐Dependent H/D Kinetic Isotope Effects and the Role of the Di(μ‐oxo)diiron(IV) Core in Soluble Methane Monooxygenase: A Theoretical Study

Soluble methane monooxygenase (sMMO) is an enzyme that converts alkanes to alcohols using a di(μ‐oxo)diiron(IV) intermediate Q at the active site. Very large kinetic isotope effects (KIEs) indicative of significant tunneling are observed for the hydrogen transfer (H‐transfer) of CH₄ and CH₃CN; however, a relatively small KIE is observed for CH₃NO₂. The detailed mechanism of the enzymatic H‐transfer responsible for the diverse range of KIEs is not yet fully understood. In this study, variational transition‐state theory including the multidimensional tunneling approximation is used to calculate rate constants to predict KIEs based on the quantum‐mechanically generated intrinsic reaction coordinates of the H‐transfer by the di(μ‐oxo)diiron(IV) complex. The results of our study reveal that the role of the di(μ‐oxo)diiron(IV) core and the H‐transfer mechanism are dependent on the substrate. For CH₄, substrate binding induces an electron transfer from the oxygen to one Fe^IV center, which in turn makes the μ‐O ligand more electrophilic and assists the H‐transfer by abstracting an electron from the C?H σ orbital. For CH₃CN, the reduction of Fe^IV to Fe^III occurs gradually with substrate binding and H‐transfer. The charge density and electrophilicity of the μ‐O ligand hardly change upon substrate binding; however, for CH₃NO₂, there seems to be no electron movement from μ‐O to Fe^IV during the H‐transfer. Thus, the μ‐O ligand appears to abstract a proton without an electron from the C?H σ orbital. The calculated KIEs for CH₄, CH₃CN, and CH₃NO₂ are 24.4, 49.0, and 8.27, respectively, at 293 K, in remarkably good agreement with the experimental values. This study reveals that diverse KIE values originate mainly from tunneling to the same di(μ‐oxo)diiron(IV) core for all substrates, and demonstrate that the reaction dynamics are essential for reproducing experimental results and understanding the role of the diiron core for methane oxidation in sMMO.     

Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate

A non‐natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non‐natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD‐dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO₂ source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When ¹³C‐isotope‐labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom‐economic strategy for CO₂ fixation where formate is used in lieu of CO₂ and offers dedicated reducing power.     

Synthesis and Biological Evaluation of Potent Bisubstrate Inhibitors of the Enzyme Catechol O‐Methyltransferase (COMT) Lacking a Nitro Group

Inhibition of the enzyme catechol O‐methyltransferase (COMT) represents a viable strategy for regulation of the catabolism of catecholamine neurotransmitters or their precursors, and is of considerable interest in the therapy of Parkinson's disease. Herein, we report the development of a new generation of potent bisubstrate inhibitors of COMT derived from nitro‐substituted ligand 1 (K_i = 28 nM , Table 1), which achieve high biological activity despite the lack of a NO₂ substituent on the catechol moiety. Their synthesis takes advantage of a convergent approach, in which a series of functionalized catechol intermediates is prepared (Schemes 2–7) and coupled to a common adenosine‐derived allylic amine building block (Scheme 8). Biological activities of the newly synthesized inhibitors, determined by in vitro enzymatic assay and kinetic studies, clearly demonstrate that high inhibitory potency of the bisubstrate inhibitors is not correlated with the pK_a of the catechol OH groups. Aromatic residues, connected to the catechol via a biaryl‐type linkage, were found to maximally benefit from additional favorable hydrophobic interactions with the enzyme and thus to be preferred replacements of the NO₂ group in 1 . A competitive kinetic inhibition mechanism (Fig. 2) with respect to the cofactor binding site was confirmed in all cases, supporting a bisubstrate inhibition mode for inhibitors 2 – 19 .     

Influence of the protein and DFT method on the broken‐symmetry and spin states in nitrogenase

The enzyme nitrogenase contains a complicated MoFe₇CS₉ cofactor with 35 possible broken‐symmetry (BS) states. We have studied how the energies of these states depend on the geometry, the surrounding protein, the DFT functional and the basis set, studying the resting state, a one‐electron reduced state and a protonated state. We find that the effect of the basis set is small, up to 11 kJ/mol. Likewise, the effect of the surrounding protein is restricted, up to 10 and 7 kJ/mol for the electrostatic and van der Waals energy terms. Single‐point energies calculated on a single geometry give a good correlation (R² = 0.92‐0.98) to energies calculated after geometry optimization, but some BS states may be disfavored by up to 37 kJ/mol. A change from the pure TPSS functional to the hybrid B3LYP functional may change the relative energies by up to 58 kJ/mol and the correlation between the two results is only 0.57‐0.72. Both functionals agree that BS7 is the most stable BS state and that the ground spin state is the quartet for the resting state and the quintet for the reduced state. With the TPSS functional, the BS6 state is the second most stable state, always at least 21 kJ/mol less stable than the BS7 state. However, with the B3LYP functional, BS10 is the second most stable state and for the protonated state it comes close in energy. Based on these results, we suggest a procedure how to consider the 35 BS states in future investigations of the nitrogenase reaction mechanism.     

The Cofactor of Tetrachloroethene Reductive Dehalogenase of Dehalospirillum multivorans Is Norpseudo‐B12, a New Type of a Natural Corrinoid

The corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans was isolated in its Coβ‐cyano form. This cofactor represents the main corrinoid found in D. multivorans cells. Analysis of the isolated cyano‐corrinoid by a combination of HPLC and UV/VIS‐absorbance spectroscopy revealed it to be nonidentical to a variety of known natural B₁₂ derivatives. From high‐resolution mass‐spectrometric analysis, the molecular formula of the corrinoid isolated from D. multivorans could be deduced as C₅₈H₈₁CoN₁₇O₁₄P. The sample of the novel corrinoid from D. multivorans was further analyzed by UV/VIS, CD, and one‐ and two‐dimensional ¹H‐, ¹³C‐, and ¹⁵N‐NMR spectroscopy, which indicated its structure to be closely related to that of pseudovitamin B₁₂ (Coβ‐cyano‐7″‐adeninylcobamide). By the same means, the corrinoid could be shown to differ from pseudovitamin B₁₂ only by the lack of the methyl group attached to carbon 176, and, therefore, it was named norpseudovitamin B₁₂ (or, more precisely, 176‐norpseudovitamin B₁₂). Norpseudovitamin B₁₂ represents the first example of a ‘complete’ B₁₂‐cofactor that lacks one of the methyl groups of the cobamide moiety, indicating that the B₁₂‐biosynthetic pathway in D. multivorans differs from that of other organisms. X‐Ray crystal‐structures were determined for norpseudovitamin B₁₂ from D. multivorans and the analogues pseudovitamin B₁₂ and factor A (Coβ‐cyano‐7″‐[2‐methyl]adeninylcobamide). These first accurate crystal structures of complete corrinoids with an adeninyl pseudonucleotide confirmed the expected coordination properties around Co and corroborated the close conformational similarity of the nucleotide moieties of norpseudovitamin B₁₂ and its two homologues.     

Functional chemical models of multielectron water oxidation in photosynthesis

The study of water oxidation by Mn _n ^IV clusters, which are functional chemical models of the manganese cofactor (enzyme that oxidizes water in photosystem II of natural photosynthesis) has demonstrated that a Mn₂^IV cluster oxidizes two water molecules to form one oxygen molecule, Mn₄^IVoxidizes four water molecules to form two O₂ molecules, and Mn₈^IVoxidizes eight water molecules to form four O₂ molecules. A Mn₆^IV cluster oxidizes six water molecules to two ozone molecules, whereas Mn₁₂^IV oxidizes twelve water molecules to four ozone molecules. The six-electron oxidation of water to ozone is also observed in photosynthesis in red and brown sea algae under conditions of water deficit. It is hypothesized that, in the case of water deficit in algae, the manganese cofactor with four manganese ions turns into the cofactor with six manganese ions.     

The Reaction Mechanism and Kinetics Data of Racemic Atenolol Kinetic Resolution via Enzymatic Transesterification Process Using Free Pseudomonas fluorescence Lipase

A thorough study on free‐enzyme transesterification kinetic resolution of racemic atenolol in a batch system was investigated to gain knowledge for (S)‐atenolol kinetics. Analyses of enzyme kinetics using Sigma‐Plot 11 Enzyme Kinetics Module on the process are based‐on Michaelis–Menten and Lineweaver–Burk plot, which give first‐order reaction and ordered‐sequential Bi–Bi mechanism, where V_max, K_{M‐vinyl acetate}, and K_{M‐(S)‐atenolol} are 0.80 mM/h, 29.22 mM, and 25.42 mM, respectively. Further analyses on enzyme inhibitions find that both substrates inhibit the process where (S)‐atenolol and vinyl acetate develop competitive inhibition and mixed inhibition, respectively. Association of (S)‐atenolol with free enzyme to inhibit the enzyme is higher than reaction of active enzyme–substrate complex with vinyl acetate.